Single nucleotide polymorphisms (SNPs) are extensively used in case-control studies of practically all cancer types. They are used for the identification of inherited cancer susceptibility genes and those that may interact with environmental factors. However, being genetic markers, they are applicable only on heritable conditions, which is often a neglected fact. Based on the data in the nationwide Swedish Family-Cancer Database, we review familial risks for all main cancers and discuss the evidence for a heritable component in cancer. The available evidence is not conclusive but it is consistent in pointing to a minor heritable etiology in cancer, which will hamper the success of SNP-based association studies. Empirical familial risks should be used as guidance for the planning of SNP studies. We provide calculations for the assessment of familial risks for assumed allele frequencies and gene effects (odds ratios) for different modes of inheritance. Based on these data, we discuss the gene effects that could account for the unexplained proportion of familial breast and lung cancer. As a conclusion, we are concerned about the indiscriminate use of a genetic tool to cancers, which are mainly environmental in origin. We consider the likelihood of a successful application of SNPs in gene-environment studies small, unless established environmental risk factors are tested on proven candidate genes. Genetic association studies on complex diseases have become very popular and most of them are case-control studies using single nucleotide polymorphisms (SNPs) as markers. There has been concern about the poor reproducibility of the results and the reasons for such discrepancies have been discussed -5. HowevHow feasible is it then to carry out SNP studies on cancer, particularly when the subjects are overwhelmingly unselected cases, among whom familial cases are rare. It is worrisome that genotyping is now almost a required standard component in epidemiological studies, without consideration of the expected heritable influence. Formal sample size and power calculations are irrelevant if there are no data on the assumed heritable component in the causation of a particular cancer. It is also worrisome that the fundamental differences between purely genetic and gene-environment studies regarding control populations and multiple testing problems are not appreciated. In this contribution, we first review study designs and aims of SNP studies, then we give data on familial risks for the main types of cancer, to give an idea of the upper bounds for the risks that can be expected in genotyping studies. Finally, we will calculate familial risks resulting from variants of assumed genotype relative risk and allele frequency. Such data are useful in the assessment of study designs and in the evaluation of obtained results. We use the term 'familial' to denote cancers in two or more first-degree relatives and 'heritable' when an inherited gene defect is known or inferred due to a high risk . InsteadIn the simplest form, a SNP with a known or assumed function is selected, and the genotypes are determined in cases and controls to test for association. Most studies published on cancer are of this kind, purely "genetic studies" testing the effect of the genotypes on the risk of cancer, without considering any other variables ,18. In ain vivo systems [in vivo functional test cannot be easily devised. Drug metabolism genes are a fortunate exception in this regard, because aberrant responses in humans have lead to the characterization of the underlying gene variants. One common feature of almost all the published studies is that patients have been collected without regard of a family history, thus sacrificing statistical power but attempting to compensate with a large sample size [Most studies have selected SNPs with assumed functional effects or they test the effects of haplotypes. For some genes, null or truncating alleles exist and homozygotes would then lack a functional protein. However, for missense types of SNPs the functional effects may be small or nil when tested in  systems . Unfortuple size ,25. Usinple size .5 = 3125 unique cells), and, secondly, the genes that are selected for analysis, are taken from a pool of tens or hundreds of potential candidate genes. No solutions have been found for this "two-dimensional" multiple testing problem. However, important for the present discussion, gene-environment interactions only exist if there is a heritable component in the particular cancer, and the likelihood of observing an effect is larger if the heritable component is large.A variant of the candidate gene approach is the "gene-environment study", which is founded on the assumption that in complex diseases environmental factors interact with heritable factors, and a strong effect can be detected when both are present -29. In eIn populations of random mating, it may be plausible that gene-environment interactions of epidemiologically measurable magnitude exit in the absence of a measurable familial risk. In the case of many exposures, some with harmful and others with protective effects, interacting with many genes, it may be possible that the familial risks are missed in spite of true gene-environment effects. Similarly, non-conventional dose-effect relationships, such as those suggested for blood vitamin D levels and prostate cancer , would bAccording to the complex disease paradigm, many relatively common alleles, interacting with environmental factors, cause susceptibility to common diseases ,32. SuchFamilial risk of a disease is a measure of its clustering in family members. Commonly, familial risk is defined between those who have a relative  with cancer compared to those whose relatives are free from cancer, given as a familial relative risk or familial standardized incidence ratio (SIR). The SIRs shown below have been obtained from the Swedish Family-Cancer Database, the largest dataset of the kind in the world . FamiliaFamilial cases constituted 15.4% of all prostate cancers, which was the largest proportion by far. The proportion was 10.1% for colorectal adenocarcinoma and 8.5% for breast cancer, but only 0.5% for connective tissue and testicular tumors. The proportions are generally higher for common cancers. Overall familial cancers (same cancer in offspring and parents) constituted 5.5% of all cancers.We have tried to estimate the degree of environmental contribution to the familial risk by comparing cancer risks betweens spouses. Spouse concordance, which does not generally exceed an SIR of 1.3, can be noted only for cancers with known strong environmental risk factors: lung and genital cancers and early onset gastric and pancreatic cancers and melanoma ,39. SpouIf environmental causes of familial clustering have been quantified or excluded, familial SIRs and proportions give estimates on the heritable effects for cancer at the level of nuclear families (here between parents and offspring). Because of low penetrance, familial proportions underestimate true heritable effects. On the other hand, the twin model assumes that the shared environmental effects of monozygotic and dizygotic twins are identical, which may not be true. If monozygotic twins share more than dizygotic twins, the estimated heritability is exaggerated. Thus, the heritability estimates for cancer are still unreliable, and, due to possible interactions, a dichotomous classification into heritable and environmental components is conceptually inaccurate . MoreoveResults from a successful SNP study can imply that the particular variant contributes to a familial risk of the particular cancer. The resulting familial risk depends on the allele frequency of the SNP, observed OR and the mode of inheritance, i.e., on the relative risks of heterozygotes compared to homozygotes. In the dominant model, the risk of heterozygotes equals that of the variant homozygotes; in the recessive model, the risk of heterozygotes equals that of the wild type homozygotes. In the additive model, the risk of heterozygotes is the mean of the two homozygotes; in the multiplicative model, the risks between the genotypes differ by a constant multiplier.BRCA1/2, ATM, p53 and CHEK2, explain about 25% of the risk [The methods for the calculation of familial risks to offspring of affected parents . According to Table BRCA1 and BRCA2 combined. The effects of metabolic polymorphisms on various cancers have been reviewed in an IARC publication [It is of interest to examine the magnitude of familial risks which would be predicted from the published ORs for candidate genes. In a review of 34 polymorphisms in 18 different genes tested for breast cancer, a large proportion of the associations were not significant and the ORs were below 2.0 . Even malication . Among mlication ,50. SomeThe poor reproducibility of candidate gene studies has most commonly been associated to small sample size, population stratification and low prior probability, i.e., poor selection of genes or SNPs; a SNP with small functional effect would also imply a low prior probability for an effect ,32. We aIt is commendable that all available molecular and environmental data are being used in attempts to understand the mechanisms of human cancer -53. With


There is considerable interest in identifying target genes and chromatin binding sites for transcription factors in a genome-wide manner. Such information may become useful in diagnosis and treatment of disease, drug target identification, and for prognostication. In cancer diagnosis, patterns of transcription factor binding to specific regulatory chromatin elements are expected to complement and enhance current diagnostic predictions of tumor behavior based on protein and mRNA analyses. Signal transducer and activator of transcription-5 (Stat5) is a cytokine-activated transcription factor implicated in growth and progression of many malignancies, including hematopoietic, prostate, and breast cancer. We have explored immunoaffinity purification of Stat5-bound chromatin from breast cancer cells to identify Stat5 target sites in an unbiased, genome-wide manner.in vitro and in vivo as tested by electrophoretic mobility shift assay and chromatin immunoprecipitation, respectively. While there was no binding to fragments lacking a Stat5 consensus binding sequence, presence of a Stat5 binding sequence did not assure binding.In this report, we evaluate the efficacy of a Stat5-bound chromatin library to identify valid Stat5 chromatin binding sites within the oncogenome of T-47D human breast cancer cells. A general problem with cloning of immunocaptured, transcription factor-bound chromatin fragments is contamination with non-specific chromatin. However, using an optimized strategy, five out of ten randomly selected clones could be experimentally verified to bind Stat5 both in vivo Stat5 chromatin binding sites in cancer, including abnormal regulatory sites in tumor-specific neochromatin.A chromatin library coupled with experimental validation may productively identify novel  Transcription factors function uniquely at the interface of the genome and the proteome. A significant portion of transcription factors serve not only as executors of gene transcription programs, but also as biochemical sensors of extracellular stimuli. For instance, members of the nuclear receptor family are directly activated by lipophilic extracellular ligands, and transcription factors of the Smad and Stat families are activated by phosphorylation in response to cytokine stimulation of cell surface receptors. Chromatin-bound transcription factors that act both as sensors of extracellular cues and as transcriptional effectors carry exceptional instructive value about the biological state of individual cells. Their high biological information value makes such factors particularly attractive for use as markers to predict disease activity and outcome, as well as predictive markers of disease-responsiveness to drugs.ENCyclopedia Of DNA Elements), has been initiated with the ambitious goal of identifying all regulatory elements of the human genome, including chromatin binding sites for individual transcription factors [Based on related and broader rationale, the second phase of the human genome project, ENCODE  and chromatin immunoprecipitation (ChIP).In contrast to transcription factors that bind to chromatin in a constitutive manner, Stat5 is a latent cytoplasmic transcription factor that is activated by tyrosine phosphorylation and binds tightly to DNA in response to extracellular cytokines, such as prolactin . We usedMethods. Before subcloning of the captured chromatin fragments, the specificity of immunocapture was verified by analysis of binding to known human Stat5 target chromatin. In particular, we took advantage of earlier work that has identified a group of Stat5 regulated genes that have been shown to contain the Stat5 consensus sequence, TTCNNNGAA, in the traditional promoter element [Cytokine-Inducible SH2 Protein (CISH), β-Casein, and α2-Macroglobulin. Due to the average chromatin fragment size of 400 bp, primers were designed to yield shorter PCR products of 200 – 300 bp.Sonication is necessary to shear genomic DNA into fragments that can be easily manipulated for PCR amplification, cloning, and sequencing. Fragments of approximately 400 base pairs (bp) allow for a complete sequencing read-through and are of sufficient size to localize the fragment within the human genome with a high degree of statistical certainty. Optimal shearing of chromatin from formaldehyde-fixed T-47D cells into approximately 400 bp fragments was established empirically Figure , lane 5. element ,15. AliqCISH gene response element in T-47D cells  insert-containing PCR products. Figure The enriched, Stat5-bound chromatin pool was then cloned into a bacterial vector to generate a chromatin library. Because sonication generates random overhangs in double stranded DNA , T4 DNA in vitro by EMSA. Stat5 binds to the consensus sequence TT(N5)AA and to relatively conservative variations, with TTC(N3)GAA considered to be optimal [in vitro was detected in seven of the ten chromatin fragments, as evidenced by prolactin-inducible DNA-binding complexes that could be supershifted by a specific anti-Stat5 antibody, but not by non-specific IgG  prolactin and (+) prolactin samples. In all cases there was no detectable product from the replicate samples that had been immunoprecipitated with a non-specific IgG antibody (data not shown), indicating a specific Stat5-mediated capture of genomic elements. All PCR amplifications were performed at least twice on at least two separate pools of immunoprecipitated genomic elements.As a second and independent means to validate the quality of the Stat5 chromatin library, the same randomly selected fragments were analyzed for inducible Stat5 binding in vitro Stat5 binding by EMSA, five  were also consistently positive for in vivo Stat5 binding by ChIP assay  was purchased from Sigma Chemical Co. .The human breast cancer cell line T-47D was grown to confluence in RPMI-1640 medium  supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin  at 37°C with 5% CO7 cells/T175 cm2 flask), T-47D cells were serum starved for 24 h and then treated with or without 10 nM hPRL for 30 min. For prodifferentiation experiments with glucocorticoid pretreatment, confluent cultures were maintained in serum-free RPMI-1640 with 1 μM Dex dissolved in DMSO or in DMSO alone for 96 hours, then stimulated with or without PRL for 30 min. Proteins were then crosslinked to the chromatin by the addition of formaldehyde  to a final concentration of 1% and incubated for 30 min at 37°C. The cells were rinsed, scraped, and pelleted in ice-cold PBS with 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A. Cell pellets were resuspended in 400 μl lysis buffer  and the lysates were sonicated using a Sonic Dismembrator , fitted with a tapered microtip set to an amplitude of 50% and were pulsed twice for 30 s. Debris was pelleted by centrifugation for 10 min at 13,200 RPM at 4°C. The supernatants were then diluted 10-fold in IP buffer  and 1% was set aside for Pre-IP or input sample. The solution was then pre-cleared with beads  for 30 min at 4°C. Next, the samples were incubated overnight with Stat5 antibody  or IgG IP-control followed by immunoprecipitation with pre-incubated beads. The samples (beads) were then washed once with each of these buffers in the following order: Wash Buffer 1 , Wash Buffer 2 , Wash Buffer 3 , then twice with Wash Buffer 4 . The samples were eluted from the beads in 1% SDS and 0.1 M NaHCO3. Next, the cross-links were reversed with 0.2 M NaCl overnight at 65°C followed by protein digestion with proteinase K for 1 hour at 45°C. The DNA was recovered by phenol:chloroform extraction and ethanol precipitation. This pool was then used as PCR template or could be blunted with T4 DNA polymerase  at 37°C for 5 minutes under standard conditions for uniform cloning. Taq DNA polymerase (New England Biolabs) was then used to add a 3' A overhang for cloning into a TOPO TA cloning kit . The ligated vectors were transformed into electrocompetent bacteria, DH-5αE (Invitrogen), using the manufacturer's recommendations for transformation in a Bio-Rad Gene-Pulser , and plated on S-Gal/Ampicillin plates . Positive clones by blue-white screening were then analyzed directly by colony PCR in a standard reaction with vector-specific M13 and T7 primers that flank the cloning site and were incubated at 94°C for 4 min and cycled at 94°C for 30 s, 55°C for 45 s, and 72°C for 30 s. Colonies with quantifiable inserts were then purified , sequenced, and localized using BLAST .After grown to confluence  = 6.67, as described previously . Paralleα32P dATP . Initially the samples were incubated at 94°C for 1 min, then cycled 36 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The reaction was then held at 72°C for 5 min following the cycling to allow for product fill-in and addition of a 3' terminal "A". After completion of the cycling, the PCR products were purified using the Qiagen PCR purification kit, according to manufacturer's instructions. The final products were eluted in 50 μl and stored at -20°C until use.Radiolabeled products were generated by PCR in a 10 μl reaction under standard conditions with the addition of 0.25 μl 32P-labeled PCR probe and incubated for 20 min at room temperature. The samples were then resolved in a 3% non-denaturing polyacrylamide gel, as previously described [The DNA-protein binding reactions were performed in a 10 μl mixture containing 3 μl of nuclear extract from the respective sample, and 1 μg of double-stranded poly dI:dC , as previously described . After 1escribed .CCF #5 forward 5' TGA CAT CAG TGA GAG TGG AGG 3', reverse 5' TGA GGC TGT AAT GTC ACT CAG AA 3'; CCF #11 forward 5' GGA CAC ATC CAC TAC TGC CA, reverse 5' AAA AGA AGT GCA GTT CAG GAT AA 3'; CCF #14 forward 5' GCC TGT GTG ATA TCA TAT GGA AAG 3', reverse 5' TTT CCA AGA ACT TAT CAG AAT GAC TT 3'; CCF #18 forward 5' TCA GTC TTC CTC TTT CCC CA 3', reverse 5' CGG CTT ACA TTC TGT GTC GC 3'; CCF #23 forward 5' CAT ATC TGT CTA CAA ATA ACA GTT CCC 3', reverse 5' AAT AGA GGG CAG AGT TAA GAA TCA AA 3'; CCF #28 forward 5' AAG CCT GAA AAG AAA AAT CTC A 3', reverse 5' CCT GAC ACA TCC AGA CTT GG 3'; CCF #29 forward 5' CAG ACG TTT GCT GGA AGA TAT G 3', reverse 5' TTG TTC CAT GAA ACC AGG G 3'; CCF #30 forward 5' AAG GCC ATT GAA GTG AGG TG 3', reverse 5' CCA TCA GCC AGT CAT TGA AG 3'. The PCR was performed with standard conditions using template immunoprecipitated from cells treated with or without prolactin to indicate Stat5 inducibility and was executed at least twice on each of 2 separate pools.As an independent validation technique, we designed primers specific to each cloned chromatin fragment (CCF) then performed PCR on a separately-generated enriched pool of immunoprecipitated Stat5-chromatin interaction sites. The following primers were used for PCR: MJL carried out the experimental studies, participated in the design of the study, and drafted the manuscript. JX participated in the design of the study and scientific discussion of the results. HR conceived and participated in the design of the study, contributed in the evaluation of the results, and in the preparation of the manuscript. All authors read and approved the final manuscript.


Functional magnetic resonance imaging offers the promise of peeking into the human mind. What signals in the human brain can we really detect and how should the technology be used? Functional magnetic resonance imaging—fMRI—opens a window onto the brain at work. By tracking changes in cerebral blood flow as a subject performs a mental task, fMRI shows which brain regions “light up” when making a movement, thinking of a loved one, or telling a lie. Its ability to reveal function, not merely structure, distinguishes fMRI from static neuroimaging techniques such as CT scanning, and its capacity to highlight the neural substrates of decisions, emotions, and deceptions has propelled fMRI into the popular consciousness. Discussions of the future of fMRI have conjured visions of mind-reading devices used everywhere from the front door at the airport terminal to the back room of the corporate personnel office. At least one “neuromarketing” research firm is already trying to use fMRI to probe what consumers “really” think about their clients' products.But will fMRI's utility in the real world ever match the power we currently imagine for it? Is fMRI likely to leave the clinic for widespread use in the courtroom or the boardroom? Are there neuroethical nightmares just around the corner? Or are all these vivid specters really just idle speculations that will never come to pass?To understand the potential, and the limitations, of fMRI, it's helpful to know how the technique works. The heart of the apparatus is a large donut-shaped magnet that senses changes in the electromagnetic field of any material placed in its center, in particular—when a head is scanned—the blood as it flows through the brain. When a region of the brain is activated, it receives an increased flow of oxygenated blood . This influx of oxygenated blood alters the strength of the local magnetic field in proportion to the increase in flow, which is detected and recorded by the imaging machinery.The resolution of the best fMRI machines—the smallest “volume picture element,” or voxel, they can distinguish and make an image of—is currently about 1.5 mm ×1.5 mm × 4 mm, the size of a grain of rice. There are approximately 150,000 of these little volumes in the typical brain, and the immense computers hooked up to the scanners record and integrate signals from all of them. In a typical experiment, a subject, lying still with his head surrounded by the magnet, does nothing for thirty seconds, then performs some task for thirty seconds, then lies still for thirty seconds. For each voxel, the signal during the task is compared to the signal at rest; those areas of the brain with stronger signals during the task are presumed to be processing the information that underlies the performance of the task . AccordiThe first caveat about fMRI's imaging power, though, and one that every neuroimager stresses, is that a voxel is a long way from a neuron. There are an estimated 100 billion neurons, so at best, an fMRI is signaling blood flow changes associated with the increased activity of tens of thousands of neurons. As a result, says Hirsch, fMRI “falls short when we want to ask about more detailed brain processes. We're not learning that much about how neurons are doing local computing.” While resolution will improve over time, it seems unlikely that fMRI will ever detect the activity of individual neurons, and so its ability to dissect the “fine structure” of thought is inherently limited. These limitations have not prevented fMRI researchers from making some major discoveries about brain function, however. Hirsch, for instance, showed in one study that minimally conscious individuals still process human speech, and in another, that those who become bilingual as young children employ overlapping language areas in the cerebral cortex, while those who learn a second language later in life use a different area for the second language. The key strength of fMRI, she says, is that it provides the ability to test these kinds of hypotheses about structure–function relationships in the normal brain.But the hypotheses that can be tested and the conclusions that can be drawn are still largely about group averages, not about the functionings of individual brains, and therein lies a second major caveat about the use of fMRI beyond the clinic. John Gabrieli, Associate Professor of Psychology at Stanford University, has shown that distinct activation patterns in the brains of dyslexic children normalize as they improve their reading skills . It seemthis scan in this individual on this day does or doesn't reflect his underlying racial attitudes.There are similar limitations to most other applications of fMRI—while conclusions can be made about aggregated data, individual scans are for the most part too hard to interpret. There is not yet any real understanding of how brain patterns change over time in an individual, or how interindividual differences should be interpreted in relation to the conclusions that are valid for groups. This makes fMRI an unlikely tool for job screening, for instance. While one study has shown a brain signature in a group of white people that is associated with racial bias, denying a particular individual a job on the basis of such a scan would likely lead straight to a lawsuit, with experts debating whether On the other hand, Hirsch has used individual scans to help locate a patient's language structures that must be spared during neurosurgery. “If you are a neurosurgeon planning a resection, you don't want an average brain at all. Millimeters matter.” But her success is precisely because she is not using group data to make inferences about the individual—she is not leaping over the chasm, but instead is toiling entirely on the other side of it. “The goal is personalized medicine,” she says.Even this kind of personalized approach with fMRI is fraught with problems when researchers attempt to apply it outside the clinic, because of limitations in the technology itself. One researcher with firsthand knowledge of these problems is Daniel Langleben, Assistant Professor of Psychiatry at the University of Pennsylvania School of Medicine. In 2002, Langleben showed that when subjects were hiding information in an attempt to deceive , they had intense activity in five distinct brain areas not seen when they were telling the truth. In effect, Langleben used the fMRI as a lie detector. It is potentially even more powerful than a standard polygraph test, he says, because there are thousands of brain regions which can be scanned for deception-triggered variation, versus only three variables—skin conductance, respiration, and blood pressure—used in the standard polygraph. Not surprisingly, Langleben got a lot of press after he announced his results, and his experiment led directly to speculation that we might eventually see fMRIs installed at airports, scanning the brains of would-be terrorists trying to deceive security screeners, or in courtrooms, catching perjurers red-handed (or perhaps red–anterior-cingulate-gyrused?).Langleben is enthusiastic about the potential for an fMRI-based lie detector, and has even applied to the Department of Justice for a grant to develop the technology . But he is also clear about how difficult it will be to get one that really works outside the highly structured confines of the research lab. “We are a long way from making a working polygraph,” he says. Even with a “Manhattan Project” type effort, he speculates it would take at least ten years. “There are still essential discoveries to make along the way,” he says, “and there's a good chance it would end in total failure.” It's not just a matter of developing the imaging technology, he stresses—“we'll need fundamental developments in semantics, too.” This is because “a lot still depends on how you ask the question”—the subtlest of differences can dramatically shift which areas of the brain respond. Given the sensitivity of the fMRI result to such seemingly minor perturbations, it's hard to imagine it could be reliably adapted to the hurly-burly of an airport security checkpoint.Even well-performed scans done in topnotch clinics may not easily find their way into the courtroom. Perhaps the least likely use of fMRI is in determining if a defendant is telling the truth, according to Hank Greely, Professor of Law at Stanford Law School, since compelling someone on trial to submit to an fMRI could be seen as a violation of the Fifth Amendment right against self incrimination, just as giving spoken testimony against oneself is. On the other hand, says Greely, DNA samples and fingerprints can be compelled—whether a brain scan is more like testifying or more like submitting to a blood test is an open question. Still, for the moment, scanning under duress simply isn't feasible, since all you have to do to ruin a good scan is move your head. Motion-correcting algorithms can be used, but they are nowhere near advanced enough to correct for large-scale movements by an unwilling subject. It's much more likely that an fMRI of a willing defendant would be introduced to convince the jury he is telling the truth, or performed before trial to rule out an innocent suspect. While to Greely's knowledge fMRI evidence hasn't yet been used in court, “it's certain to be tried,” and the barrier to its admission will fall as both the reliability and the ease of administration increase. “The easier, the cheaper, the more pleasant a technique is, the more likely it is to be used in the legal system.”Other forensic uses of fMRI are likely to arrive sooner rather than later. Could scans showing diminished impulse control—a function controlled by several regions of the brain, including the striatum and the ventromedial prefrontal cortex—be used to support more lenient sentencing, or even acquit a defendant, because he couldn't control his violent impulses? Or alternatively, will those same scans be used to argue for harsher sentences, since the defendant is clearly “hardwired” to commit similar crimes again? Courts already consider other factors, such as a history of child abuse, in an attempt to more fully understand the psychological state of the defendant. Will brain scans be seen as the ultimate “objective” look into the mind of the person on trial?Deciding all these issues of admissibility will be judges who will need to weigh competing claims from lawyers with competing interests, backed up by expert witnesses with competing theories. Here, the desire to apply the science may rush ahead of its demonstrated validity.Langleben, for one, doesn't think fMRI will be legitimately ready for the courtroom for a long time. On the other hand, he says, “if you want to abuse this technique and claim that it works, you can create tests that will produce results—I can see how it could be done. We know enough to rig it.” But still, he says, “we have all the tools we need to prevent this—there are enough people who are sufficiently honest [who would counter the premature use of fMRI in these contexts].”For now, at least, given the problems inherent in current fMRI technology, the neuroethical nightmare scenarios of widespread brain scanning seem unlikely to come to pass, at least until radical advances make it far cheaper, much less invasive, far less sensitive to subtle perturbations, and with a much more robust ability to legitimately extrapolate from a finding about a group to a prediction about an individual. Where fMRI is concerned, “a penny for your thoughts” is currently more like “a million pennies for a group-averaged hemodynamic response to highly constrained stimuli under entirely artificial conditions.”In light of this, bioethical concerns about fMRI applications should perhaps be viewed not as predictions of a certain future but rather as worstcase scenarios, a reminder of what we want to avoid. “It's a funny thing about the bioethics field,” says Greely. “The general approach is to look for bad news.”While many of these “worst cases” seem highly unlikely to come to pass, Judy Illes, of the Stanford Center for Biomedical Ethics, thinks some action is warranted now, if only to generate a better understanding of the ethical dimensions of fMRI research. She notes that “bioethicists are often viewed as the ethics police,” but she doesn't see regulations as the right path to shape the future uses of fMRI. Instead, she thinks a coalition of involved parties—scientists, lawyers, ethicists, politicians—should work together to develop guidelines that all will find acceptable. “I'm not in the business of stopping anything.”What everyone apparently already agrees on is the need for carefully designed experiments and cautious interpretation of the data. “A huge message in imaging is that you really have to look at the experimental setup at the common-sense level,” says Gabrieli, and avoid the tendency to “pick the most dramatic interpretation.” “The public needs to be reminded of the limitations of these findings,” agrees Hirsch. And as Langleben puts it, expressing his skepticism that there will ever be a one-size-fits-all, foolproof fMRI mind reader: “I don't think we'll ever be able to be stupid about it.”


Plasmodium falciparum multiple epitope (ME)– thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model.Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. P. falciparum. Analysis was per protocol.A total of 372 Gambian men aged 15–45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity.DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, −22% to +34%; DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell–inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults. Inducing an immune response is just one part of what a vaccine needs to do before it will protect against episodes of malaria. Plasmodium falciparum parasites [Anopheline spp. vectors [P. falciparum [P. falciparum malaria, were manufactured to explore this approach [The disease burden of malaria has increased in recent years partly because of the rise of drug-resistant arasites  and inse vectors . There ilciparum ,6,7,8,9.lciparum ,11. DNA approach . γ-interapproach .DNA and modified vaccinia virus Ankara (MVA)'s excellent safety profiles in malaria-naïve and semi-immune volunteers have been discussed previously . In seve8 plaque-forming units, produced very strong vaccine-induced CD4+ and CD8+ T cell responses in previous phase I studies in the United Kingdom [Priming with three 2-mg intramuscular DNA ME-TRAP vaccinations at 3-wk intervals, followed by boosting with one intradermal MVA ME-TRAP vaccination of 1.5 × 10 Kingdom . The imm Kingdom . DNA ME- Kingdom .P. falciparum infection. We chose a two-DNA prime, one-MVA boost regimen with 3-wk between doses because this is a three-dose regimen that would be amenable to integration with the World Health Organization/United Nations Children's Fund Expanded Program on Immunization, with the necessary supporting safety and immunogenicity data both from adults in the United Kingdom and Gambia. We used 3-wk intervals because 4-wk intervals had not been evaluated in phase I trials previously, hence bridging studies would be necessary to bridge to the three-dose, 4-wk interval Expanded Program on Immunization schedule.To follow up these encouraging findings in volunteers, we have conducted a randomised, controlled trial of DNA ME-TRAP/MVA ME-TRAP in a rural part of Gambia to explore whether this vaccine combination could provide protection against natural P. falciparum antigens. It also contains two non-malarial CD4+ T cell epitopes [P. falciparum TRAP [P. falciparum used to produce the vaccine construct is T9/96. The candidate malaria vaccines were manufactured to Good Manufacturing Practice by contract manufacturers . DNA ME-TRAP was supplied as a single dose of2 mg in 2-ml vials. MVA ME-TRAP was supplied as two-dose vials, each containing 3 × 108 plaque-forming units in 0.8 ml. The rabies vaccine  was supplied as a lyophilised single-dose vial with accompanying diluent and syringe. This vaccine was chosen because of its public health benefit in Gambia.The malarial DNA sequence is known as ME-TRAP. The ME string contains 14 CD8+ T cell epitopes, one CD4+ T cell epitope, and two B cell epitopes from six pre-erythrocytic epitopes . The ME rum TRAP ,24,25. Trum TRAP . The strApproval was obtained from the Joint Gambian Government/Medical Research Council Ethics Committee, the Oxford Tropical Research Ethics Committee, and the London School of Hygiene and Tropical Medicine Ethics Committee. An independent Data and Safety Monitoring Board provided oversight for the trial. In addition, independent clinical trial monitors monitored the trial for adherence to International Committee on Harmonisation Good Clinical Practice guidelines.Malaria incidence is highly seasonal in Gambia. The climate is typical of sub-Sahelian Africa, with a long dry season followed by a relatively short rainy season from July to October. Rainfall averages about 600 mm per year. Morbidity and mortality from malaria both occur more frequently during the rainy season. However, in 2002, Gambia experienced a drought, and the malaria season was delayed, with few disease episodes before October. The entomological inoculation rate varies between less than 1 and greater than 100 in Gambia . Based oBefore recruitment, meetings were held with village heads and elders, followed by general village meetings at which the study was explained. Volunteers received information sheets and consent forms translated into the three local languages in Arabic script, as well as in English. After written informed consent was obtained by a study physician, age and identity were checked, pre-test HIV counselling occurred, and potential volunteers underwent clinical evaluation, including a full medical history and clinical examination. They were screened for haematological (full blood count), renal (plasma creatinine), and hepatic  dysfunction, and duplicate malaria smears were made. Exclusion criteria included any chronic illness detected by clinical evaluation, ALT greater than 42 , creatinine greater than 130 (micromoles/litre), packed cell volume less than 30%, positive antibody ELISA to HIV-1 or HIV-2, simultaneous participation in another clinical trial, blood transfusion in the month prior to vaccination, previous experimental malaria vaccination, administration of another vaccine within 2 wk of vaccination, previous rabies vaccination, allergy to any previous vaccine or to sulphadoxine/pyrimethamine, history of splenectomy, and any treatment with immunosuppressive drugs. Eligible volunteers were given a unique study number and a photographic identity card. Parental written informed consent was obtained for volunteers aged 15–17 y.A member of the Data and Safety Monitoring Board generated and held the randomisation code that associated each study number with a specific vaccine. A blocking procedure was used, and whole villages were enrolled with sequential study numbers to ensure balanced numbers in each group. During the course of the study investigators and volunteers did not know the size of the blocks, nor were they aware of which vaccine preparation was administered to a particular volunteer. Opaque sealed envelopes were used for vaccine allocation. Study numbers were not preprinted on vials but were written on vials at vaccination. Used vials were checked for correct allocation off-site. Vaccination was performed by nurses who played no other part in the trial.8-plaque-forming-units dose of MVA ME-TRAP or (2) three doses of rabies vaccine; injections were given on days 0, 21, and 42, timed to coincide with the start of the rainy season. The first two doses of vaccine consisted of two intramuscular injections, one into each deltoid muscle. DNA ME-TRAP was given as 1 ml and rabies vaccine as 0.5 ml into each arm. The third dose of vaccine was given as four intradermal injections into the skin overlying the deltoid muscle, with two injections into each arm. The malaria vaccine group received MVA ME-TRAP as four 0.1-ml injections, whereas the control group received four 0.05-ml injections of rabies vaccine. Two weeks before administration of the third dose, all volunteers received three tablets of sulphadoxine/pyrimethamine to clear blood-stage P. falciparum infections [Volunteers were randomly assigned to receive either (1) two 2-mg doses of DNA ME-TRAP followed by a single 1.5 × 10fections .After each vaccination volunteers were observed for 1 h and visited at home on the first, second, and seventh day postvaccination for assessment of local adverse events , systemic adverse events , and unsolicited adverse events. One week and 13 wk after the third vaccination, venous blood was collected for repeated measurement of full blood count, ALT, and creatinine.P. falciparum parasites at any parasitaemia with either an axillary temperature of 37.5 °C or more or one or more of the following symptoms: headache, myalgia, arthralgia, malaise, nausea, dizziness, or abdominal pain.Since vaccination with MVA causes a characteristic local reactogenicity in some subjects, specific steps were taken to ensure that the participants were evaluated in a double-blinded manner. Field workers who assessed reactogenicity after the third dose were different from those who undertook surveillance during the parasitological follow-up period. During the surveillance period, starting 2 wk after the third dose of vaccine, volunteers were visited twice weekly and asked whether they had attended a health centre. At weekly visits blood smears and axillary temperatures were taken. At midweek visits, blood smears and temperature were taken if symptoms compatible with malaria were present. Investigators and field supervisors made random visits to ensure accurate data collection. This active case detection was supplemented by passive case detection by study nurses to whom volunteers had 24-h access at three of the study villages and by a clinic at Medical Research Council Farafenni (20 km from the study villages). Symptomatic malaria was defined as the presence of asexual P. falciparum parasites was confirmed by a supervisor before a slide was declared positive. The arithmetic mean of the A and B slides was used to determine parasite density. If parasite densities for A and B slides were markedly discrepant, a third read was performed by the supervisor and this read was used for analysis. Parasite density was expressed per microlitre . Full blood counts were performed and packed cell volumes were measured in a CA620 cell analyser . ALT  and creatinine (micromoles/litre) were measured in a Visual analyser .When blood smears were obtained, two sets of duplicate blood smears  were made. A Field's stain was performed on films obtained from subjects with possible clinical malaria and the films read immediately. Two further smears (“A” and “B” slides) were stained with Giemsa after overnight drying; 100 high-power fields were read by two slide readers before a film was declared negative. The presence of 5 peripheral blood mononuclear cells (PBMCs) were assayed as described [P. falciparum.Effector T cell responses were assessed in ex vivo γ-interferon enzyme-linked immunospot (ELISPOT) assays for 98 volunteers randomly selected from a substudy list containing a 3:1 ratio of participants receiving malaria vaccines to participants receiving rabies (control) vaccines. For this assay, 4 × 10escribed , using MP. falciparum, defined as the number of days from the start of the surveillance period to the date of the first positive slide. Vaccine efficacy was calculated from the hazard ratio estimated by Cox's regression, adjusting for the effects of prognostic variables. Volunteers who received fewer than three doses or who were parasitaemic both prevaccination and at the beginning of surveillance without an intervening negative blood smear were excluded from the primary analysis but included in a secondary analysis. Observations on individuals who were lost to follow-up or were missing from trial data for 3 wk were censored. Analyses were done with Stata version 7 . ELISPOT responses were analysed as follows. After subtraction of medium-alone values from each pooled peptide response, responses were summed across T9/96 and 3D7 pools. Geometric means were calculated for T9/96 TRAP, 3D7 TRAP, and ME string responses. Responses in the two groups were compared using the Mann-Whitney test.An analysis plan, written before unblinding, specified exclusion criteria, statistical methods, and important covariates . Ethnic group, though not specified as a covariate in the analysis plan, was found on analysis to be associated with the risk of infection, and was included as a covariate. The primary endpoint was time to first infection with asexual In total, 489 volunteers were screened , of whomNo clinically significant differences in packed cell volume, ALT, or creatinine were seen in either vaccine group. One volunteer who received rabies vaccine had a history of breathlessness and chest pain several years prior to enrolment, experienced a relapse of symptoms, and deteriorated and died, probably from heart disease, 3 mo after he received the last dose of vaccine. This event was regarded as unrelated to vaccination. There were no other serious adverse events. Adverse events were rare after first and second doses and were not increased in the DNA ME-TRAP group compared to the rabies vaccine group (data not shown). Injection site pain, limited arm motion, headache, and malaise in the first 24 h after vaccination  were more common after MVA ME-TRAP vaccination than rabies vaccination . Some vop <0.001, range 6.25–2148.75). A large cross-reactive T cell response to 3D7 TRAP, a strain with 6% amino acid variance to T9/96, and a weaker response to the ME string were also seen at this timepoint  per million PBMCs for T9/96 and 3D7 strains of TRAP and the ME string, respectively. In the malaria vaccine group, the effector T cell frequency to the vaccine strain of the TRAP antigen, T9/96, was geometric mean 251.1 SFCs per million PBMCs . Similar results were obtained when all participants who received at least one dose of vaccine were included in the analysis .By the end of the study, 171 participants developed parasitaemia, 80/141 (57%) in the malaria vaccine group and 91/155 (59%) in the rabies vaccine group. The distribution of time to first infection was similar in the two groups . VaccineP. falciparum densities in first infections were similar in the two groups .Geometric mean During surveillance, there were ten episodes of symptomatic malaria in the malaria vaccine group and 13 in the rabies group. The risk of malaria-related symptoms during an episode of parasitaemia was similar in both vaccine groups. Percentage mean packed cell volume at the end of the trial was similar in both groups .Within the ELISPOT substudy group, the risk of developing parasitaemia was not associated with effector T cell response to the 3D7 strain of TRAP. The 80 men from the substudy group who received three doses of either malaria vaccine (55 men) or rabies vaccine (25 men), completed 11 wk of surveillance, and had complete ELISPOT data after dose three were divided into four quartiles. Men with the highest effector T cell responses had hazard ratios for infection similar to those with the lowest after adjustment for age, bednet use, and village of residence.The incidence of parasitaemia decreased with increasing age, and was decreased in those of Fula ethnicity compared to those in the Mandinka and Wollof ethnic groups . The useP. falciparum infection rate in a semi-immune adult African population. This provides a second comparison between protection in malaria-naïve and malaria-experienced adults. RTS,S/AS02, a circumsporozoite protein malaria vaccine based on a hepatitis B surface antigen virus-like particle formulated in a proprietary adjuvant, provided about 40% sterile protection in the artificial challenge model [This trial demonstrated that vaccination with two doses of DNA ME-TRAP followed by a single dose of MVA ME-TRAP is safe and highly immunogenic for effector T cell induction but that it did not reduce the ge model  and 71% ge model . The lacge model , whereasge model . The preAnother possible reason for the observed low efficacy is that the frequency of the effector T cell response declines from 7 d after boost, and so efficacy would be prevented if very high frequencies of circulating effectors are needed for protective efficacy. Alternatively, a suboptimal regional memory T cell pool in the liver may be responsible , or, lesIn previous studies in East and West Africa, summed T cell responses to TRAP in unvaccinated semi-immune adults by ex vivo γ-interferon ELISPOT were geometric mean less than 20 SFCs per million PBMCs. The candidate regimen represents a new method for induction of unprecedented effector T cell frequencies, which are about 50-fold higher than those induced by lifelong natural exposure. Estimates of the reduction in liver-stage parasite burden induced by these vaccines in the human challenge model are of the order of 80%–90% of infected hepatocytes ,33. It iSecond-generation prime-boost vaccine strategies for malaria currently in or near to clinical evaluation include the following: use of a different viral vector as the priming agent that may lead to proportionately greater CD8+ rather than CD4+ T cell induction , as is the case with fowlpox-MVA immunisation; the use of a different antigen, the circumsporozoite protein, or polyprotein constructs  to addreWe were unable to obtain a useful estimate of the likely efficacy of the DNA ME-TRAP/MVA ME-TRAP vaccination regime against clinical disease. Even for an adult population, the incidence of clinical disease was lower than expected. Sulphadoxine/pyrimethamine was administered 4 wk before the start of surveillance in this study and in an RTS,S field efficacy study . There iThis study highlights North Bank Division in Gambia as an excellent malaria vaccine field trial site both for adults and, by extrapolation, for children. In a low-transmission year, cumulative incidence overall in men aged 15–45 y was 72% over 11 wk, which was higher than expected. Also, compliance was good despite a demanding study design, and migration from the study area was acceptably low.This paper adds to the body of data detailing the very gradual acquisition of anti-infection immunity in adults resident in sub-Saharan Africa . While sImmunological analysis of the high level of protection inducible by immunisation of humans and animals with irradiated sporozoites has encouraged attempts to generate protective immunity by subunit vaccines that induce strong cellular immune responses. To date the induction of high-level protective T cell responses against malaria and some other infectious pathogens has generally required two-component prime-boost vaccination approaches . We repoMalaria kills 1–2 million people a year, mostly children under the age of five who live in sub-Saharan Africa. Scientists are trying to develop cheap, safe, and effective vaccines that could be given to people living in regions where malaria is very common to prevent them from developing the disease.The researchers enrolled 372 Gambian men aged 15–45 years into the study. They injected half the men with two malaria vaccines, one after the other, and half the men with a rabies vaccine that does not protect against malaria  just before the rainy season, when malaria is especially prevalent. The scientists took blood smears from the men once a week and checked to see if they had been infected with the parasite that causes malaria. They found that the men who had been vaccinated became infected just as quickly as those who had not. Although the two malaria vaccines in concert did not work, neither did they cause any serious side effects. The men given the malaria vaccines did produce an immune response to the vaccines, though not one that was clinically useful.Plasmodium falciparum, the parasite that causes malaria. However, there are other vaccines in development that have not been tested yet.It looks as though the combination of these two vaccines is not effective at preventing infection with http://www.lshtm.ac.uk/gmp/Gates Malaria Partnership (which co-funded the study): http://www.malariavaccine.org/Malaria Vaccine Initiative: http://www.mmv.org/pages/page_main.htmMedicines for Malaria Venture: http://rbm.who.int/partnershipRoll Back Malaria Partnership: http://www.wellcome.ac.uk/en/malaria/The Wellcome Trust (which co-funded the study): http://www.controlled-trials.com/isrctn/trial/1/0/05221133.html.This trial has been submitted for registration in the International Standard Randomised Controlled Trial Number (ISRCTN) Register. The ISRCTN is ISRCTN05221133; Web site Table S1(55 KB DOC).Click here for additional data file.


Polyketides, natural products from microorganisms, have been a main source of antibiotics. Understanding the 'programming' of the enzymes that produce these complex molecules has opened a new field of drug discovery For half a century, natural products from microorganisms have been the main source of medicines for treating infectious disease. The most important chemical class of these antibiotics, apart from the penicillins, is the polyketides. They are made by the stepwise building of long carbon chains, two atoms at a time, by multifunctional enzymes that determine the chain length, oxidation state, and pattern of branching, cyclisation, and stereochemistry of the molecules in a combinatorial fashion to produce an enormous variety of structures. Recent elucidation of the genetic ‘programming’ of the enzymes has opened a new field of drug discovery based on rationally engineering the enzymes to produce ‘unnatural natural products’ with novel properties.Streptomyces is the best-known genus. These organisms make an amazing array of so-called secondary metabolites that have evolved to give their producers a competitive advantage in the complex soil environment, where they are exposed to stresses of all kinds  is simply the most discussed of a series of threats , which are related to the fatty acid synthases that make the lipids essential for the integrity of cell membranes, but they carry out much more complex biosynthetic routines. Repeated rounds of carbon chain building and modification use a series of independently variable reactions selected according to a ‘program’ characteristic of each PKS . Recent  threats . How do The heart of PKS function is the synthesis of long chains of carbon atoms by joining (condensing) together small organic acids, such as acetic and malonic acid, by a so-called ketosynthase function. This uses the building units in the form of activated derivatives, called coenzyme A (CoA) esters, so we speak of acetyl-CoA and malonyl-CoA, for example. The special form of condensation that joins them is driven by loss of carbon dioxide. Thus, when acetyl-CoA, with two carbon atoms, joins with malonyl-CoA, with three carbons, one of the latter is lost and a chain of four carbon atoms results . FurtherChoices of the number and type of the building units are variables in determining polyketide structure. Another concerns the keto groups (C=O) that appear at every alternate carbon atom in the growing chain as a result of the condensation process (accounting for the name polyketide). They may remain intact. Alternatively, some may be modified or removed by a series of three steps , any of Streptomyces coelicolor (S. coelicolor itself) and tetracenomycin. The main variable in their structure is carbon chain length, with few choices of different building units or keto group modification, so the programming would (in principle) be simple. The DNA sequences responsible for such PKSs revealed sets of genes encoding proteins, including ketosynthases, ketoreductases, and acyl carrier proteins (ACPs)  and filled the need for spare parts for the engineering of countless new polyketides . SeveralMeanwhile, the programming of the aromatic PKSs was harder to understand. They had been found to contain only a single ketosynthase, which has to operate a specific number of times to build a carbon chain of the correct length, so how is this determined? How does a single reductive enzyme know which keto groups to modify? And how is the starter unit for building the carbon chain selected ? Considerable progress had been made in constructing novel compounds by mixing and matching PKS subunits, but this was largely based on empirical knowledge about which components to put together . A speciIn the first paper , the autWhat about the choice of starter unit? Most aromatic polyketides start with acetyl-CoA. An important earlier publication by Leadlay and colleagues  had showPloS Biology, is that the PKSs for these compounds consist of two modules of active sites. The components of each module are not activities carried on the same protein, as in the PKSs for the complex polyketides, but are all separate proteins. They form functional modules nevertheless. The newly recognized modules in the producers of compounds that start with nonacetate units have a dedicated ACP and a special ketosynthase that carries out a first condensation, joining the unusual starter unit to the first malonyl-CoA extender unit. The starter module then hands the resulting ‘diketide’ on to the second module  for typical extension by successive condensation with malonyl-CoA units to complete the chain. If the starter module is not present, the second module defaults to its typical habit of decarboxylating malonyl-CoA to acetyl-CoA and starts the chain with that.What The excitement of the work for biotechnology is that it offers the prospect of engineering promising drug candidates by making novel combinations of starter and extender modules and perhaps of feeding the starter modules with a whole range of unnatural substrates . It is e


Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos.We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products. Oryzias latipes) are the analyses of mutants, miss-expression of wild type genes or their variants by mRNA injection and gene specific translational inhibition by morpholino injections . DNA was prepared using a Qiagen Maxiprep kit  and dialyzed using nitrocellulose filters . DNA was injected through the chorion into the cytoplasm of one-cell stage embryos. Heat-shock treatment was performed in small volumes (100–200 μl) using a waterbath at 37°C. Animals used in the study were kept according to national and international ethical provisions for animal husbandry as implemented at EMBL.Embryos were observed and scored using a MZFLIII dissecting microscope with a 436/20 nm (EF); 480/40 nm (BF) filter set for CFP, a 510/20 nm (EF); 560/40 nm (BF) filter set for YFP and a 360/40 nm (EF); 420 nm (BF) filter set for UV/Brightfield. The stereomicroscope was equipped with a DC500 digital camera for imaging .Transgenic embryos were heat-shocked and subsequently kept at 28°C to recover for different periods of time. Total RNA was isolated from individual embryos as described . Total RCG designed and performed all experiments and drafted the manuscript. JW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Currently there is a lot of interest in the flexible framework offered by item banks for measuring patient relevant outcomes, including functional status. However, there are few item banks, which have been developed to quantify functional status, as expressed by the ability to perform activities of daily life.This paper examines the psychometric properties of the AMC Linear Disability Score (ALDS) project item bank using an item response theory model and full information factor analysis. Data were collected from 555 respondents on a total of 160 items.Following the analysis, 79 items remained in the item bank. The remaining 81 items were excluded because of: difficulties in presentation (1 item); low levels of variation in response pattern (28 items); significant differences in measurement characteristics for males and females or for respondents under or over 85 years old (26 items); or lack of model fit to the data at item level (26 items).It is conceivable that the item bank will have different measurement characteristics for other patient or demographic populations. However, these results indicate that the ALDS item bank has sound psychometric properties for respondents in residential care settings and could form a stable base for measuring functional status in a range of situations, including the implementation of computerised adaptive testing of functional status. It is now widely accepted that examining quality of life is an important aspect in the treatment and evaluation of many conditions. Functional status is seen as an important determinant of quality of life. A wide variety of instruments have been developed to quantify functional status . These iThe AMC Linear Disability Score (ALDS) project item bank was developed to quantify functional status ,9. The AThis paper examines these five aspects of the ALDS item bank using the responses given by residents of supported housing schemes, residential care and nursing homes in and around Amsterdam, the Netherlands. This, mainly elderly, population has been chosen because they generally experience some level of functional restriction and consume a large amount of health care services.A and B have half their items in common, as do item sets B and C, item sets C and D and item sets A and D. The items in common between two sets of items are known as 'anchors' and allow all items and patients to be calibrated on the same scale. The patterns of missing data in this type of design are, in statistical terms, ignorable [A), 143 respondents (item set B), 138 respondents (item set C) and 124 respondents (item set D).This paper considers 160 items, which were considered to be applicable in a residential care setting. Each item has two response categories: 'I could carry out the activity' and 'I could not carry out the activity'. If a respondent had never had the opportunity to experience an activity 'not applicable' was recorded. In the analysis, responses in the category 'not applicable' were treated as if the individual items had not been presented to the individual respondents . It was A total of 555 residents of supported housing, residential care and nursing homes were interviewed. The median age was 84 years (range 37 to 101 years), while 444 (80%) were female. Since the respondents were interviewed 'at home', accurate data on medical conditions were not available. All respondents gave informed consent. The study was approved by the medical ethics committee in our hospital.Pik, that patient k responds to item i in the category 'can' is modelled usingIn this paper the data were analysed using the two-parameter logistic item response theory model ,9,17,18.θk denotes the ability of patient k to perform activities of daily life. The discrimination parameter (αi) and difficulty parameter (βi) describe the measurement characteristics of item i. The larger the value of βi, the more difficult item i is. In addition, the larger the value of αi, the better an item is a distinguishing between abilities above and below βi. If the values of αi are constrained to be equal for all items, the model in equation 1 becomes the one-parameter logistic item response theory model [βi for, say males and females, are significantly different. If the values of βi for different groups of respondents are significantly different, then there is evidence of differential item functioning. Full-information factor analysis also uses an extension of the model in equation 1. These approaches are described in mathematical terms in the Appendix. In this paper, estimates of αi and βi were obtained using a marginal maximum likelihood based procedure [θk) follow a Normal distribution and can account for incomplete designs, as described in the Appendix. Expected a posteriori methods were used to estimate θk [where ry model . The modrocedure . This meimate θk .βi) was similar for male and female patients and for patients younger than 85 years and those aged 85 or older. The model is described in depth in the Appendix. Items were excluded from further analysis if the value of the item difficulty parameter was significantly different (1% level) between gender or aged based groups. In this step, the fit of the model to the data from each item was not assessed. In step (c), estimates of the item parameters (αi and βi) were obtained. The fit of the model to the data from each item was assessed using G2 statistics [p-value of less than 0.01, were excluded from the item bank. In addition, the stability of the estimates of the item parameters over different sets of items was examined using the model from step (b). Items were excluded from further analysis if the value of the item difficulty parameter was significantly different (1% level) between item sets A and B, B and C, C and D or A and D. Furthermore, a Kolmogornov-Smirnov test was carried out to examine whether the ability parameters (θk) were Normally distributed. In step (d), the dimensionality of the item bank was examined using item response theory based full information factor analysis [A and B (293 respondents), B and C (281 respondents), C and D (262 respondents) or A and D (274 respondents). A fifth, confirmatory, factor analysis was carried out on the whole data set (555 respondents). In addition, Cronbach's coefficient alpha was calculated for each anchor and the whole data set [χ2 test. The analysis in steps (a), (b), (c) and (e) was carried out in Bilog, version 3.0 [To achieve the objectives of this study, there were five steps in the statistical analysis. In step (a), the amount of variation in the response categories used for each item  was consatistics . Items, analysis ,22,23. Tdata set ,25. In sβi) for for males and females and/or for younger and older respondents. Of these 26 items, 19 had different measurement characteristics for females and for males, 5 items had different measurement characteristics for those aged under 85 and for those aged 85 or over, and 2 items had different measurement characteristics for both males and females and for older and younger respondents. In step (c), 23 items had an item fit statistic p-value of less than 0.01. In addition, 3 items were excluded from further analysis because the value of the item difficulty parameter (βi) was significantly different between two item sets of items. Hence, 79 psychometrically sound items remained in the item bank. A short description of the content of the 79 items in the final version of the calibrated item bank, together with estimates of the dispersion (α) and difficulty (β) parameters and their standard errors, are given in Tables θ do not follow a Normal distribution . In step (d), the full information factor analysis indicated that, for three of the four anchors between the item sets, there was only one latent root of the correlation matrix larger than 1. In the fourth item set, a second latent root was marginally above 1. The percentage of the variance explained by the first factor varied between 67% and 72%. The values of Cronbach's alpha coefficient for the four anchors were between 0.86 and 0.93. The confirmatory factor analysis carried out on the whole data set indicated that 70% of the variance was explained by the first factor. Cronbach's alpha coefficient for the whole data set equalled 0.98. In step (e), the one-parameter logistic item response theory model was fitted to the 79 items remaining after step (c). This model fitted the data significantly less well than the two-parameter model . For 3 items, the item fit statistic had p-value < 0.01. After removal of these items, the two-parameter model was still significantly better than the one-parameter model .Of the 160 items included in the item bank, one was removed because it was worded differently in two different item sets. Of the 159 remaining items, 77 were removed from the item bank. This process is described in Table In this study, the psychometric properties of the item bank have been examined using a sample of 555 respondents and an incomplete calibration design. Each item was presented to between 262 and 293 respondents. These figures are above the minimum, of 200 respondents, regarded as necessary to implement the models used in this paper . It coulθ). This has been examined using item response theory based full-information factor analysis. Part of the full-information factor analysis was performed on sub-sets of the data, as exploratory analyses on incomplete designs may lead to instable results. However, the confirmatory factor analysis was performed on all data. The results, together with the high level of internal consistency, as measured by Cronbach's alpha, and the acceptable fit of the two-parameter logistic item response theory model to the data indicate that the items presented in this paper probably represent a unidimensional construct in a population of respondents requiring residential care.One of the major assumptions underlying the use of the item response theory models described in this paper is that the items reflect a single latent trait (θ) follow a pre-specified, usually Normal, distribution. In this study, there was no evidence that these values did not follow a Normal distribution. This is in contrast to many previously published studies into health and quality of life outcomes, where a strongly skewed distribution was found. The authors feel that there are two reasons for this contrast. Firstly, in this study, the respondents all had some level of restriction in their ability to perform activities of daily life. Secondly, the item bank includes items well above and well below the level of functional status enjoyed by the respondents. This means that the item bank did not have a ceiling or floor effect with respect this this population.Another important assumption when using item response theory models in conjunction with marginal maximum likelihood estimation procedures is that the values of the latent trait (βi) for males and females or for younger and older respondents. This may seem complicated, but is straightforward in the framework of a computerised item bank.In this study, 81 (51%) of 160 items were removed from the item bank because they did not conform to the psychometric standards required of the item bank. This is a much higher level than would be expected in the calibration of an item bank for use in educational measurement. However, when the results are examined more carefully, 28 items were removed because they were too difficult or too easy for the population in this study. In addition, 26 items were removed because they had different item parameters for different groups of respondents. These problems would have been identified much earlier in an educational item bank. Hence, only 26 (25%) of 106 items were removed due to item misfit. The number of items retained in the item bank may have been higher if a more flexible model, based on, for example, non-parametric smoothing techniques had been used [This paper has concentrated on the two-parameter logistic item response theory model. However, the one-parameter logistic item response theory model was also fitted to the 79 items remaining in the item bank. This model fitted the data significantly less well than the two-parameter model, even after 3 items demonstrating misfit at the item level were removed. This confirms the choice of the two-parameter model. This model was chosen because it allows the probability of responding in the category 'can' to be modelled more flexibly than when the one-parameter logistic model is used. This enables a more realistic model for the data to be built than when the more restrictive approach associated with the one-parameter model is chosen .This paper has examined the calibration of the ALDS item bank in a population requiring residential care. It has been shown that the item bank has sound psychometric properties and could form a stable base for a wide range of applications. However, it is possible that the items will have different measurement characteristics for patients requiring treatment for specific chronic conditions or in other countries. Hence, it is important that the ALDS item bank is tested carefully before it is used to assess the functional status in other groups of respondents or in other countries.Now that the measurement properties of the ALDS item bank have been examined carefully, the item bank can be used as a foundation for quantifying functional status. If modern algorithms, such as computerised adaptive testing, are implemented, it will be possible to obtain accurate measurements, whilst keeping the burden of testing on respondents and interviewers to a minimum. Items can be selected for use in further research, for allocation individuals to appropriate care settings and for calculating institutional funding based on the actual care load. It is hoped that the ALDS item bank will play an important part in the implementation of computerised adaptive testing of functional status.βi. This is known as differential item functioning. For instance, if interest is in possible differences in βi between males and females, then the probability, Pik, that patient k responds to item i in the category 'can' is written asThe model in equation 1 can be extended to test whether different groups of respondents to have different values of βiM is the item difficulty for male respondents, βiF-M is the difference between the item difficulty for males and for females and Ik is an indicator variable taking the value 0 if respondent k is male and the value 1 if respondent k is female. The hypothesis H0 : βiF-M = 0 can be tested to examine whether item i has the same measurement characteristics for males and for females.where αi and βi) were estimated using marginal maximum likelihood methods. The likelihood, L, over n items and K (K = 555) respondents can be written asIn this study, the item parameters , can be calculated usingThe value of the standard difficulty parameter, (αim and δi are estimated using marginal maximum likelihood methods.Generally, the parameters ALDS = AMC Linear Disability ScoreNone declared.RH and RL were supported by a grant from the Anton Meelmeijer fonds, a charity supporting innovative research in the Academic Medical Center, Amsterdam, The Netherlands.RL conceived the study and supervised the data collection. RH prepared the first draft and carried out the analyses. RL, MV and RJdH critically reviewed the manuscript. RH prepared the final version.


The Australian Health Care Agreements for the five years 1 July 2003 to 30 June 2008 were signed in August 2003 after vituperative debate and intransigence from the Commonwealth that vitiated the negotiation process. The new Agreements, which were not as generous as the Agreements they replaced, increase accountability on the States, requiring States to match increases in Commonwealth funding, and de-emphasise the prospects for further reform in Commonwealth-State relations during the course of the Agreements. This paper describes the new Australian Health Care Agreements and the process which led to them. The most significant Australian health policy event for 2003 was the signing of the five-year Australian Health Care Agreements. The Agreements were preceded by an ultimatum to the States and Territories from the Commonwealth indicating that there would be no changes from the offer on the table. This led to bitter political recriminations, but the Agreements were eventually signed.In fact important preparations for Agreement renewal occurred in April 2002 with the Commonwealth and State Ministers, in a display of remarkable amity and accord, endorsing a new approach to the Agreements that:• Commonwealth/State relations in the health arena should focus on the provision of optimal care and health outcomes, regardless of jurisdictional boundaries.• It is in the best interests of all Australians for the Commonwealth, Stats and Territories to work co-operatively to improve the health and wellbeing of the community and the way in which health services are provided;• The 2003–08 Agreements should contain the principles, objectives and proposed health outcomes designed to achieve those objections.The Ministers also agreed to establish nine reference groups to address key issues in health reform which would feed into the Agreement "negotiation" process . The refThe reference groups were co-chaired by senior Government officials and non-Government clinical experts involving participants from the bureaucracies, people who work in health agencies, and consumers. The reference groups created great expectations amongst the neophyte health policy contributors who believed the rhetoric of the Commonwealth about being prepared to consider wide ranging changes to the health sector.Seasoned commentators also called for reform -4. AlthoOn 23 April 2003 the Commonwealth produced a non-negotiable offer with severe penalty clauses if States refused to sign by the Commonwealth's arbitrary deadline of 31 August 2003. An Australian Health Reform Alliance was formed to put pressure on the Commonwealth to respond to the reference group reports and to attempt to ensure that the 2003–2008 Agreements did not waste yet another opportunity to improve the efficiency, equity and quality of the health system. The Alliance's National Health Summit, which met at Old Parliament House, presented its final communiqué to non-Government politicians following a march up the hill to New Parliament House . The ComThe Commonwealth's confrontationalist stance effectively destroyed relationships between the Minister for Health and Ageing, Senator Kay Patterson, and her State colleagues, and she was replaced as Health Minister by Tony Abbott MP in the Ministerial reshuffle of October 2003.There have been five Health Care Agreements since Medicare was introduced in 1984. The emphasis, orientation and priorities of these Agreements have changed over time • funding for palliative care, mental health, and safety and quality initiatives.The most contentious difference between the 1998–2003 and 2003–2008 Agreements related to the indexation provisions : its perception that when the Commonwealth increased expenditure on hospital services, this often had no discernible impact on hospitals as State Governments withdrew funding concomitantly. As Deeble points out, the reality is more complex, but the evidence is that an increased Commonwealth share is associated with growth in spending . The newWhy were the processes so acrimonious and what shaped the Agreement outcome? To some extent the shape of the 2003–08 Agreement negotiations was inevitable. The political context, where all state and territory governments were of the opposite political colour from the Commonwealth, meant that harmonious negotiations were probably never seriously in contemplation. Commonwealth governments of both persuasions have tightened up accountability on states with successive Agreements and so tighter control was also probably inevitable. Two important political choices did exacerbate the tensions and inflamed the processes.First, the 2003–08 framework was more parsimonious than predecessor Agreements. As mentioned above, this represented a saving to the Commonwealth of about $1 billion on the Forward Estimates. A contemporary political issue was the decline in bulk billing. The Commonwealth's response to this involved an injection of around $1 billion. The link between the two policy debates within the Health portfolio is clear. Cabinet probably judged the political costs of finding a $1 billion saving from the states as low, as state premiers complaining about Commonwealth cuts and meanness is a regular part of the political landscape. Further, States would probably have criticised the Commonwealth position regardless of the offer made.The second choice that shaped the process was the Commonwealth's intransigence after the drafts were released. The Commonwealth's position here may have been based on a recognition that, eventually, all the states would have to sign the Agreements as they were politically committed to Medicare and free access at point of admission to hospitals, and that the states could not afford to suffer the cash flow consequences announced by the Commonwealth if the Agreements weren't signed by their deadline. The Prime Minister probably took a strong hand in this decision and left no room for his Health Minister to manoeuvre. The Minister's failure to attend meetings exacerbated an already difficult situation.A positive of the process was the extensive involvement of practitioners in the lead-up to the draft Agreements through the Reference Groups. Commonwealth-state negotiations had hitherto been an arcane process involving bureaucratic insiders. This widening of participation was welcomed by those involved and has set a precedent for future negotiations.The 2003–08 Agreements commit the Commonwealth and states to work towards reform in a number of areas including the interface between hospitals, primary care, and aged care; continuity of care particularly in cancer care and mental health services; and continued work on pharmaceuticals reform. A subtle shift from the predecessor Agreement model is the more sceptical and parsimonious approach to the potential for health care reform. Despite the aspirations implicit in establishing the nine reference groups, the language of the 2003–08 Agreements reflects a much more hard-nosed approach to reform with a strong emphasis on efficiency. This approach is most clearly articulated in Clause 18: "The Commonwealth considers that for its part such reforms can take place within existing funding parameters".Although predecessor Agreements also made provision for reform to Commonwealth/State relations, the progress in designing and implementing reform has not lived up to expectations. The most important shift that occurred during the course of the 1998–2003 Agreements was the rationalisation of hospital provision of outpatient pharmacy services, a long overdue response to a significant frictional issue in Commonwealth/States relations .laissez faire attitude by a federal government to its state politically-allied counterparts. A Commonwealth Labor government would be just as keen as its Liberal predecessor to ensure that states are held accountable for maintaining spending and access.It is unclear whether the dynamic, facilitatory aspects of the 2003–08 Agreements will lead to any reform, especially given the acrimonious exchanges prior to signature of the Agreements. However it is important to note that, with a Federal election due at the end of 2004, there is a possibility that a Labor Government will be administering the remainder of the 2003–08 Australian Health Care Agreement. A new Government may be more committed to reforming and strengthening Medicare. However, despite the fact that a Labor would then hold political office throughout Australia, at all levels, this would not necessarily presage a more The author(s) declare that they have no competing interests.