Homo erectus, appears to have shared her island home with dwarf elephants and giant rats.The recent discovery of a Hobbit-like hominid on the Indonesian island of Flores was startling in some respects—its rather modern existence, for one—but it represents a classic case of Darwinian evolution. For reasons that are not entirely clear, when animals make their way to isolated islands, they tend to evolve relatively quickly toward an outsized or pint-sized version of their mainland counterpart. Following this evolutionary script, the Flores woman, presumably a downsized version of Harpagornis moorei), which had a wingspan up to 3 meters. Though Haast's eagle could fly—and presumably used its wings to launch brutal attacks on the hapless moa—its body mass (10–14 kilograms) pushed the limits for self-propelled flight.Perhaps the most famous example of an island giant—and, sadly, of species extinction—is the dodo, once found on the Indian Ocean island of Mauritius. When the dodo's ancestor (thought to be a migratory pigeon) settled on this island with abundant food, no competition from terrestrial mammals, and no predators, it could survive without flying, and thus was freed from the energetic and size constraints of flight. New Zealand also had avian giants, now extinct, including the flightless moa, an ostrich-like bird, and Haast's eagle (As extreme evolutionary examples, these island birds can offer insights into the forces and events shaping evolutionary change. In a new study, Michael Bunce et al. compared ancient mitochondrial DNA extracted from Haast's eagle bones with DNA sequences of 16 living eagle species to better characterize the evolutionary history of the extinct giant raptor. Their results suggest the extinct raptor underwent a rapid evolutionary transformation that belies its kinship to some of the world's smallest eagle species.Hieraaetus. Surprisingly, the genetic distance separating the giant eagle and its more diminutive Hieraaetus cousins from their last common ancestor is relatively small.The authors characterized the rates of sequence evolution within mitochondrial DNA to establish the evolutionary relationships between the different eagle species. Their analysis places Haast's eagle in the same evolutionary lineage as a group of small eagle species in the genus Without the fossils to directly determine divergence times, Bunce et al. relied on molecular dating techniques that use the rate of sequence evolution in the genes studied to establish the relative evolutionary ages of the eagles. Proposing a divergence date of roughly 0.7–1.8 million years ago, the authors acknowledge that while this is the “best available approximation of the ‘true’ date,” additional molecular data could help refine the estimate. Whatever the date of divergence, the extinct giant eagle is clearly an anomaly among the eagles studied here. The increase in body size—by at least an order of magnitude in less than 2 million years—is particularly remarkable, Bunce et al. argue, since it occurred in a species still capable of flight.Hieraaetus species, the authors recommend reclassifying the New Zealand giant as Hieraaetus moorei. This study shows how quickly morphological changes can occur in vertebrate lineages within island ecosystems. Could it be that anthropologists might some day uncover evidence of a giant version of the Flores woman?The absence of mammalian competitors facilitated the evolution of much larger eagles and owls on Cuba and may have likewise precipitated the rapid morphological shift seen here. Haast's eagle, the authors write, “represents an extreme example of how freedom from competition on island ecosystems can rapidly influence morphological adaptation and speciation.” Given its similarity to the smaller


Despite some high-profile setbacks in gene therapy over the past few years, scientists have not lost hope that targeted gene transfer will one day treat a wide range of acquired and congenital diseases. After two young gene therapy patients developed a leukemia-like disorder last year—apparently because the viral vector used to carry the corrective gene activated a cancer-causing gene—researchers redoubled their efforts to develop safer, more effective retroviral gene delivery methods. Such efforts depend on understanding how and where retroviral vectors integrate into the genome.In a new study, Cynthia Dunbar and colleagues describe a nonhuman primate model that mimics gene therapy protocols in humans, and report the integration biases of two classes of retroviral vectors being developed for clinical trials. The retroviruses, the authors show, display clear preferences that not only suggest different genomic integration mechanisms but also have different implications for safety.Retroviral gene therapy exploits a retrovirus's skill at entering a cell, infiltrating its genome, and hijacking its molecular machinery for its own reproductive advantage. In gene therapy, therapeutic genes largely take the place of viral genes, and so the “infected” cell churns out beneficial gene products, not viruses. Before a retrovirus can integrate into a host cell, however, it must copy its genome—which is encoded in RNA—into DNA, so the cell's copying machinery will recognize it. After generating this “pre-integration complex,” the virus must access the cell's chromosomal DNA, which lies behind a nuclear barrier. Different retroviruses accomplish this task in different ways.Lentiviruses—which include AIDS and SIV (simian immunodeficiency virus)—can infect nondividing cells simply by slipping through nuclear pores. Oncoretroviruses—such as murine leukemia virus, or MLV, the vector type used for the vast majority of previous clinical trials, including the trial complicated by leukemia in two patients—must wait until the nuclear membrane dissolves during cell division. Once integrated into the host genome, the provirus—and its therapeutic gene—will persist through each new cell division—a trait that underlies its usefulness as a vector as well as its risk. Retroviruses that insert near proto-oncogenes can activate these genes and set the cell on the path to tumorigenesis. Until recently, researchers assumed this risk was extremely low because retroviral integration was thought to be random—an assumption recently undercut by a number of studies that mapped retroviral integration in different cell lines.Dunbar and colleagues take these studies a step further by mapping the integration patterns of MLV and SIV vectors in hematopoietic stem cells (HSCs) of rhesus monkeys. HSCs are the cells typically used to carry these vectors for therapeutic applications involving any of the cell types, such as red blood cells, produced by the bone marrow. The monkeys had received infusions of HSCs carrying either the SIV or MLV vectors between six months and six years earlier. Two types of white blood cells (granulocytes and mononuclear cells) were harvested from the monkeys and evaluated for proviral insertion sites.Of nearly 1,000 integration sites identified, 760 could be mapped to unique corresponding sites in the human genome (432 MLV and 328 SIV). While both MLV and SIV vectors tended to integrate within genes, MLV showed a strong preference for the starting end of genes, most likely to result in gene activation. In contrast, SIV showed a preference for genomic regions of high gene density, but not for specific sites within a gene. Surprisingly, MLV targeted one gene—known previously to be involved in spontaneous leukemias and in murine retroviral oncogenesis—seven times, a “highly nonrandom” result suggesting that such insertions may occur far more often than previously thought. About 40 genes, including seven known oncogenes, were targeted more than once by one or both vectors. Such differences, Dunbar and colleagues note, “likely reflect the vectors' distinct mechanisms for accessing DNA and integrating,” which could in turn affect their risk of causing insertional mutagenesis. Even though the vectors tend to integrate nonrandomly and can target oncogenes, however, none of the monkeys showed signs of ill effects such as leukemia.But before any widespread applications of retroviral gene therapy can proceed, Dunbar and colleagues argue, potential risks of proviral insertion must be assessed in the specific cell types associated with different gene therapies. And with a model for long-term, genome-wide retroviral integration analysis that mimics human gene therapy protocols, the authors have made an important contribution toward that end.


The Campanulaceae (the "hare bell" or "bellflower" family) is a derived angiosperm family comprised of about 600 species treated in 35 to 55 genera. Taxonomic treatments vary widely and little phylogenetic work has been done in the family. Gene order in the chloroplast genome usually varies little among vascular plants. However, chloroplast genomes of Campanulaceae represent an exception and phylogenetic analyses solely based on chloroplast rearrangement characters support a reasonably well-resolved tree.rbcL sequence data. The results suggest some interesting and unexpected intrafamilial relationships. For example fifteen of the taxa form a derived clade; whereas the remaining three taxa – Platycodon, Codonopsis, and Cyananthus – form the basal clade. This major subdivision of the family corresponds to the distribution of pollen morphology characteristics but is not compatible with previous taxonomic treatments.Chloroplast DNA physical maps were constructed for eighteen representatives of the family. So many gene order changes have occurred among the genomes that characterizing individual mutational events was not always possible. Therefore, we examined different, novel scoring methods to prepare data matrices for cladistic analysis. These approaches yielded largely congruent results but varied in amounts of resolution and homoplasy. The strongly supported nodes were common to all gene order analyses as well as to parallel analyses based on ITS and Our use of gene order data in the Campanulaceae provides the most highly resolved phylogeny as yet developed for a plant family using only cpDNA rearrangements. The gene order data showed markedly less homoplasy than sequence data for the same taxa but did not resolve quite as many nodes. The rearrangement characters, though relatively few in number, support robust and meaningful phylogenetic hypotheses and provide new insights into evolutionary relationships within the Campanulaceae. Allied to the Campanulaceae are the Lobeliaceae, Cyphiaceae, Cyphocarpaceae, Nemacladaceae, Pentaphragmataceae, and Sphenocleaceae; at times, all of these taxa have been included in the Campanulaceae at varying taxonomic rank by different authors . Forty-two of the derived character states were unique to a single taxon and 37 were phylogenetically-informative. Six trees of 97 steps were obtained with consistency indices of 0.81 with autapomorphies included and 0.67 with autapomorphies excluded (CI = 0.81/0.67). Ten nodes were common to the six shortest trees Fig. . Eight oTo construct Matrix 2 and Matrix 3, we interpreted endpoints as events where possible. Under our interpretation, several types of rearrangements contributed to cpDNA evolution in the family, including multiple inversions (scored primarily as endpoints), five IR expansion or contraction events, eight transpositions, two deletions, and 14 large insertions greater than 5 kb in size . The strict consensus of the 241 trees includes six resolved nodes Fig.  all six Codonopsis, Platycodon, and Cyananthus are basal within the family. Analyses on Matrix 2 and 3 support a Codonopsis + Cyananthus sister group relationship and a monophyletic basal clade whereas the Matrix 1 analysis supports a Codonopsis + Platycodon sister group and a paraphyletic basal grade. Neither outcome is very well supported; the alternative scenarios each require only a single additional step in the other data set. Within the fifteen derived taxa some of the relationships are not resolved or resolved but weakly supported. However, some groupings are well supported in all analyses. The South African taxa, Merciera, Prismatocarpus and Roella, form a clade . Wahlenbergia is the sister to these three taxa in all analyses with varying levels of support . Other groupings include a Symphyandra + Edraianthus clade (BS = 86-91) and Legousia + Asyneuma + Petromarula + Triodanis (BS = 94-100).All results Figs. ,3,4,5 inCampanula + Adenophora, whereas Matrix 2 supported Adenophora + Jasione; Matrix 1 supported Codonopsis + Platycodon (BS = 50), whereas Matrix 2 and 3 supported Codonopsis + Cyananthus (BS = 94-99); and Matrix 1 supported  the placement of Cyananthus at the base of the derived clade, whereas Matrix 2 and 3 analyses supported the monophyly of the basal group (BS = 56-68). One clade, Legousia + Asyneuma (BS = 87), was recovered only by the Matrix 1 analysis within a clade not further resolved by the other gene order analyses.The five analyses had somewhat different characteristics Table . For exarbcL data from the same eighteen taxa (Table Musschia and Jasione varies between the rbcL and ITS results (the placement of these taxa is largely unresolved by the gene order data); 2) In both sequence-based trees, Campanula and Adenophora are separate lineages (rather than sister taxa) basal to the Legousia-Asyneuma-Triodanis-Petromarula clade, whereas in the gene order analyses they are allied to Symphyandra-Edraianthus, and Trachelium; 3) Matrix 1 supports a Legousia-Asyneuma clade, whereas a Legousia-Triodanis clade occurs in the sequence-based trees; and 4) Matrix 1 and ITS support a Codonopsis-Platycodon grouping within the basal clade, whereas rbcL and Matrix 2 and 3 analyses support Codonopsis-Cyananathus. Among these instances of disagreement between weakly supported nodes, there is no general pattern of disagreement between the sequence data and the gene order analyses. And among strongly supported nodes, again, there is complete agreement, among all analyses-sequence and gene order.We included sequence data here mainly to allow for a comparison with gene order data in terms of phylogenetic utility. The xa Table  providedxa Table , thirteexa Table  providedxa Table  was genexa Table . The ITSxa Table . In the The relatively large number of gene order mutations that have occurred in the Campanulaceae chloroplast genomes causes difficulties when interpreting their phylogenetic significance. The phylogenetic analysis of such a complex set of cpDNA rearrangements within a group of plants is without precedent. The first problem was simply defining individual mutational events. Although the ideal way to analyze rearrangement data is to determine presence or absence of specific events, in the Campanulaceae, this was not possible in many cases given our present knowledge. Where multiple overlapping rearrangements have occurred between genomes, the two specific endpoints that define a particular inversion may not be determinable. Because of the inherent complexity of the data, we felt a new method of character analysis of the rearrangement data was warranted. Our approach involved coding endpoints, along with more easily defined rearrangements, as characters for different cladistic analyses. Endpoints were defined as two non-contiguous tobacco regions that are now adjacent in the genomes of one or more species.Using endpoints as characters is advantageous. It allows for the incorporation into the analysis of data that could not be used if only unambiguously interpreted events were included. However, using endpoints as characters has several drawbacks, including the inadvertent weighting of certain events over others. Inversions necessarily produce two endpoints, and transpositions three, whereas gene losses and IR boundary changes produce a single endpoint. Therefore inversions would be included twice and transpositions three times if scored as "independent" endpoints rather than events. Plus, if both endpoints of an inversion are still intact in a genome, the inversion is scored twice, if only a single endpoint remains the inversion is counted only once, and if both endpoints have been lost (through further mutation) the inversion will not be included at all. This may represent a problem in the Campanulaceae analyses because there appears to be a mixture of event types and at least some endpoint reuse .Asyneuma, the 53–56 region has been secondarily removed from the IR and returned to near its original location leaving behind small portions of 53 and 56 in the IR, detectable using southern hybridization. In Wahlenbergia, Merciera, Prismatocarpus and Roella, the 53, 54 portion of the 53–56 block has moved from the IR back to the LSC. One explanation for the high level of rearrangement apparently associated with this segment is that the region contains a transposable element. Third, a possible duplicative transposition is suggested . Other rbcL. In matrix 3, only three endpoint characters (8.8% of parsimony-informative characters) are homoplastic, each changing one extra time over the tree. In contrast, within ITS, 18 characters (9.2% of parsimony-informative characters) change three or more extra times over the tree, and 71.9% of characters are homoplastic. Presumably because of this high level of homoplasy, only two nodes are retained in the consensus of all ITS trees from the shortest to 1% longer, whereas seven nodes are retained in matrix 3 trees "to 1% longer" – the highest number of any of the analyses. Matrix 3, the matrix in which characters are most interpreted as mutational events, is especially strong in its performance, exceeding both sequence data sets in average bootstrap value per resolved node and CI (in addition to those characteristics just discussed). This suggests that the closer we can get to scoring the actual mutations the stronger gene order data will perform. Although the endpoint only matrix provides useful insights on relationships, we would argue that the extent to which these gene order characters cannot recover the phylogeny is directly related to our ability to define individual mutational events.Not only do these data support a well-resolved phylogeny but they provide robust support of several nodes. Matrix 3 supports as many nodes at BS ≥ 90 as ITS and more than Codonopsis, Platycodon, and Cyananthus are basal in the family as supported by both gene order and sequence data. Takhtajan's system [Cyananthus (in its own tribe Cyanantheae) as the most primitive member of the family, placing Platycodon and Codonopsis in other tribes  and Codonopsis (colpate) are more closely related to each other than either is to Platycodon (colporate), which is also supported by the gene order tree . The gene order data indicate that the affinities of Jasione and Edraianthus lie with Northern Hemisphere species rather than with Wahlenbergieae. The gene order data also are compatible with other available nucleotide data in addition to those reported here [[Trachelium and Campanula. They also agreed in the grouping of Asyneuma and Petromarula. The only discrepancy was in the placement of Legousia; the serological study placed this genus basal to all others surveyed [In contrast, studies of pollen ultrastructure have indicated that nulaceae ,38. Thesnulaceae -41. The ed here [,11,42, Led here [ and are ed here [. Althougsurveyed .Wahlenbergia is primarily a Southern Hemisphere Old World genus [W. gloriosa, mapped for this study, is Australian [Roella, Merciera, and Prismatocarpus are all endemic to South Africa [Trachelium and Legousia clades are primarily European to Eurasian, although Triodanis is endemic to North America and Campanula has a few North American representatives [Musschia is endemic to the island of Madeira [The groups delimited by cpDNA rearrangements also exhibit geographical integrity. ld genus ; W. glorstralian . Roella,h Africa -47. The ntatives ,48-50. M Madeira .Cyananthus. Studies of pollen ultrastructure indicated that the Asian genera Codonopsis, Cyananthus, and Platycodon are basal members of the Campanulaceae [rbcL sequence data position the Campanulaceae sister to the North American family Nemacladaceae [Merciera, Prismatocarpus, Roella, and Wahlenbergia) are in a much more derived position in the cpDNA tree.There has been considerable debate regarding the relationships among the four centers of taxonomic diversity of the Campanulaceae: Asia, Europe , South Africa, and western North America. Bentham  hypothesnulaceae ,38. Recenulaceae ,53,54 inladaceae ,54. Our Musschia and Platycodon as Platycodinae, clearly incompatible with both our results and pollen evidence. Musschia is placed in the derived clade (A), although its exact placement varies among all the analyses, including rbcL and ITS. De Candolle [Merciera's taxonomic position because its four basal ovules and single-seeded (by abortion) unilocular capsule [Merciera with Wahlenbergia, Roella and Prismatocarpus in his Wahlenbergieae but also included other genera forming a polyphyletic group according to our results.In addition, the gene order data suggest affinities of several controversial genera Fig. . SchönlaCandolle  was unsu capsule  are uniq capsule . This ge capsule , but is  capsule  placed MAdenophora and Symphyandra have been segregated from Campanula based on the presence of a conspicuous tubular nectariferous disc and connate anthers, respectively. Adenophora and Campanula are sister taxa in the gene order analyses (except those based on Matrix 2) and Adenophora's chloroplast genome is derived relative to Campanula's  belong to Triodanis. As expected, Triodanis and Legousia belong to the same cpDNA clade (A2), united by an unusual mutation that transferred a large segment of the large single copy (LSC) region to the SSC region [Legousia has a putative transposition not found in Triodanis, whereas Triodanis has a unique large insertion [Much controversy surrounds the taxonomy of the genera ia e.g. ,48). McV. McVTrioia  the stronger will be the results. Even in cases such as this, with high levels of gene order complexity, cpDNA gene order mutations make excellent phylogenetic markers.Despite the difficulties in interpreting such a complex set of rearrangements, the systematic utility of chloroplast DNA in the Campanulaceae is evident. Our results support the division of the family into two groups previously unrecognized in any taxonomic treatment. In addition, numerous groupings within the larger, derived clade are strongly supported. The data indicate that traditional classifications based on fruit and ovary characters are unnatural, and suggest affinities of several difficult genera. Additional sampling within large genera, such as BamHI, BglII, EcoRI, EcoRV, HindIII, and SstI, and double digests were carried out using HindIII and the remaining five enzymes. Hybridization probes consisted of 106 small tobacco cpDNA probes (average size 1.2 kb) provided by J. Palmer [HindIII cpDNA fragments from Trachelium caeruleum of the Campanulaceae were also used as hybridization probes [Jasione and Roella [Roella because hybridization signals became increasingly weak in later rounds of hybridization. In Jasione, rearrangements involving the IR/SSC junction and SSC region prohibited the complete resolution of the map. The restriction site maps were then interpreted as linear "number" maps representing the hybridization patterns of 106 consecutively numbered tobacco cpDNA probes for the 18 taxa  seemed readily interpretable. For example, if a region of the genome was simply reversed in order  we assumed that an inversion had taken place to result in the different arrangements of gene segments. Likewise if genomes differed in content of the IR, we assumed that single duplication or loss events were responsible. Making such inferences, we constructed Matrix 2 that is composed of 31 events and 53 endpoints. We then went further and constructed hypotheses of rearrangement events to account for the differences among the genomes of the three major clades delimited among the fifteen derived taxa . If thesCladistic analyses were performed on each of the three data sets using equal weighting of all included characters. The second and third matrices were also analyzed giving weights of two to all non-endpoint characters. This weighting represents an attempt to compensate for the unintentional weight given to inversions in which both endpoints are present. This, of course, results in down-weighting inversions in which only one endpoint remains and fails to include inversions whose endpoints are both absent.rbcL data. The ITS sequences were generated and aligned by Eddie et al [rbcL data to add to the two taxa already available [rbcL sequence data from all of the eighteen mapped taxa. Exactly the same DNAs were used.Finally, to allow for a direct comparison of performance between gene order data and sequence data over the same taxa, we conducted maximum parsimony analyses of ITS and ie et al . We detevailable  so that rbcL sequences, we PCR-amplified about 1370 bp of the gene in 50 μl reactions containing: 1 μl unquantified total genomic DNA, 0.2 mM each dNTP, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.4 μM each primer, and 1 unit Taq polymerase. Cycling conditions were as follows: 1 95°C denaturation step for 3 minutes 30 seconds, 30 cycles of 1 minute at 95°C, 1 minute at 55°C, and 1 minute 30 seconds at 72°C, and finally a 7 minute 72°C step. The PCR primers plus two internal primers were used for sequencing; the forward amplification primer and two internal primers were designed by G. Zurawski . The Zurawski primer commonly used as the reverse amplification primer did not work in many Campanulaceae; we designed an alternative: 5'-GTATCCATTGCGCAAACTC-3'. For sequencing, two successful PCR reactions were combined and then cleaned (and concentrated) using the Qiagen QIAquick PCR Purification Kit . Depending on the concentration of the recovered product, 0.5–2 μl of this template was cycle sequenced and resolved on an ABI Prism 377 Automatic DNA Sequencer. Electropherograms were inspected, and then sequences were edited and assembled using Sequencher, vers 3.1 (Gene Codes Corp.) The sequences have been deposited in GenBank  . Tobaccoie et al  is availie et al . The strie et al  using 10rbcL sequence data, analysed ITS and rbcL data, and modified the thesis chapter for publication. RKJ assisted in all aspects of the work.MEC performed the DNA isolations and Southern hybridizations, mapped the genomes, performed character codings and analyses for Matrix 2 and Matrix 3, and wrote up her work as a chapter of her Ph.D. thesis. LAR confirmed genome maps and character codings, updated MEC's analyses, added MATRIX 1 and its analysis, generated the rbcL data, concatenated for each taxon. PAUP statements in the file identify each individual data matrix and the characters in Matrix 2 and 3 that are weighted in some analyses.One additional file is provided – data file 1. This file is in NEXUS format and includes data for Matrix 1, Matrix 2, and Matrix 3 encodings and the Click here for file


Identification and treatment of unrecognised asthmatics in the community is important for improving the health of the individual and minimising cost and quality of life burden. It is not practical to offer clinical diagnostic assessment to whole communities, and a simple tool such as a questionnaire is required to identify a smaller target group. Conventional questionnaire screening methods which separate individuals into positive and negative categories have resulted in large numbers of individuals requiring clinical assessment. This study has therefore developed and tested a weighted scoring system that prioritises those most urgently in need, based on their questionnaire responses.A stratified random sample of adult respondents to a general practice postal questionnaire survey were categorised 'asthmatic' or 'non-asthmatic' according to three expert physicians' opinions. Based on this categorisation, logistic regression was used to derive weights reflecting the relative importance of each question in predicting asthma, allowing calculation of weighted scores reflecting likelihood of asthma. Respondents scoring higher than a chosen threshold would be offered diagnostic examination.Age and presence of wheeze were most influential (weight 3) and overall weighted scores ranged from -1 to 13. Positive predictive values (PPV) were estimated. For example, setting the threshold score at nine gave an estimated PPV for asthma diagnosis of 93.5%, a threshold score of seven corresponded to PPV 78.8%. PPV estimates were supported by examining 145 individuals from a new survey.Weighted scoring of questionnaire responses provides a method for evaluating the priority level of an individual 'at a glance', minimising the resource wastage of examining false positives. There are individuals in the community who are asthmatic but are not receiving treatment because they are unknown to the medical services -5. DetecDefining additional categories according to the possible numbers (0 to 6) of 'yes' answers to the 6 key questions is too simplistic as it assumes equal weights for each question and takes no account of variables such as age and gender. The present paper describes the development of a more refined method, which includes additional questions and uses question weights to calculate a score for each respondent. This weighted score reflects probability of asthma, allowing clinical resources to be targeted to those most likely to have the disease. A score is chosen as a threshold and the questionnaire is given in an opportunistic fashion to patients consulting a medical practice for any reason. The questionnaire score is calculated 'at a glance' and those whose score is higher than the chosen threshold, and who are not already receiving asthma treatment, are offered diagnostic examination. The threshold can be adjusted to take resource limitations into account.The study described in this paper was based on data collected as part of the WYCAP investigation. WYCAP is a long term prospective investigation of the natural history of asthma in two general practice populations. It is based on a postal respiratory questionnaire adapted from the European Community Respiratory Health Questionnaire (ECRHQ) . Four poIt was necessary to select an asthma-enriched sample in order to determine response patterns of both asthmatic and non-asthmatic respondents . RespondThe weights were derived based on a linear logistic regression model, designed to take as input the questionnaire responses 1 to 11 and predict the probability of asthma for any individual from the wider community. The model associates each question on the questionnaire with a coefficient reflecting the relative importance of that question in determining probability of asthma. A positive value for a coefficient influences towards a decision that the individual is classified asthmatic, the higher the value the greater the effect. Negative coefficients influence away from a decision that the individual is classified asthmatic. In order to provide a model representative of the wider community, a cross-validation technique was used  in whichThe full set of postal responses to the 1995 questionnaire survey was used to assess effectiveness by estimating PPV of the scoring system in detecting undiagnosed asthmatics in the community. Weighted scores were calculated for all 1995 survey respondents and evidence of either 'asthma diagnosis ever' or 'prescription for asthma medication in the last 12 months' was obtained from their medical records. The percentage of undiagnosed asthmatics with a threshold score or higher, who could be expected to be confirmed clinically asthmatic, was estimated using a technique which referenced the probabilities of asthma of sample respondents, (based on the combined expert opinions), their population strata (based on number of 'yes' answers to the six key questions) and weighted scores (based on questionnaire responses) [see Additional file The estimates of PPV for the various score thresholds depend on the fact that higher scores correspond to higher probability of asthma. The weighted scoring system is valid if it can be shown to retain this property when applied to a new data set, and this was tested by comparing scores with clinical diagnosis of asthma in a subset of data collected in 2001. Sophisticated modelling techniques had been used to rank the 2001 survey questionnaires in order of probability of asthma as part of another study ,18. The The method of combining the three independent physician opinions differed from that used in phase 1. This time, the aim was not to produce diagnoses as the basis of a mathematical model, but rather to emulate as far as possible diagnosis in a clinical setting. Each physician was therefore asked, on the basis of the clinical review information, to classify each individual as 50% or greater probability of obstructive airways disease (OAD), or less than 50% probability. For those 50% or greater probability (more likely than not to have OAD), the physician was asked to state whether the disease was asthma, chronic obstructive pulmonary disease (COPD) or mixed disease (asthma and COPD). Individuals in whom at least two physicians diagnosed asthma or mixed disease were designated clinically asthmatic.As part of the 2001 population survey, each reviewed individual had already completed a postal questionnaire, and a weighted score was calculated based on questionnaire responses. For each possible score, the proportion of reviewed individuals with that score or higher, who had been designated clinically asthmatic was calculated. These percentages reflect the association between score and probability of asthma and were used as an indication of the validity of the postal questionnaire and scoring system.For comparison, unweighted scores (number of 'yes' answers to the six key questions) were also calculated for respondents from the top 10% of the ranked population list, and the percentage of asthmatics associated with each of the unweighted score thresholds was similarly calculated.In the 1995 WYCAP survey, 10429 individuals were sent a postal respiratory questionnaire with a 72.7% response rate, leaving 6825 after exclusion of incomplete questionnaires. Four hundred and twenty individuals were selected for the stratified random sample and invited for clinical review. Of these, 201 (48%) attended, leaving 180 after exclusion of those whose screening questionnaire was incomplete. The ages of the 180 respondents with fully completed questionnaires ranged from 16 to 83 years, with a median age of 50.5 years, and 41.7% were male. Eighty four individuals were designated clinically asthmatic and 96 non-asthmatic based on the combined opinions of the three physicians.The weights derived for each question are shown in table Weighted scores were calculated for all postal questionnaires n = 6825) in the 1995 WYCAP population and ranged from -1 to 13. Table 5 in the The third phase confirmed that higher scores correspond to higher probabilities of asthma in the independent test subset selected from the 2001 survey respondents. Two hundred and eighty three individuals comprised the top 10% of the probability ranked list and were invited for review along with 39 low probability individuals selected from the end 200. In total, 145 individuals attended for review, their ages ranged from 16 to 91 years with a median age of 50 years, and 40.7% were male.One hundred and twenty six high probability and 19 low probability individuals attended. For the 126 high probability asthmatics from the top 10% of the ranked list, scores ranged from six to 12, compared with a maximum score of eight in those respondents not in the top 10%. The age range and median age associated with each score threshold is reported in table In general, agreement between the experts was good with pair-wise kappa statistics of 0.62, 0.64 and 0.65 respectively. All three experts agreed the same diagnostic category for 99 out of the 145 reviewed individuals (82 from the high probability group and 17 from the low probability group) and two out of three experts agreed for a further 38 (37 high probability and one low probability). The percentages of individuals diagnosed as having asthma or mixed disease associated with each score threshold are shown in table Table In general, questionnaire surveys for detecting individuals likely to have a disease partition respondents into 'positive' and 'negative' categories. This is appropriate when a community-wide survey can be undertaken and the medical services are able to provide clinical assessment for all those in the 'positive' category. This study has developed a method for optimising limited resources by targeting expensive clinical examinations to those most likely to be at risk, minimising the chance of needlessly examining a healthy individual and allowing resource availability to be taken into account.Developing a model which uses a questionnaire to identify likely asthmatics in the general population crucially depends on having a subset of questionnaires from respondents reliably classified as 'asthmatic' or 'non-asthmatic' on which to build the model. In a condition such as asthma there is inherent uncertainty in the diagnosis, and even detailed clinical review information inevitably produces disagreement between experts as to the diagnostic category for some of the reviewed individuals. Other studies have produced questionnaire models for predicting high risk asthmatics ,9,10,19 Since the aim of a screening system is generally to identify individuals with a high probability of the condition being tested for, the performance of the screening system on high probability individuals is most important. For this reason it was necessary in this study to identify a set of high probability asthmatics for clinical review in the phase 3 validation stage. It was also necessary that these high probability asthmatics were identified by a system which was independent of the weighted scoring system being tested. For this reason, a neural network was used to rank the 2001 survey questionnaires in order of probability of asthma. The neural network is a sophisticated statistical technique that was used to model complex relationships between questions on the questionnaire to predict probability of asthma of a respondent based on questionnaire responses. The neural network model was validated [The question weights were consistent with clinical explanations. Wheeze in the last twelve months was the most important symptom and the weights for each age band decreased with increasing age which is consistent with the explanation that wheezing in older people can be explained by reasons other than asthma, for example, heart disease, malignant lung disease or one of the chronic obstructive lung conditions collectively referred to as COPD. The interpretation of the low weighting given to respiratory symptoms other than wheeze, for example, night cough or shortness of breath, is that these symptoms are highly correlated with wheezing, and as such provide no additional information over an above the high 'wheeze' weighting. Whereas any of these symptoms viewed in isolation would be highly correlated with a diagnosis of asthma, when viewed in the context of a multivariate model such as this weighted scoring system, it is the interaction between the variables which defines the final model. The small negative coefficient for smoking is consistent with the explanation that those with asthma tend to stop smoking or never to begin. Alternatively, asthma-like symptoms in some smokers may be caused by conditions other than asthma, for example malignant lung disease, COPD, or heart disease, or smoking itself may cause symptoms.The intended application of weighted scoring is to find individuals who are likely to have asthma but are not already receiving appropriate treatment. Superficially, it may appear that the questions relating to an attack of asthma in the last twelve months and current medication for asthma should be excluded from the model as they may produce higher scores in asthmatics who are already diagnosed and receiving treatment, effectively disadvantaging the undiagnosed individuals. However, the relationship between these two questions and practice records of diagnosis is far from definitive. For example in the 1995 survey, of the 6570 respondents who had no record of either diagnosis of asthma nor medication prescription in the last year, 175 individuals answered 'Yes' to the current medication question on the questionnaire and 128 reported an asthma attack in the last twelve months. The scoring system reflects an accurate ranking of the whole population and the questions relating to attack of asthma in the last 12 months and current medication for asthma were found to be important in the whole population model. In general, higher scores reflected higher PPV estimates, but the final column of table One possible source of error in the weighted scoring system is under or over self-reporting of symptoms, such as the individual ranked as low probability who was found clinically asthmatic in the absence of reported symptoms on the questionnaire. Another possible source of error is differences between the development data sample and the intended target population due to non-response in the postal survey, exclusion of incomplete questionnaires and non-attendance at clinical review. Analyses revealed no evidence of sample bias from these sources in terms of gender or numbers of 'yes' answers to the six key questions on the questionnaire. Those who attended for review had a higher median age (50 years) than those invited but not attending (32 years). It is also important to acknowledge that the system here was tested using postal questionnaires, whereas in practice the patient may well complete the questionnaire in a surgery or clinic environment.The question weights were derived from a logistic regression model and commonly, scarcity of data means that this modelling technique is applied as a single model fitted to the entire data subset  with nonAssessing likelihood of asthma and ranking by simply counting the number of 'yes' answers to key questions is a simple but relatively crude method since it assumes equal weights for the questions. However, some responses may exert a stronger influence than others, there may be interactions between responses and features such as age and gender cannot be included at all. For example, an elderly person with four 'yes' answers to the key questions may be considered less likely to be asthmatic than a young person with three, since respiratory symptoms in the elderly may sometimes be explained by conditions other than asthma. Hence, ranking according to the number of 'yes' answers may not produce the required probability ordering. This is illustrated by the small differences in proportions of clinical asthmatics associated with the various simple score thresholds shown in table Where the availability of resources is a limiting factor, the relevant measure of effectiveness in targeting clinical review is the 'true positive' rate, that is, the percentage of individuals scoring above the threshold in whom the diagnosis is confirmed. The other commonly reported test measures of sensitivity, specificity and negative predictive value have no equivalent in a technique based on population ranking where the aim is to target expensive clinical examinations to those most at risk and minimise the 'wasted' examination of false positives.When resources for diagnostic examination are limited prioritisation may be necessary. The use of weighted scoring and priority levels rather than the more conventional binary separation into positive and negative allows the score threshold to be adjusted to balance patient need with available resourcesCOPD Chronic obstructive pulmonary diseaseOAD Obstructive airways diseasePPV Positive predictive valueWYCAP Wythenshawe Community Asthma ProjectThe author(s) declare that they have no competing interests.SH performed the statistical analysis and developed the weights. TF and PF performed the clinical reviews. JS advised on statistical aspects of combining expert opinions. MH managed the data collection. All authors participated in study design and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:The Respiratory Questionnaire. The respiratory questionnaire used in the postal surveyClick here for fileEstimating PPV for weighted scores. Details of method used for estimating PPV based on postal questionnaire responsesClick here for file


One of these methods is Domain Fusion, which has proven to be effective in predicting functional links between proteins.Several Saccharomyces cerevisiae proteins.Analyzing the structures of multi-domain single-chain peptides, we found that domain pairs located less than 30 residues apart on a chain are almost certain to share a physical interface. The majority of these interactions are also conserved across separate chains. We make use of this observation to improve domain fusion based protein interaction predictions, and demonstrate this by implementing it on a set of We show that existing structural data supports the domain fusion hypothesis. Empirical information from structural data also enables us to refine and assess domain fusion based protein interaction predictions. These interactions can then be integrated with downstream biochemical and genetic assays to generate more reliable protein interaction data sets. Networks of interacting molecules drive every process in biological cells. Proteins dominate these networks, some of which involve transient interactions such as signal transduction cascades and ligand-receptor interactions, while others form more permanent molecular machineries such as ribosomes and polymerases. Unraveling these networks and interactions will not only help us better understand complex cellular processes, but also enable us to make inferences about the function of individual proteins through 'guilt-by-association' .Saccharomyces cerevisiae, in which networks of its interacting proteome where constructed using genome-wide screens. [Over the last few years, high-throughput interaction detection assays have been introduced and refined to complement the traditional genetic and biochemical techniques. High-throughput mass spectrometry protein complex identification (reviewed by Pandey and Mann ), and yescreens. -7in silico methods have recently been developed to predict associations from these data.The wealth of genomic and protein sequences, the increase of 3D structures of protein complexes, together with the deluge of microarray expression data, has provided researchers with an overwhelming body of information that can be used to infer both functional as well as interaction linkages. Clearly, bioinformatics and computational biology are necessary tools for delineating this information. In response to the data explosion, several Phylogenetic profiles focus on the co-occurrences of genes across several organisms. By studying the pattern of evolutionary conservation between sets of genes in different organisms (phylogenetic distribution), it has been shown that these phylogenetic profiles can be successfully used to infer both localization as well as functional association between proteins -10. Protab initio categorization and prediction of protein interactions [Structural data has also been mined and analyzed for residue patterns within interfaces between pairs of interacting proteins. These are then used to train learning models for ractions ,14. Jansractions  illustrain silico experiments [A number of groups have compared and reported on the protein interaction datasets that are emerging from the various genome-scale biochemical, genetic and eriments -18. All in silico methods have higher coverage and higher accuracy than the majority of biochemical/genetics methods, second only to high-throughput mass spectrometry. The use of sensible strategies and filters has allowed in silico analyses to provide better performance. On top of that, these methods are less biased towards abundant proteins. In silico analyses are indispensable, and further improvements of these methods to make them more accurate will provide a cleaner set of data for downstream biochemical/genetic studies.The study by von Mering and co-workers  showed tIn this study, we make use of an empirical observation that domain pairs, which lie in close proximity on a protein chain tend to interact, to refine the domain fusion analysis. This way, we aim to improve the accuracy of the domain fusion analysis.The basis for domain fusion (or gene fusion) is the observation that certain proteins (termed the Rosetta stones) in a given species are found to consist of a fusion between two separate proteins in another species. Through fusion, the entropy of dissociation between the two proteins is reduced, and it is hypothesized that in all likelihood, these two separate proteins share a functional association, if not a physical interaction ,12.Domains have been described as the primary building blocks of proteins , recombiIn this paper, we make use of existing structural data to support the domain fusion hypothesis. We interrogated known 3D structures for evidence of inter-domain physical interactions on the same chain. We investigated and concluded that there was an association between the distances at which domains are spaced apart on the chain, and the propensity for a domain-pair to interact.We also show that domain pairs, located in close proximity on a protein chain, are likely to interact even when found residing on different chains, hence proving that the domain fusion hypothesis is valid.Finally, we demonstrate that peptide chains with closely spaced domains are likely to make better Rosetta stones, and we make use of this observation to improve domain fusion based protein interaction predictions.The available structural data indicate that intra-chain domain pairs, which lie in close proximity on a peptide chain, tend to physically interact with one another. The mean distances of interacting intra-chain domain pairs are smaller than ones which do not interact; interacting pairs are on the average 50 residues apart, while non-interacting pairs have a mean distance of 166 residues between them.H0): Domains pairs separated by no greater than a predefined distance and their tendency to interact were independent. The p-values indicate the probability of having the chi-squared test statistic as extreme as, or larger than observed when H0 is true.In order to verify the correlation between distance and interaction, we made use of contingency tables and the chi-squared test statistic. For a set of inter-domain distances ranging from 5 residues to 200 residues, we constructed 2 × 2 contingency tables that classified domain pairs according to two criterions; 1) whether or not they are separated by a distance no greater than a threshold, and 2) whether or not the domain pair is interacting. The chi-squared value of each table was used as a statistic to test the null hypothesis . In other words, there is a correlation between domain-pairs spaced less than 30 residues apart on a single peptide chain and their tendency to interact across separate chains of a polypeptide complex.S. cerevisiae was downloaded from Swiss-Prot, and domain fusion based protein interactions were predicted as described in the Methods section. After filtering for promiscuous domains, a total of 9279 protein interactions remained, of which 28% or 2629 were supported by a Rosetta stone with no more than 30 residues between the fused domains.We wanted to verify that the 30 residues criteria could be used as a measure to filter and improve predictions made using the domain fusion methodology. A set of proteins for the budding yeast The functional category assigned to each protein in an interacting pair was used to gauge the plausibility of the interaction; if two different proteins were found physically interacting, one would expect the two proteins to have overlapping functional categories. 62% of the interacting protein pairs, supported by a 30 residue Rosetta stone, have both partners belonging to the same functional category. The same proportion for interacting pairs not supported by a 30 residue Rosetta stone is 48%. This 14% difference is significant with a p-value of less than 0.001, using a two-sample t-test.In silico methods for predicting protein interactions are not only able to match the accuracy of the other genetic, biochemical and biophysical techniques, but also have the added advantage of providing higher coverage [in silico methods, domain fusion is an attractive technique because it enables a functional link to be drawn between two proteins based solely on their primary sequence. Still, large-scale sets of high-throughput protein interaction data available today are spurious, more than half of them proving to be false positives [coverage . Among tositives , the chaProtein interactions can be classified as either permanent or transient interactions. The data from this study were taken from the PDB, where most of the submitted structures are results from x-ray crystallography experiments. Consequently, we believe that the vast majority of our deduced domain and protein interactions are physical, permanent interactions.Our study of multi-domain, single and multiple-chain protein structures in the PDB gave us two results. First of all, it supports the domain fusion hypothesis suggested by Marcotte and Enright. Secondly, it allows us to conclude that single chain peptides with closely spaced domain pairs make better Rosetta stones, and hence better predictors of protein interactions.Evident from the set of PDB structures we studied, is a correlation between the distance separating a pair of domains on a protein chain, and their tendency to physically interact with one another. As described by Marcotte and co-workers when they constructed the domain fusion hypothesis for evolution of protein interactions, affinity between interacting pairs of domains may be enhanced when the domains are fused together on the same chain . ConsequPreviously, Park and co-workers had obserThe data from multi-chain PDB structures provide additional support to the domain fusion hypothesis, by showing that most of the intra-chain domain interactions are similarly represented across separate chains of a complex. This provides additional mechanistic evidence that the interaction between the two domains is most probably functional and conserved.To our knowledge, this is the first time structural data has been used to support the domain fusion hypothesis.We tried to uncover a pattern within the set of closely spaced, yet non-interacting domain pairs. We wanted to detect if there was an over-representation of domains from a specific molecular functional category in this non-interacting list. This list is displayed in Table Furthermore, since the interactions we can detect from structural data are more likely to be permanent interactions, it is possible that the reason no physical contact is witnessed between these proximal domains in structural data is because the domains form transient interactions that are not captured in the x-ray crystallography data.We also looked for a relation between protein disorder and interacting domain pairs. We wanted to see if protein domain pairs which interact on the same chain, tend to be linked by a disordered region. To this effect, we used DisEMBLto do theS. cerevisiae proteins indicate that Rosetta stones with domains separated by less than 30 residues do indeed make better domain interaction (and hence protein interaction) predictors.Our results from predicting interactions among The set of protein interactions inferred from these Rosetta stones are enriched with more reliable interactions, as judged by using similar function as a criteria. The total number of interactions is reduced to nearly a quarter when employing this method. This allows us to conclude that the number of false positives is reduced, increasing the accuracy of the prediction. Without needing to employ a hard filter, protein interactions predicted using the domain fusion methodology may be ranked according to the quality of the Rosetta stones each interaction is inferred from, allowing one to identify a much smaller subset of more reliable interactions, and use them for downstream analyses.We have successfully demonstrated the use of current structural data as a resource for refining current protein interaction predictions, in particular domain fusion predictions. Our data strongly suggests that domain pairs separated by less than 30 residues on a peptide chain are almost certainly to physically interact, and this criterion is useful in accessing protein interactions predicted from Rosetta stone proteins.Going forward, the availability of a large number of structures through structural genomics programs will facilitate a larger sampling of the domain structure space. New patterns may emerge as use of this data becomes available, allowing better predictions to be made.We used domain models from the Protein Family database (PFAM)  which weWe used the coordinates within the PDB data files to calculate the distances between domains, and to determine if they are interacting. Two domains are judged to be interacting if they share at least five contacting residue pairs, where contacting residues are residue pairs with less than 6Å between their respective alpha-carbon atoms.Using a similar approach to the above, we obtained a set of multi-chain PDB structures in which the previously determined domain-domain interactions can be observed across separate peptide chains within a complex. The ASALIST from the PQS server was used PFAM domains were categorized into Gene Ontology (GO) molecular functions and cellular processes using the PFAM2GO data provided by the GO consortium .Saccharomyces cerevisiae found within the Swiss-Prot database, and gauged the quality of the interactions by looking at the function of each interacting protein.In order to assess how distance between domains could be used to improve the domain fusion based protein interaction predictions, we predicted interactions between 6918 proteins from the organism The steps taken to predict protein interactions based on domain fusion are as follows. Swiss-Prot (release 42.9) and Trembl (release 25.9)  protein S. cerevisiae proteins, by looking at the complement of PFAM domains between each and every pair of proteins, and seeing if there were any Rosetta stone determined domain interactions between the domains of each protein. The protein interactions were sorted into two groups; one group inferred from domain interactions supported by the existence of a Rosetta stone protein with no more than 30 residues between the domain pair, and the other group with no support from a 30 residue Rosetta stone.This domain interaction set was then used to predict pairwise protein interactions between the To validate these protein interactions, we mapped the proteins to the MIPS comprehensive yeast genome database , and looAll the data was stored in a relational database schema implemented in MySQL, with a set of perl modules written for data transaction and manipulation. The Bioperl bioinformatics tool kit  was usedJMC participated in the design of the study, performed the data and statistical analysis, as well as drafted the manuscript. PRK conceived of the study, participated in its design and coordination, and edited the final draft of the manuscript. Both authors read and approved the manuscript.


Bmal1 and Clock, genes that function in the core molecular clock, exert profound control over recovery from insulin-induced hypoglycaemia. Furthermore, asynchronous dietary cues may modify glucose homeostasis via their interactions with peripheral molecular clocks.Circadian timing is generated through a unique series of autoregulatory interactions termed the molecular clock. Behavioral rhythms subject to the molecular clock are well characterized. We demonstrate a role for Bmal1 and Clock in the regulation of glucose homeostasis. Inactivation of the known clock components Bmal1 (Mop3) and Clock suppress the diurnal variation in glucose and triglycerides. Gluconeogenesis is abolished by deletion of Bmal1 and is depressed in Clock mutants, but the counterregulatory response of corticosterone and glucagon to insulin-induced hypoglycaemia is retained. Furthermore, a high-fat diet modulates carbohydrate metabolism by amplifying circadian variation in glucose tolerance and insulin sensitivity, and mutation of Clock restores the chow-fed phenotype.  Besides regulating various behavioral rhythms, the molecular clock plays a role in the control of glucose homeostasis The master clock, which, in mammals, resides in the hypothalamic suprachiasmatic nucleus (SCN), is thought to synchronize multiple peripheral oscillators to ensure temporal coordination of behavior and metabolism. Peripheral clocks amplify or dampen central rhythms or exhibit autonomous behavior to facilitate local adaptive responses . The cenThe variation in sleep and wakefulness (activity) is perhaps the most well-known circadian rhythm. Surgical ablation of the SCN in mice  and ratsi).Genes relevant to the molecular clock are also expressed in peripheral tissues  where apBmal1 and Clock—in regulating recovery from insulin-induced hypoglycaemia. Furthermore, the impact of a high-fat diet (HF) was to amplify the diurnal variation in glucose tolerance and Si in a manner dependent on the Clock gene. These studies suggest that the temporal distribution of a caloric load may influence the response to insulin and that circadian variability in glucose homeostasis may be subject to modulation by asynchronous dietary cues.Our studies revealed a profound role for core clock genes—mut)(Clock or deficient −/−) mice, peaking at approximately circadian time point 4 (CT4) and CT28  (We examined the role of the molecular clock in glucose homeostasis by using mice in which core clock genes are impaired at 7 PM) A and 1B,at 7 PM) . We alsoat 7 PM) C, which at 7 PM) , and adiat 7 PM) D, which at 7 PM) , oscillaat 7 PM) .Bmal1−/− and mutClock mice , insulin secretion, and Si. Si and insulin secretion, but not Sg, exhibited a diurnal variation in WT mice fed a regular chow diet (RC)  on high-fat feeding. Again, fat composition was not significantly altered by the presence of the Clock mutation (The frequent sampling intravenous glucose tolerance test (FSIGT) was performed to assess more precisely the impact of the molecular clock on sensitivity to insulin. This test provides an estimate of Sic clamp . Additioiet (RC) . Circadisyndrome , and a ddrome   A. Simila< 0.001) B, the HF< 0.001) B. Thus, < 0.001) . The mut< 0.001) .i that were imperceptibly low . However, Sg, insulin secretion, and Si were restored to a WT phenotype in mutClock mice that were also HF-fed for 11 mo . Long-term hyperlipidemia is known to induce frank diabetes with impaired release of insulin , in contor 11 mo . This isBmal1−/− and mutClock mice, where the counterregulatory hormone response is unimpaired. Thus, steroids, epinephrine, and glucagon appear to facilitate recovery from insulin-induced hypoglycaemia in a manner distinct from, but complementary to, the molecular clock.Maintenance of blood glucose levels within a narrow range is critical to mammalian survival, and environmental cues can trigger appropriate tissue disposition of glucose through adaptive behaviors such as in hibernation  or the “Per1  or Per2  (unpublished data). In addition, many of their mRNAs are at lower levels in Bmal1−/−  (unpublished data), consistent with a direct role of Bmal1 in their transcription. Thirdly, these metabolic parameters are disrupted in both circadian mutants with the same rank order of potency as the locomotor activity phenotypes −/− > Clockmut), and subjective night beginning at 7 PM (CT12).mutClock mice were placed on an HF  and compared to age-matched WT mice on a regular chow diet (RC). Mice were on RC for 8 wk except for those subjected to FSIGT where they received RC for 11 mo. Body mass composition was measured by dual energy X-ray absorptiometry at 10 mo.WT and Tests were performed as described  with a di, Sg, and acute insulin response to glucose, which measures insulin secretion. Si is the ratio of insulin delivery rate to the interstitium to insulin extraction rate from the interstitium. Long-term feeding of HF to WT mice resulted in imperceptibly small insulin sensitivity values. This could be the consequence of impaired delivery of insulin to the interstitium, exacerbated extraction rate, or a combination of both factors. Insulin secretion is derived from area under the insulin curve, above basal, from 0 to 10 min after glucose infusion; and disposition index, which equals the product of insulin sensitivity multiplied by insulin secretion and measures the degree to which insulin sensitivity can be compensated for by elevated insulin secretion , ICN Biochemicals , and Linco Research . Plasma glucose was measured by the glucose oxidase method using a glucose analyzer machine for FSIGT and by glucometer for the intraperitoneal tolerance test. PEPCK activity was quantitated by a bioluminescent method .−/−Bmal mice and between WT and mutClock mice. Plasma samples for glucagon analysis were pooled and were thus not compared by a formal statistical analysis. Results are presented as mean ± standard error of the mean (SEM), except for the FSIGT data (p < 0.05.The significance of differences amongst the tolerance test curves was assessed by distribution-free two-way ANOVA with a Bonferroni correction. FSIGT data were tested by one-way ANOVA with the Kruskal-Wallis test. Paired Student's t-tests were used to perform comparisons of corticosterone levels before and after insulin injection in IGT data , present


Making choices is a fundamental aspect of human life. For over a century experimental economists have characterized the decisions people make based on the concept of a utility function. This function increases with increasing desirability of the outcome, and people are assumed to make decisions so as to maximize utility. When utility depends on several variables, indifference curves arise that represent outcomes with identical utility that are therefore equally desirable. Whereas in economics utility is studied in terms of goods and services, the sensorimotor system may also have utility functions defining the desirability of various outcomes. Here, we investigate the indifference curves when subjects experience forces of varying magnitude and duration. Using a two-alternative forced-choice paradigm, in which subjects chose between different magnitude–duration profiles, we inferred the indifference curves and the utility function. Such a utility function defines, for example, whether subjects prefer to lift a 4-kg weight for 30 s or a 1-kg weight for a minute. The measured utility function depends nonlinearly on the force magnitude and duration and was remarkably conserved across subjects. This suggests that the utility function, a central concept in economics, may be applicable to the study of sensorimotor control. Economists use the concept of a utility function, which increases with increasing desirability of the outcome, to characterize human decision making. This concept is shown here to apply to the control of movement In real world situations we often have to choose between possible actions that lead to different outcomes. To provide a computational framework for such a decision process, the notion of a utility function is often used . A utiliIn sensorimotor control, utility functions that depend on several variables occur frequently. Consider, for example, unpacking a car after a snowboarding vacation. We could carry all the suitcases at the same time, reducing the time to unpack but maximizing the weight we have to lift concurrently. At the other extreme we could transport each item individually, which would minimize the magnitude of the force required at the expense of a long unpacking duration. The chosen solution is likely to lie somewhere between these two extremes and may reflect an optimal decision based on a utility function that depends on duration and magnitude of the forces. Once a utility function is specified, the decision problem becomes one of solving an optimal control problem, finding the actions that maximize the utility.A number of studies in the field of optimal sensorimotor control have proposed loss functions (the negative of utility) and derived the optimal actions given these proposed loss functions. For example, the minimum jerk model  suggestsT, and the maximum value of the force, F. On each trial, subjects experienced two force profiles that differed in both T and F. They then had to choose which of the two force profiles they would experience again. They were told to choose the force that required the least effort. In this two-alternative forced-choice experiment subjects thus indicated their preference for one combination of F and T over another combination of F and T. This allowed us to infer indifference curves: Given the choice of two combinations of F and T that are on the same indifference curve, subjects will have no preference. The associated utility of these force profiles is thus identical. To obtain a full utility function from a set of utility curves we additionally needed to determine the utility of one indifference curve relative to another. This was achieved by finding “doubling points.” A doubling point is a point on one indifference curve that subjects show no preference for when compared to experiencing a point on another indifference curve twice . This predicts hyperbolas as indifference lines (2 × T)(F ((F) (Various hypothesized utility functions predict different choices and thus different indifference lines. The first model we hypothesized was that subjects would minimize the integrated force they are using ce lines A. Altern(F2 × T) B. In thi T) ((F) C.A single subject's results are shown in We furthermore measured how much smaller a force profile needed to be  so that experiencing it twice had the same utility as experiencing the unscaled profile once. For the four open-circle reference points in F/T = 44.6). Fitting a model of the form Loss = (FT)α to the data from the double-hump experiments leads to an α of 1.1 ± 0.15 (mean ± SEM over subjects). This α, when fit to the data from all the subjects for each of the four lines, is approximately constant . The shape of the loss function is highly conserved over the set of subjects. In particular, the effect that indifference curves increase for both very short and long durations is found over the set of subjects.F and T. However, it is highly conserved across the subjects, suggesting a common underlying mechanism is at work. Moreover, our results suggest that the opposite approach—first hypothesizing a loss function and then predicting human decision making—is likely to miss interesting aspects of the behavior and underlying processes. We are therefore hopeful that the application of economic methods to the study of the nervous system, referred to as neuroeconomics  participated in this study. The experiments were carried out in accordance with institutional guidelines. A local ethics committee approved the experimental protocols.While seated, subjects held the handle of robotic manipulandum with two degrees of planar freedom. This was a custom-built device (vBot) consisting of a parallelogram constructed mainly from carbon fiber tubes that were driven by rare earth motors via low-friction timing belts. High-resolution incremental encoders were attached to the drive motors to permit accurate computation of the robot's position. Care was taken with the design to ensure it was capable of exerting large end-point forces while still exhibiting high stiffness, low friction, and also low inertia.The robot's motors were run from a pair of switching torque control amplifiers that were interfaced, along with the encoders, to a multifunctional I/O card on a PC using some simple logic to implement safety features. Software control of the robot was achieved by means of a control loop running at 1,000 Hz, in which position and force were measured and desired output force was set.A virtual reality system was used that prevented subjects seeing their hand, and allowed us to present visual images into the plane of the movement  approximated a square profile, but with smooth onset and offset:The experiment consisted of trials in which the robot generated force profiles on the subjects' hands. The force profiles experienced were parameterized by their duration On each trial the subjects experienced two different force profiles and then could choose which of the two profiles to experience for a second time. Using such a forced-choice procedure allowed us to determine the indifference curves.1F. The subjects then returned the cursor to the starting sphere. (2) The other selection sphere turned green, and subjects were required to place the cursor in that sphere, where they experienced a force profile 2F. Subjects then returned the cursor to the starting sphere. (3) Both selection spheres turned green, and subjects were required to choose which of the two spheres to move to, where they would experience the same force associated with that sphere, either 1F or 2F. Therefore, subjects could decide which force profile, 1F or 2,F to experience a second time.Subjects saw a starting sphere and two selection spheres see A. Each tT of 200, 300, 400 and 500 ms. The maximal force F was chosen for each reference so that the ratio T/F had the value 44.6. This gave a maximal force that ranged from 4.5 N for the shortest duration reference to 11.2 N for the longest duration reference. These reference points lie along a straight line in time–force space , 85.4, 142.1, and 203.0, with the aim of finding the point along the line at which subjects would choose between the reference and test force indifferently . We used an adaptive fitting protocol . This condition was run interleaved with the other conditions. We assumed that the utility of experiencing the double hump was twice the utility of a single hump (a linearity assumption). This assumption allowed us to link the reference point to a point of half its utility, further allowing us to linearly interpolate log(utility) between these points to obtain estimates of the loss function between the lines.The above procedure allowed us to obtain indifference lines—where the utility has equal value. However, to obtain a full utility function we need to join up these lines and determine the relative utility of one indifference line to another. To achieve this we performed a two-alternative forced-choice paradigm in which the reference force was as before, but the test force was selected from the


In vitro studies imply that BHRF1 is dispensable for both virus replication and transformation. However, the fact that BHRF1 is highly conserved not only in all EBV isolates studied to date but also in the analogous viruses Herpesvirus papio and Herpesvirus pan that infect baboons and chimpanzees respectively, suggests BHRF1 may play an important role in vivo.Epstein-Barr virus (EBV) latently infects about 90% of the human population and is associated with benign and malignant diseases of lymphoid and epithelial origin. BHRF1, an early lytic cycle antigen, is an apoptosis suppressing member of the Bcl-2 family. Herpesvirus papio BHRF1 has been shown to function in an analogous manner to EBV BHRF1 in response to DNA damaging agents in human keratinocytes. In this study we show that the heterologous expression of the previously uncharacterised Herpesvirus pan BHRF1 in the human Burkitt's lymphoma cell line Ramos-BL provides similar anti-apoptotic functions to that of EBV BHRF1 in response to apoptosis triggered by serum withdrawal, etoposide treatment and ultraviolet (UV) radiation. We also map the amino acid changes onto the recently solved structure of the EBV BHRF1 and reveal that these changes are unlikely to alter the 3D structure of the protein.These findings show that the functional conservation of BHRF1 extends to a lymphoid background, suggesting that the primate virus proteins interact with cellular proteins that are themselves highly conserved across the higher primates. Further weight is added to this suggestion when we show that the difference in amino acid sequences map to regions on the 3D structure of EBV BHRF1 that are unlikely to change the conformation of the protein. Apoptosis is a genetically programmed form of cell death employed to regulate cell number, spatial organisation and remove infected and damaged cell populations that may compromise the integrity of the organism . AberranIn vivo, EBV is associated with a number of benign and malignant proliferative diseases, such as infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma [in vitro leads to outgrowth of apoptosis resistant lymphoblastoid cell lines (LCLs), which phenotypically resemble activated B-cells. Although B cells can produce progeny virus, epithelial cells are also known to be permissive for viral replication. Studies have shown that EBV has at least two mechanisms of suppressing apoptosis. During latent infection latent membrane protein 1 (LMP1) is the key mediator of protection. LMP1, a CD40 mimic, up-regulates cellular Bcl-2 [Many viruses have evolved multiple strategies to counteract host-mediated apoptosis in order to facilitate infection, replication or persistence ,4. Epstearcinoma ,7. Latenar Bcl-2  and A20 ar Bcl-2  via the ar Bcl-2 ,11 and tar Bcl-2 ,13 whichar Bcl-2 , EBV andin vitro [in vivo remains unclear. BHRF1 is thought to act in lytic cycle to delay apoptosis during replication in terminally differentiating epithelium [in vitro [Despite being considered dispensable for EBV replication or cellular transformation in vitro ,16, BHRFin vitro , and itsithelium . Howeverithelium  and lateithelium  and in Ein vitro . Additioin vitro , DNA damin vitro  and cytoin vitro . It is pHerpesvirus papio (H. papio) and Herpesvirus pan (H. pan) infect baboons and chimpanzees respectively. H. papio encodes a BHRF1 homologue that shares 65% amino acid identity (81% similarity) with EBV BHRF1, whilst the H. pan BHRF1 is even more similar at 82% identity and 90% similarity [H. papio BHRF1 has been shown to confer resistance to cisplatin induced apoptosis in human keratinocytes akin to EBV BHRF1 [H. pan BHRF1 exhibits similar anti-apoptotic activity to that of EBV BHRF1 in the human Burkitt's lymphoma cell line Ramos-BL in response to apoptosis induced by either serum withdrawal, etoposide treatment and the previously untested trigger, ultraviolet radiation. These results show that H. pan BHRF1 is biologically active in human B-lymphocytes in vitro suggesting a conserved role for BHRF1 during in vivo infection of B-lymphocytes.Analogues of EBV are found in the higher primate species. milarity . H. papiBV BHRF1 , but thiH. pan BHRF1 and EBV BHRF1 to regulate serum reduction induced apoptosis in the EBV negative Ramos-BL cell line to ascertain whether the genes are functional, as well as structural, homologues. In order to produce expression vectors, the H. pan and EBV BHRF1 reading frames were amplified by PCR from template vectors pHVPanTW4 and pDH222 respectively using primers incorporating XhoI restriction sites. The PCR fragments were introduced into the pCR2.1Topo® vector and the XhoI fragment was released by restriction digestion of the TopoBHRF1 constructs and cloned into the XhoI restriction site of the pcDNA4/HisMax mammalian expression vector (InVitrogen), which produces an N-terminal fusion protein with 6His and Xpress epitopes.As the senior author has previously shown that EBV BHRF1 suppresses apoptosis in response to serum reduction in various BL cell lines , we initH. pan BHRF1 and cloned by limiting dilution in medium containing 150 μg/ml zeocin. There was no obvious difference in the numbers of drug resistant clones that were produced by transfection with the vector or either of the BHRF1 constructs. Drug resistant clones were screened by RT-PCR for BHRF1 expression and approximately 50% of the clones were shown to be expressing detectable BHRF1. The RT-PCR profiles of two clones each of the three transfected populations which were selected for further analysis, are shown in Figure H. pan BHRF1 (E and F), indicative of BHRF1 expression.Ramos-BL cells were electroporated with either control vector, EBV BHRF1 or H. pan clones Pan 4 and Pan 5 and BHRF1 null vector control clones His C12 and His C13 were seeded in medium containing either 10% or 0.1% serum and the percentage of apoptotic cells was determined at 24-hourly intervals, using acridine orange to distinguish between apoptotic and viable cell nuclei. Necrotic nuclei that appeared after several days due to secondary necrosis of cells already lost by apoptosis were excluded from the counts. Apoptotic death was also confirmed by DNA laddering (not shown). We found that when maintained in medium containing 10% serum  induces apoptosis within hours. The EBV and H. pan BHRF1s afforded significant protection against the induction of apoptosis by all three triggers, compared to the vector controls. There was no statistical difference between the EBV and H. pan BHRF1s, (except for clone EBV1B at 96 hours in 0.1% serum), suggesting that H. pan BHRF1 is indeed a true functional homologue of EBV BHRF1. The difference between clone EBV1B and the other BHRF1 clones at 96 hours in low serum could possibly be attributed to slightly higher levels of BHRF-1 expression in this clone, although it is difficult to quantify absolute expression levels from the fluorescence images shown in Figure In this study we present evidence that the BHRF1 protein encoded by poptosis  and delain vitro . The priin vitro ,25 and hWhen one looks more closely at the regions of the protein that differ between the human and chimpanzee viruses Figure , it is pH. pan) and EBV BHRF1 in a human lymphoid cell background. Both proteins protect against apoptosis induced by two previously described triggers and also a new trigger, UV radiation, against which BHRF1 has never been reported to provide protection. Comparison of the EBV and H. pan proteins at the 3D structural level reveals that none of the changes is likely to significantly alter the structure of the protein.Our results are the first demonstration of functional homology between a primate BHRF1 , 55°C (30 sec) and 72°C (60 sec). The PCR products were introduced by Topo TA cloning into pCR2.1-Topo® (Invitrogen) and the XhoI fragments were released by digestion with XhoI and cloned into the Xho1 site of pcDNA4/HisMaxC (Invitrogen). The plasmids were sequenced to verify that the sequences had joined in frame and contained no PCR induced errors.The 573 bp coding sequences of EBV BHRF1 and m pDH222  and pHVPm pDH222  respecti6 Ramos-BL cells were independently transfected with 20 μg endotoxin-free EBV BHRF1, H. panBHRF-1 or vector control constructs  by electroporation in a BioRaD Gene Pulser II at 0.45 kV, 125 μF. The cells were seeded in 96-well plates at a density of 5 × 104 cells/ well in RPMI 1640 supplemented with 10% FetalClone III calf serum (HyClone) 2 mM L-glutamine (Sigma) 0.4% gentamycin (Sigma) and 200 μg/ml Zeocin (Invitrogen). Wells showing outgrowth of drug resistant cells were cloned by limiting dilution and clones were expanded into 2 ml cultures and maintained in medium containing 150 μg/ml Zeocin. All cultures were fed twice weekly by replacing 90% of the culture with fresh medium.6 × 106 cells using the RNA Safekit (Q Biogene) according to manufacturers instructions. Reverse transcription of 100 ng RNA with oligo dT primer using the Improm II kit (Promega) in a total volume of 20 μl according to manufacturer's instructions was followed by PCR (parameters as above) using 5 μl of the template cDNA and 50 pmols each of the forward and reverse primers CTGTACGACGATGACGATAAG and GTGTCTTCCTCTGGAGATA respectively, to amplify a 655 bp product.Total RNA was purified from 5 × 10Cells were suspended in PBS and spotted onto multiwell glass slides and air dried for 60 minutes. Slides were fixed for 20 minutes in methanol at -20°C followed by 5 minutes in acetone at -20°C. The cell spots were rehydrated in 20% normal rabbit serum (in PBS) for 20 minutes then incubated with a 1 in 500 dilution of the monoclonal anti-poly histidine antibody  for 1.5 hours at room temp. After three 5 minute washes with PBS, the cells were incubated with a 1 in 50 dilution of FITC goat anti-mouse IgG (Biomeda) for 1 hour at room temp. Thereafter the slides were washed thrice in PBS, a drop of DABCO was added and coverslips were placed over the cell spots. The cells were viewed with by fluorescence microscopy and photographed with a Leica digital camera using a 1.27 sec exposure time and 15× magnification.6 cells (BHRF1 or vector clones) were seeded in either medium containing 10% serum or 0.1% serum and the percentage of viable versus apoptotic cells was determined by acridine orange flourescence at 24 hour intervals. For the etoposide assays, 0.5 × 106 cells (washed 3 times) were seeded in either routine medium containing DMSO  or medium containing 200 ng/ml etoposide (diluted from a 1 mg/ml solution in DMSO) and the % of apoptotic cells versus viable cells was determined every 24 hours by acridine orange fluorescence. For the UV induction assays, 1 × 106 cells in 10 ml of medium were placed in a Petri dish and exposed to 30 Jm-2 UV. The cells were then pelleted by centrifugation and resuspended at 2 × 105cells/ml in medium and returned to culture flasks.For the serum reduction assay cells were washed 3 times in PBS and then 0.5 × 10Each clone was tested in triplicate and each experiment was repeated 3 times.10 μl acridine orange (100 μg/ml) was added to 100 μl of cell culture containing the individual clones. At least 200 cells were counted using a haemocytometer at 15 × magnification and the number of viable versus apoptotic cells in the population noted.H. pan – Herpesvirus pan, PBS phosphate buffered saline, PCR – polymerase chain reaction, UV – ultraviolet, v-Bcl-2 – viral Bcl-2 homologueEBV – Epstein Barr virus, MH did the RT-PCR and UV assay, TW constructed the expression vectors and carried out some of the statistical analyses and SAH produced the transfected clones, carried out the serum withdrawal and etoposide assays, some of the statistical analyses and did the structural comparisons.


Drosophila.Cocaine addiction wreaks profound changes on the brain, hijacking reward circuits and depressing inhibitory loops to the point that drug seeking and taking become central drivers of behavior. Lying at the core of these behavioral changes are molecular ones; at its most basic level, addiction alters the sensitivity of neurons. While primates and rats are useful for mapping out the neural complexity of these behavioral manifestations, insights into the molecular basis of drug abuse can be garnered more easily from simpler models, such as the fruitfly, The reigning model of cocaine's effects on the brain has highlighted its ability to block reuptake of dopamine by cells of a brain region called the nucleus accumbens. But numerous experiments show this is not the whole story. Ulrike Heberlein and colleagues describe their discovery of a new gene that modulates sensitivity to cocaine within the cells of the fruitfly's internal clock. They further show that the cells' role in regulating cocaine sensitivity is distinct from its function as a timekeeper.Drosophila is loss of “negative geotaxis,” or wall climbing, in response to startle. Using this behavior to screen 400 different mutants, the researchers identified seven with an increased response to cocaine, and for two of these, the disrupted gene was the same, Lmo. The Lmo protein, whose levels were reduced by the mutations, is known to regulate certain transcription factors during development. Despite this, no developmental defects were detected in the loss-of-function mutants that might explain the cocaine effect. The researchers also found that a third mutation in the same gene, previously associated with disruption in wing formation, increased levels of the Lmo protein, and decreased response to cocaine. Thus, Lmo appears to play a central role in regulating cocaine sensitivity.One known effect of cocaine on Lmo is found throughout the body, it is enriched in the brain, and by expressing normal Lmo in oversensitive mutants, Heberlein and colleagues discovered that its cocaine-related effects were localized to the ventral lateral neurons (LNvs). Comprising about ten cells per hemisphere, these neurons provide the fly with an internal clock, driving circadian activities even in the absence of light. Not surprisingly, Lmo mutants had weaker circadian rhythms than normal flies.While v is a small peptide called PDF, and PDF mutation causes circadian disruptions. It does not, however, alter cocaine sensitivity. Furthermore, completely obliterating the LNv, or blocking its ability to fire, disrupted circadian rhythmicity but reduced cocaine sensitivity, rather than increasing it. These results indicate than the LNv normally enhances sensitivity to cocaine, a function enhanced further by Lmo mutants, and does so independently of circadian regulation.But is increased cocaine sensitivity a simple consequence of a broken clock? Apparently not. To date, the only known output of the LNLmo's role in modulating cocaine sensitivity. Drawing on recent evidence that a subset of LNv cells possess dopamine receptors, they suggest that Lmo expression normally regulates the density of these receptors on LNv cells. Loss of Lmo would raise the number of receptors, thereby increasing the sensitivity to cocaine. A key prediction from their findings is that the LNv has another output, as yet undetected, in parallel with PDF that mediates responsiveness to cocaine.Based on their results, Heberlein and colleagues propose a possible model for Lmo-related proteins are found in key areas of mammalian brains, these results may have important implications for understanding innate differences in sensitivity to cocaine in humans, and potentially provide targets for development of drugs to treat or prevent addiction.Because