Juvenile test period  or Young-adult test period .Administration of the N-methyl-D-aspartate (NMDA) antagonist ketamine during the perinatal period can produce a variety of behavioral and neuroanatomical changes. Our laboratory has reported reliable changes in learning and memory following a single dose of ketamine administered late in gestation. However, the nature of the drug-induced changes depends on the point during embryonic development when ketamine is administered. Embryonic day 18 (E18) rat fetuses pre-treated with ketamine  and taught a conditioned taste aversion (CTA) learn and remember the CTA, whereas E19 fetuses do not. The current study sought to determine if long-term behavioral effects could be detected in animals that received ketamine or a saline control injection on either E18 or E19. Rat behavior was evaluated on two different measures: spontaneous locomotion and water maze learning. Measurements were collected during 2 periods: Water maze performance of ketamine-treated rats was similar to that of controls when tested on P18. Likewise, the age of the animal at the time of ketamine/saline treatment did not influence learning of the maze. However, the young-adult water maze test (P81) revealed reliable benefits of prenatal ketamine exposure – especially during the initial re-training trial. On the first trial of the young adult test, rats treated with ketamine on E18 reached the hidden platform faster than any other group – including rats treated with ketamine on E19. Swim speeds of experimental and control rats were not significantly different. Spontaneous horizontal locomotion measured during juvenile testing indicated that ketamine-treated rats were less active than controls. However, later in development, rats treated with ketamine on E18 were more active than rats that received the drug on E19.These data suggest that both the day in fetal development when ketamine is administered and the timing of post-natal behavioral testing interact to influence behavioral outcomes. The data also indicate that the paradoxical age-dependent effects of early ketamine treatment on learning, previously described in fetuses and neonates, may also be detected later in young adult rats. Administration of ketamine or other NMDA receptor blocking drugs  may brinThe toxic effects of NMDA receptor blockade are apparently dependent on the dose of the drug, administration regimen, and the age of the animal treated. For example, vulnerability to MK-801-induced cortical vacuolization is not evident in fetal animals but rather begins at approximately the time of puberty . FurtherRecent experiments from our laboratory have focused on age-dependent effects of a single dose of ketamine on fetal learning and memory. Rat fetuses can learn conditioned taste aversions (CTAs) and exhibit taste-mediated conditioned motor responses (CMRs) ,20, whicThese previous studies have only tested the durability of the ketamine paradox over a period of up to 2 weeks  and haveThe current study extends our original observations and reports how a single injection of ketamine on either E18 or E19 modulates spontaneous locomotor activity and performance in a water maze. We tested the rats as juveniles and then as young adults. Our data suggest several age-dependent effects of early ketamine treatment – effects that depend on not only the length of time between drug administration and behavioral testing but also on the day in embryonic development when the NMDA receptor antagonist was administered.Over the10 trials of the water maze test, subjects significantly reduced their latencies to mount the hidden platform  indicating the gradual learning of the maze. However, as Figure  = 4.233;Time spent treading water at the side of the tank may be interpreted as an alternative, futile, escape strategy and not necessarily as an indicator of learning the position of the hidden platform. Subsequent analyses subtracted out stop-times in order to provide the most accurate portrayal of maze learning during the first 3 trials. With stop-time removed, the declining time-to-platform  and swimming distance  over the first 3 trials indicated a learning of the maze during this initial exposure to the apparatus. However the behavioral changes induced by drug or age manipulations were not statistically significant. The number of trials in which the rat failed to mount the platform within 90 seconds was not significantly different among the 4 treatment groups. Likewise, swim speed did not decrease significantly over the first 3 maze trials indicating that the animals were not fatiguing as they undertook multiple swims.Once the rat mounted the hidden platform we timed how long the subject remained there before it was removed (maximum of 30 seconds). An analysis of these data during the first 3 maze trials revealed neither a significant influence of subject age nor drug treatment.An analysis of the second water maze test indicated that there was a significant reduction in time  to mount the hidden platform over the 10 trials , P81 rats that were treated with ketamine on E18 did not swim significantly faster than any of the animals in the other treatment groups. For example, the swim speeds for trial 1 were: . Fatigue did not seem to play a role in the group differences since swim speed remained stable in all groups over the first 3 water maze trials on P81.A single ketamine treatment during the perinatal period had long-lasting effects on spontaneous locomotor movements. Ketamine's effects depended on the age of behavioral testing as well as the age of the drug treatment. Horizontal movements  were more prominently influenced by perinatal ketamine than were vertical movements (rearing).P11 rats treated prenatally with ketamine showed habituation to the open-field test chamber and exhibited reduced horizontal movements overall  = 7.79, p = 0.007]. However, there was a significant interaction between drug treatment and the block of time in which the locomotor measurement was made . At the end of 5 minutes, P11 rats reduced their spontaneous locomotion to approximately 20% of its original level. For this reason, we performed a minute-by-minute analysis of the first 5 minutes in the open field apparatus , rats treated with ketamine on E18 found the hidden platform more quickly than did animals receiving the same treatment on E19. Moreover, they exhibited enhanced maze performance compared to both groups of saline-treated rats. It is important to note that ketamine treatment on E18 did not induce faster swim speeds. Rather, the animals swam more direct routes to the hidden platform.in utero reduced subsequent spontaneous movement. This effect was subtle  and did not depend on the subject's age at the time of the drug's administration. However, when these animals were re-tested on P60, the rats that received ketamine on E18 both moved more than the rats that received ketamine on E19 and also moved more than saline-injected controls.Effects of ketamine on spontaneous open-field locomotion were also age-dependent. In neonatal animals (P11), ketamine administration in utero. Drug-induced effects on water maze learning were observed over 11 weeks after birth and locomotor effects were documented 9 weeks post partum. These findings are consistent with others indicating that early NMDA receptor blockade may produce behavioral alterations that are detectable in adulthood  MK-801 (a potent/selective NMDA receptor antagonist) in synaptosomal membranes is differentially altered by glutamate (and other) agonists during various periods of development. These data build on previous findings  The variable number of subjects/group was due to different litter sizes and several logistical constraints that did not always allow the testing of all rats in each litter. Statistical adjustments were made in order to compensate for the unequal Ns in the treatment groups (see below). The date of conception  was designated as "embryonic day 0" (E0). Postnatal day 0 (P0) was the day of birth . The pregnant animals from which our subjects were derived were individually housed in plastic 'shoe-box' cages (44.45 cm long × 21.59 cm wide × 20.32 cm high). After birth, perinatal rats were housed with the dam until they were sexed and weaned between P21-25  . These methods produced swim distances that were not significantly different  between observers. For some of our swim speed analyses, we subtracted out any time that the animal stopped swimming mid-trial, and treaded water at the side of the tank. Dividing the swim distance by the adjusted time to mount the hidden platform produced swim speed.Two observers, blind to the experimental condition of the animals, evaluated each of the tapes of animals locomoting in the open field by counting line crossings and rears. The counts of these 2 observers were then averaged and this data point was used in our statistical analysis. There was high degree of correlation between the ratings of our 2 observers. P11 locomotor test: r(85) = 0.91, p < 0.01; P60 locomotor test: r(72) = 0.90, p < 0.01.Unless otherwise stated, locomotor data for the two different test periods (P11 and P60) and water maze data for the 2 different test periods (P18 and P81) were analyzed via separate repeated-measures, three-way ANOVAs [Drug  X Treatment age  X Time] with time blocks as the repeated factor and compensation for unequal Ns.We used several rats from each litter and employed statistical corrections in order to avoid spurious inflation of sample size . Since aAn initial inclusion of subject sex as a factor in our statistical analyses indicated no significant differences between male and female subjects. Therefore, the subsequent analyses reported here were run without this factor.Platform navigation for juvenile rats might be more challenging than the task presented to older rats. A small water maze was used in these studies and the same start-to-platform distance was used for all tests. Still, the P18 rat may have found it significantly more challenging than the P81 rat to traverse this distance. For this reason, we intentionally avoided comparing the water maze data of our juvenile and young-adult rats. Likewise, we did not make statistical comparisons between the locomotor responses of animals run at the 2 different ages since the use of different-sized apparatuses would presumably influence these data.a priori hypotheses regarding possible behavioral differences between rats treated with ketamine on E18 versus E19  was run to analyze the group differences during a particular trial. Individual group comparisons were accomplished by using either the Tukey-Kramer Multiple Comparisons Test or t-tests  using thrsus E19 . When a GAM designed the studies, performed some of the behavioral work, conceptualized the statistical analyses and drafted the manuscript. CK helped design the studies, performed most of the behavioral work, assisted with statistical analyses and the drafting of the manuscript. CM supervised most of the behavioral work, assisted with statistical analyses and the drafting of the manuscript. AS supervised and performed much of the behavioral work. AY performed most of the behavioral work. DL-F assisted with the statistical analyses. EV assisted with the behavioral work. BW assisted with the behavioral work. JMB performed aspects of the behavioral work and assisted with the drafting of the manuscript.


Heterochromatin is usually thought of as a stable and inactive region of the genome. Not so, according to a study published earlier this year Science  . HP1 is Science, both groups generated cell lines stably expressing HP1 fused to green fluorescent protein (GFP) so that they could watch the behaviour of HP1 in living cells. Specifically, they used photobleaching techniques to study the in vivo mobility of HP1. In a defined region of a cell, fluorescently tagged proteins are bleached by a laser pulse. Recovery of fluorescence in the bleached area can then only occur if bleached molecules are replaced with unbleached molecules from regions outside the bleached area. The technique is called fluorescence recovery after photobleaching (FRAP) and provides information about the mobility and stability of the cellular structures and proteins. For HP1–GFP, the speed at which fluorescence recovers depends on how tightly it is bound within heterochromatic regions. Heitz (and many others) might have expected that heterochromatin-bound HP1 shows little turnover and therefore recovery should take place very slowly.In the two articles in As discussed by the authors of both studies, several important conclusions can be drawn. In striking contrast to previous models, HP1 appears to be a very mobile molecule. The formation of heterochromatin appears not to be based on a stable oligomeric network of HP1 molecules. Furthermore, heterochromatin is accessible. There is no obvious constraint shielding these transcriptionally inactive compartments from factors residing outside. Given the rapid exchange of HP1 in heterochromatic clusters, any other soluble nuclear protein, such as a transcription factor, should be able to gain access, compete with silencing factors, and potentially activate genes located within heterochromatin. Taken together, heterochromatin appears to be a surprisingly dynamic compartment even though it forms morphologically stable entities. This dynamic situation could imply that heterochromatic silencing is not just a switch, but rather a continuous and active process.Although the new work suggests that heterochromatin is more dynamic than was thought, some caveats remain. It is still possible that a stable “mark” of heterochromatin does exist. As I discussed above, this mark might be an undetected fraction of immobile HP1 molecules. In addition, one cannot exclude that HP1 is a downstream factor that is dynamically tethered to a stable binding site, the most likely candidate being the methylated histone tail. Perhaps the role of HP1 in heterochromatic silencing has simply been overinterpreted. The formation of facultative heterochromatin by X inactivation in mammals, for example, does not involve HP1 even though appropriate histone methylation marks are set. This indicates that heterochromatin formation does not always follow the same rules and suggests that our definitions of heterochromatin must be refined. In any case, it could well be that a silent state is marked by signals that have a slow turnover. In fact, histone methylation is believed to generate a quite stable “code” . Until tDrosophila  have also been proposed to consist of dynamic collections of components . Personaosophila . This pr


Scientists debating how to assess global fisheries are now including studies of long-term ecosystem effects and options for recovery efforts. But is it possible to both conserve and farm the sea? It is becoming increasingly apparent that the vast blue expanse of ocean—the last frontier—is not as inexhaustible as it once seemed. While we have yet to fully explore the reaches of the sea, technology has granted humans the ability to harvest its wealth. We can now fish anywhere, at any depth, for any species. Like the American frontier range's bison and wolf populations brought to the brink of extinction swordfish and sharks are the ocean's most pursued prizes. The disadvantages associated with the depth and dimensions of this open range, however, have long obscured the real consequences of fishing. Indeed, scientists have the formidable challenge of assessing the status of species whose home covers over 75% of the earth.Three recent highly publicized papers—a trifecta detailing troubled waters—call attention to overfishing's contributions to the dramatic declines in global fisheries. Delving into the past, Recent reports by the United Nation's Food and Agriculture Organization (FAO) which maintains the world's most complete global fisheries database, appear to validate the conclusions of these studies. The most recent FAO report states that 28% of global stocks are significantly depleted or overexploited, and 47% are either fully exploited or meet the target maximum sustainable yield. Only 24% of global stocks are either under- or moderately exploited. As the sea is increasingly harvested, many ecologists wonder how the ecosystem will continue to function . AlthougThe FAO began keeping fisheries records in 1950. Unfortunately, an enormous amount of data comes directly from each country's fishing industry, which is often biased as a result of unreported discarding, illegal fishing, and the misreporting of harvests. For example, mid-level Chinese government officials seeking promotions systematically enhanced China's fisheries numbers in recent years—which inflated and skewed international catch rates.www.worldfishcenter.org/trawl/) confirms that the region's commercial species have been depleted to 10%–30% of what they were 30–40 years ago.The FAO data show that catches, excluding a recent surge in anchoveta and China's suspect numbers, reached a peak of 80 million metric tons in the late 1980s and have since begun to decline. Regional studies validate these trends. “Most of the line fish around the coast of South Africa are depleted to 5%–15% of pristine levels,” says George Branch, a marine biologist from the University of Cape Town . Meryl Williams, Director General of WorldFish in Penang, Malaysia, notes that the Asia-specific database called TrawlBase . And that is where fisheries have adequate access to current monitoring programs. “With the recent expansion of the Taiwanese and Chinese fleets, we don't have the kind of sampling programs needed for those kinds of fisheries,” says Rick Deriso, a fisheries scientist with the Inter-American Tropical Tuna Commission (IATTC) .Couple these inadequacies with previously unknown bycatch rates  and illegal catches, and it is easy to see that the task is formidable. The FAO estimates that roughly one-quarter of the marine commercial catch destined for human consumption—some 18–40 million metric tons of fish—is thrown back in the sea, a harvested catch that is never utilized or counted. It is estimated that the illegal, unreported, and unregulated (IUU) fisheries surpass allowed fishing quotas by 300%. IUU fishers operate in areas where fishing is not permitted, use banned technologies or outlawed net types, or underreport catches. “The IUU fishery for Patagonian toothfish expanded rapidly in the mid-1990s, likely on the order of 20–30 vessels,” says Andrew Constable, an ecological modeler at the Australian Antarctic Division , who also works with the Scientific Committee of the Commission for the Conservation of Antarctic Marine Living Resources . “These rates of IUU fishing could reduce stocks to threshold levels in some areas in two to five years,” he adds.Often overlooked is the inescapable fact that even sustainable harvest rates reduce fish populations quickly. “If the goal is a productive fishery, we're automatically talking about up to a 70% decline in population across the board,” says Deriso. The FAO's Chief of Marine Resource Services, Jorge Csirke, states that “from a stock point of view, there is no way to preserve integrity of wild stocks and exploit them at the same time.” Indeed, the United States' National Marine Fisheries Service (NMFS) considers optimal harvest rates to be between 40%–60% of virgin levels. But once fish populations dip below the 10%–20% mark, declines are of serious concern.Atlantic cod in Canadian waters suffered a total population collapse and are now on Canada's endangered species list . From 2 Given the task of compiling data on only the economically important species, fisheries biologists developed a single-species management approach in the 1960s, which assumed that fisheries affect each species in isolation. This approach, although now rife with problems, served the community and the politicians well during the decades of abundant resources. “They brought the approach of single-species management to near-perfection,” says Boris Worm, a marine ecologist at the Institute for Marine Science in Kiel, Germany. A growing discontent with the model, in addition to greater awareness of ecological interactions, however, prompted Worm and his Dalhousie University colleague Ransom Myers to question the sustainability of the single-species approach. Attempting a comprehensive assessment, their widely cited recent paper  indicateHowever, this new approach to assess fish stocks is not without its critics. Fisheries biologists point out that the nuances of management contained in fisheries data—such as altered fisher behavior, the variable “catchability” of individual species, and altered gear use—were discounted in the Despite the controversy, most agree that the large predators, particularly sharks, skates, rays, and marlin, are in the most dire straits. Unlike other lower-trophic order species, the wholesale removal of top predators has enormous effects on the rest of the ecosystem. One consequence is that overall reproduction rates can potentially suffer. Fish size, gender, and age at maturity have a substantial impact on individual species' reproduction rates. Since larger fish are the most susceptible to fishing, the population's age structure can shift as individuals, particularly females, are fished out. For example, a 23-inch (59-cm) female vermilion rockfish can produce 17 times the young of a 14-inch (36-cm) fish. Given uncertainties with population dynamics, the fact that basic biological data are missing makes the job even harder. While knowledge of these components is still quite spotty, tuna inventories, for example, have started collecting gender data on catches.Daniel Pauly, a fisheries biologist at the University of Vancouver , has shown that increased fishing has caused the industry to “fish down the food web,” or systematically move to lower trophic levels over time as higher ones were depleted . The impFisheries science has taken steps to increase the quality of data in recent years. “Traditional fishery models assumed that a fishery was a homogenous thing—like bacteria in a bottle—rather than a spatially diverse system,” says Pierre Kleiber, a fisheries biologist with the Pacific Islands Fisheries Science Center of the NMFS . He adds that recent work accounts for spatial diversity. In addition, fisheries are now dealing with the inherent uncertainty of their work and are factoring that into models and decision-making. “Uncertainty didn't used to be dealt with at all in formulating fishery management advice,” confirms Keith Sainsbury, a marine ecologist with the Commonwealth Scientific and Industrial Research Organisation (CSIRO) , adding that its absence gave rise to an awful lot of troubles. “Traditional models tended to assume perfect data with no holes in it,” says Kleiber. “Now we've tried to craft a model to fit the realities of missing data.”As well as incorporating spatial diversity and uncertainty, researchers are beginning to comprehend the ecological damage caused by different types of fishing gear. Indeed, trawling the bottom of the seafloor for groundfish can destroy a half-acre footprint of habitat . Detaile“Science is getting more realistic, but it is getting more difficult,” says Branch. Ecological models are far more complex than traditional fisheries models, says Csirke, adding that more model variables make it more difficult to apply to fisheries, an industry whose focus is, understandably, not conservation. Despite its incorporation into national fisheries policies, ecosystem-based management remains a loosely defined term. It is not a well-defined concept because it is not possible to optimize every species, says Deriso.An additional concern to scientists is that of biomass resilience in the face of environmental changes. Francisco Chavez, a biologist with the Monterey Bay Aquarium Research Institute , recently demonstrated that over a 25-year period, warmer and cooler Pacific waters tilt the distribution of anchoveta versus sardines, both open-ocean dwellers . Indeed,In the end, many scientists have their doubts about the influence of science on decision-making. “My personal view is that it's naïve to think that modifying and improving models will necessarily lead to improved natural resource management,” says Simon Jennings, a fisheries biologist with the United Kingdom's Centre for Environment, Fisheries and Aquaculture Science in Lowestoft. Indeed, the International Council for the Exploration of the Seas  recently recommended a total ban on North Sea and Irish Sea cod stocks, based on single-species assessment. Although the more intensive ecosystem-based models could not have produced a more stringent recommendation, politicians allowed harvests at roughly half of last year's catch.While lowering fisheries' effort seems the most logical approach to the recovery of depleted fisheries, social and economic concerns often stymie political action. Yet demand for seafood continues. Therefore, scientists also are investigating both conservation and alternative production options.Given the social, economic, and political problems associated with that, managers have often used closures to help a hard-hit species recover. In many cases, however, the recovery time for exploited species is longer than once thought . “Based One management strategy to recover species is to create marine protected areas (MPAs), zones that restrict all removal of marine life A review of 89 studies of MPAs by Ben Halpern, a student at the University of California, Santa Barbara , demonstrated that the average number of fish inside a reserve increases between 60%– and 150% . In addiIn terms of simply producing fish for global food needs, aquaculture  is another, increasingly popular, option. In 2001, the European Union produced 17% of total fishery's production via aquaculture. These numbers are projected to steadily increase, but some question whether aquaculture would be sufficient to supply what has been lost by overexploited fisheries.Concentrated in coastal areas, aquaculture has aroused numerous concerns. Indeed, in developed countries, most operations grow carnivorous fish, which necessitates growing fish to feed fish. While the process has become more efficient in recent years, due in part to a growing reliance on vegetarian diets, it still takes about 3 pounds (1.36 kg) of fish to create 2.2 pounds (1 kg) of desirable meat . Yet, thTo alleviate many of these concerns, open-ocean aquaculture is now being considered. Indeed, the NMFS is set to propose a Code of Conduct for Offshore Aquaculture, which would open up the 200-mile (322-km) United States Exclusive Economic Zone to net pens seaward of coastal state boundaries and authorities. The Sea Grant program in conjunction with interested business, is also currently assessing the carrying capacity of open-water pens as well as their potential environmental impact. Given increased industrial interest and unchanging demand for seafood, many think farming the sea may be around the corner.Undoubtedly, scientific effort will continue to inform both conservationists and industry about fisheries' capacity and potential recovery options. As attitudes towards fisheries continue to change, increased understanding of the ecological underpinnings should help strike a more informed balance between fisheries' conservation and production. “The big mistake is suggesting that you can manage fish stocks,” says Niels Daan, a biologist with the Netherlands Institute for Fisheries Research . “In my opinion, we can only manage human activity.”While industrial-scale fishing is a growing concern to fisheries biologists, the management of subsistence fishing in developing countries is equally complex. Indonesia alone has 1.3 million fishers. Given the lack of alternative economic options for subsistence fishers, it is much more difficult to reduce fishing because it meets immediate food and resource needs. Local scientists, often lacking in resources, have a much more difficult time assessing the effects and offering advice to governmental fisheries regulators, who have limited political influence. Kenyan researcher Tim McClanahan notes that a main problem is a lack of coordination and respect between traditional and national programs of management. Therefore, he focuses on the fishing gear used. By reconciling the impact of certain fishing gear with traditional knowledge, McClanahan has developed a basis for suggested restrictions deemed acceptable to the local community.While MPAs are heavily touted as one of the best management tools to address both conservation and fisheries management, few have been enacted. In 2001, following a strong mandate by the Australian Minister to the Environment and overwhelming political will, the Great Barrier Reef Marine Park Authority (GBRMPA) in Australia established a network of marine protected, or no-take, areas as an ecosystem-based management approach.In setting up the reserve networks, scientists determined the most effective areas to protect biodiversity with little impact to productivity. “We tried to avoid peak use areas, while protecting at least one-third of each bioregion and minimizing the impact to users of the Great Barrier Reef Park,” says Phil Cadwallader, Director of Fisheries at the GBRMPA.Off the coast of California, the Channel Islands network of marine reserves, established in April 2003, consists of 13 areas designed to protect biodiversity and critical habitat for breeding fish and to maintain biodiversity. The area has suffered serious declines of red snapper, angel sharks, and abalone, once plentiful off the California coast, over the past decade. Scientists designed the network to protect those productive habitats that would help ensure that larval dispersal was maintained between the individual reserves. Totaling 132 nautical square miles , 11 of the areas are no-take reserves—allowing no fishing or harvest of any kind.


Carcinoma of the esophagus is an aggressive malignancy with an increasing incidence. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy. A clinical practice guideline was developed based on a systematic review investigating neoadjuvant or adjuvant therapy on resectable thoracic esophageal cancer.A systematic review with meta-analysis was developed and clinical recommendations were drafted. External review of the practice guideline report by practitioners in Ontario, Canada was obtained through a mailed survey, and incorporated. Final approval of the practice guideline was obtained from the Practice Guidelines Coordinating Committee.The systematic review was developed and recommendations were drafted, and the report was mailed to Ontario practitioners for external review. Ninety percent of respondents agreed with both the evidence summary and the draft recommendations, while only 69% approved of the draft recommendations as a practice guideline. Based on the external review, a revised document was created. The revised practice guideline was submitted to the Practice Guidelines Coordinating Committee for review. All 11 members of the PGCC returned ballots. Eight PGCC members approved the practice guideline report as written and three members approved the guideline conditional on specific concerns being addressed. After these recommended changes were made, the final practice guideline report was approved.In consideration of the systematic review, external review, and subsequent Practice Guidelines Coordinating Committee revision suggestions, and final approval, the Gastrointestinal Cancer Disease Site Group recommends the following:For adult patients with resectable thoracic esophageal cancer for whom surgery is considered appropriate, surgery alone  is recommended as the standard practice. Carcinoma of the esophagus is an aggressive malignancy with an increasing incidence. Its virulence, in terms of symptoms and mortality, justifies a continued search for optimal therapy. The large and growing number of patients affected, the high mortality rates, the worldwide geographic variation in practice, and the large body of good quality research warrants a clinical practice guideline.This clinical practice guideline was developed by the Gastrointestinal Cancer Disease Site Group (DSG) of Cancer Care Ontario's Program in Evidence-based Care (PEBC), using the methods of the Practice Guidelines Development Cycle . This prThe systematic review on neoadjuvant or adjuvant therapy for resectable esophageal cancer, which forms the basis for this clinical practice guideline, is available in a companion document . Based oA systematic review with meta-analysis on neoadjuvant or adjuvant therapy for resectable esophageal cancer was developed by the Gastrointestinal Cancer DSG of Cancer Care Ontario's Program in Evidence-based Care . The eviIn discussions regarding the completed systematic review, the Gastrointestinal Cancer DSG agreed that the evidence did not support a recommendation for neoadjuvant or adjuvant chemotherapy or radiotherapy for resectable thoracic esophageal cancer. A recommendation that surgery alone should be the standard of care for this patient population was drafted, and it was recommended that the draft practice guideline be sent out to Ontario practitioners for external review.Combined modality radiotherapy and chemotherapy in the non-surgical management of localized carcinoma of the esophagus [The role of radiotherapy alone and chemoradiation alone without surgery is addressed in a separate Gastrointestinal Cancer DSG Clinical Practice Guideline: sophagus .Practitioner feedback was obtained through a mailed survey of 163 practitioners in Ontario . The survey consisted of items evaluating the methods, results, and interpretative summary used to inform the draft recommendations and whether the draft recommendations should be approved as a practice guideline. Written comments were invited. Follow-up reminders were sent at two weeks (post card) and four weeks (complete package mailed again). The Gastrointestinal Cancer DSG reviewed the results of the survey.Eighty-six surveys (58%) were returned. Twenty-nine respondents (34%)  indicated that the report was relevant to their clinical practice and completed the survey. Key results of the practitioner feedback survey are summarized below.1. Number surveyed: 163 practitioners in Ontario, Canada involved in the care of cancer patients2. Return rate: 58% 3. Written comments attached: 10%4. Agreement with the summary of evidence: 90%5. Agreement with the recommendation: 90%6. Approval of the recommendation as a practice guideline: 69%Three (10%) respondents provided written comments. One practitioner hypothesized that preoperative chemoradiation might have a role in adenocarcinoma of the lower third of the esophagus  esophageal cancer for whom surgery is considered appropriate.• If surgery is considered appropriate, then surgery alone  is recommended as the standard practice for resectable thoracic esophageal cancer. for a complete list of current and on-going projects.This Clinical Practice Guideline report is based on work completed in October, 2003. All approved PEBC Clinical Practice Guideline reports are updated regularly. Please see the PEBC's web site The author(s) declare that they have no competing interests.In order of appearance:DSG, Disease Site Group; PEBC, Program in Evidence-based Care; PGCC, Practice Guidelines Coordinating Committee.RM, RW, and LZ created the initial drafts of this clinical practice guideline with input from other members of the Gastrointestinal Cancer DSG. RM, RW, and BR created the final draft of this clinical practice guideline. Creation of the submitted manuscript was performed by BR and RM.The pre-publication history for this paper can be accessed here:


Lipoprotein lipase (LPL) has a central role in the catabolism of triglyceride-rich lipoproteins. The enzyme is anchored to the vascular endothelium through interaction with heparan sulphate proteoglycans and is displaced from this interaction by heparin. When heparin is infused, there is a peak of LPL activity accompanied by a reduction in triglycerides (TG) during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increase towards and beyond baseline. This suggests that tissue stores of LPL become depleted. It has been argued that low molecular weight (LMW) heparins cause less disturbance of the LPL system than conventional heparin does.We have followed LPL activity and TG during a dialysis-session with a LMW heparin  using the same patients and regime as in a previous study with conventional heparin, i.e. a primed infusion.The shape of the curve for LPL activity resembled that during the earlier dialyses with conventional heparin, but the values were lower during dialysis with dalteparin. The area under the curve for LPL activity during the peak period (0–180 minutes) was only 27% and for the plateau period (180–240 minutes) it was only 36% of that observed with conventional heparin (p < 0.01). These remarkably low plasma LPL activities prompted us to re-analyze LPL activity and to measure LPL mass in frozen samples from our earlier studies. There was excellent correlation between the new and old values which rules out the possibility of assay variations as a confounding factor. TG increased from 2.14 mmol/L before, to 2.59 mmol/L after the dialysis (p < 0.01). From 30 minutes on, the TG values were significantly higher after dalteparin compared to conventional heparin (p < 0.05).These results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does. Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) in circulating lipoproteins ,2. This et al. found that on a weight basis, decasaccharides released more LPL from perfused rat hearts than conventional heparin did -oleic acid-labelled triolein, 100 mg soybean TG and 10 mg egg yolk phospholipids per mL, prepared by Fresenius-Kabi, Uppsala, Sweden. Hepatic lipase was inhibited by pre-incubation of the plasma samples with immunoglobulins from a rabbit antiserum to human hepatic lipase. The assay medium contained a relatively high concentration of heparin, and possible differences in the heparin concentration or type in the sample would not affect the activity. All assays were made in triplicate and the mean value was used. A standard sample of human post-heparin plasma was run on each assay day and the value was used to calibrate for between-assay-variations. LPL protein mass was determined with an enzyme-linked immunosorbent assay, as previously described [®, Chromogenix AB, Mölndal, Sweden).Blood samples for measurement of LPL activity were collected in heparinized tubes. They were immediately chilled in ice water and centrifuged in a cooling centrifuge within 15 minutes. The plasma was frozen at -20°C and then stored at -70°C until analyses. LPL activity was measured as described  using anescribed , using iescribed . AntifacThe values are expressed in terms of median and range and were examined for significant differences by paired Wilcoxon signed-rank test. Simple linear regression and the Spearman rank correlation test were used to evaluate relationships between variables. Two-tailed P values below 0.05 were considered to be statistically significant.Table During the dialysis with dalteparin, the anti-Factor Xa activity was between 0.52 and 0.87 IU/mL. The target value recommended by the manufacturer is 0.5–1.0 IU/mL. Hence, the plasma dalteparin concentration remained well within the range for effective anticoagulation throughout the dialysis-session.The LPL-activity rose rapidly when dalteparin was administered. The highest value was at 15 minutes, median 15 mU/mL (range 9–32). The activity remained high at 30 minutes, but then decreased so that at 120 minutes the median was only 9 mU/mL (range 5–15)  at the start, to 2.59 mmol/L (range 1.49–5.04) at the end of the dialysis represents a 21 % increase (p < 0.01). Compared to the values during the earlier dialysis with conventional heparin, there was no statistically significant difference at the start of dialysis, but from 30 minutes and through the remaining session TG values were significantly higher during the dialysis with dalteparin (p < 0.05)  at start, to 6.1 mmol/L (range 3.8–8.4) at the end . This differs from the earlier dialysis with conventional heparin, when total cholesterol did not change from baseline .HDL-cholesterol did not change from baseline. Again, this differs from the dialysis with conventional heparin when HDL-cholesterol increased from 1.09 mmol/L (range 0.67–2.06) at start to 1.19 mmol/L (range 0.67–2.31) at the end (p < 0.05). No correlation was found between LPL activity and total or HDL-cholesterol changes during any of the dialysis-sessions.The median LDL-cholesterol, calculated by the Friedewald formula, was 2.9 mmol/L (range 2.1–4.9) before the dialysis and increased to 3.75 (2.1–5.6) at 240 min. This corresponds to an increase of 29% (p < 0.05).This study shows the same pattern of plasma LPL activity in HD patients given dalteparin as observed in previous studies with control subjects given dalteparin  or conveet al. found that the early LPL activity was only about half as high compared to values observed after conventional heparin [There are several earlier studies that show that administration of LMW heparin results in lower plasma levels of LPL than conventional heparin -30. LMW  heparin ,29. This heparin .et al. have, however, found that the amount of LPL released by a bolus of heparin is restored within 24 hours [The LPL activities were lower in HD patients than observed in controls given dalteparin . A simil24 hours . In rats24 hours  and chyl24 hours . A more 24 hours . Yet ano24 hours ,35.et al. found that the clearance of injected radioactively labeled chylomicron TG was dramatically increased five minutes after rats had been given conventional heparin or LMW heparin [Administration of heparin causes a temporary derangement of lipoprotein metabolism. In our studies with control subjects given conventional heparin or dalteparin the TG concentration decreased after heparin and then gradually increased again so that at the end of the study period TG exceeded the baseline level  declare that they have no competing interests.BN participated in the design of the study, carried out the patient studies, assembled the data, did the statistical analyses, and participated in writing of the manuscript; BS and GO conceived of the study and coordinated the work. TO participated in the design of the study and in writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Conventional total hip replacement (THR) may be felt to carry too high a risk of failure over a patient's lifetime, especially in young people. There is increasing interest in metal on metal hip resurfacing arthroplasty (MoM) as this offers a bone-conserving option for treating those patients who are not considered eligible for THR. We aim to evaluate the effectiveness of MoM for treatment of hip disease, and compare it with alternative treatments for hip disease offered within the UK.A systematic review was carried out to identify the relevant literature on MoM published before 2002. As watchful waiting and total hip replacement are alternative methods commonly used to alleviate the symptoms of degenerative joint disease of the hip, we compared MoM with these.The data on the effectiveness of MoM are scarce, as it is a relatively new technique and at present only short-term results are available.It is not possible to make any firm conclusions about the effectiveness of MoM based on these early results. While the short-term results are promising, it is unclear if such results would be replicated in more rigorous studies, and what the long-term performance might be. Further research is needed which ideally should involve long-term randomised comparisons of MoM with alternative approaches to the clinical management of hip disease. The treatment of younger people with disease of the hip joint presents a difficult clinical problem. Conventional total hip replacement (THR) may be felt to carry too high a risk of failure over a patient's lifetime. Overall, long-term results of THR in younger patients with a variety of underlying conditions indicate that 25–30% may require revision by 15 years . Data onMetal on metal hip resurfacing arthroplasty (MoM) offers a bone-conserving option for treating those patients who are not considered eligible for THR. MoM may also represent a more attractive alternative to other procedures such as osteotomy, bone fusion and arthroscopy, which have previously been used or been advocated as means of delaying or preventing the need for a THR. MoM involves the removal and replacement of the surface of the femoral head with a hollow metal hemisphere, which fits into a metal acetabular cup. This technique conserves femoral bone , maintains normal femoral loading and stresses, and may not therefore compromise future total hip replacements. Data on the use of MoM within the NHS in England and Wales could not be obtained in this review. Never the less, because of increasing interest in MoM, we conducted a systematic review of the evidence of effectiveness aiming to compare it with THR and watchful waiting.Initial searches failed to identify any randomised or comparative observational studies comparing MoM with any of the chosen alternatives. A structured search was conducted to identify evidence relating to the clinical effectiveness and cost-effectiveness of MoM for treatment of hip disease. The search strategy comprised of: (1) A free text search to identify any potentially relevant papers evaluating MoM (free text search terms were used because of the anticipated scarcity of published literature); and (2) A search for RCTs and systematic reviews of RCTs for THR using a modified version of the search strategy used for a recent review . The seaAll identified abstracts were assessed for subject relevance independently by two reviewers. Full papers were then obtained and formally assessed for inclusion. It was agreed at the outset of the review that the search strategy would not be limited by language. It was agreed that non-English studies would be identified, but due to time and resource limitations would not be translated and assessed for their relevance to the review. No restrictions on the type of patient were imposed. Comprehensive systematic reviews of THR was carried out in Health Technology Assessment in 1998. These reviews were updated by the National Institute of Clinical Excellence (NICE) in 2000. Therefore, in this review a search for systematic reviews and RCTs published subsequent to the completion of the systematic reviews was carried out. Table Two reviewers independently abstracted data and quality assessed the included studies. Where a difference in opinion occurred, an arbiter was consulted. A data abstraction form was developed to record details of trial methods, participants, interventions, patient's characteristics and pre-specified outcomes  therapy, physiotherapy and treatment of side effects of medications) would be about £640 . WhetherThe use of MoM in the UK is still relatively rare. However, there has been increasing interest from younger people with hip disease who are not currently considered eligible for THR and amongst surgeons who strive for better ways to treat the patients whom they see. However, only very limited evidence are currently available on MoM and although the procedure does appear promising the lack of robust comparisons with the other treatment options and of long term data make it virtually impossible to draw robust conclusions about its relative effectiveness. Given the early promise shown by MoM there is a real need for more rigorous research. Such research would be challenging, not least because of ethical considerations, but should attempt some form of prospective, preferably randomised, comparison of MoM with a policy of delayed selective surgery. These studies should preferably be large-scale, long-term, and use standard outcome measures, both pre- and post-operatively.The author(s) declare that they have no competing interests.LW carried out the critical appraisal of the included studies and assisted in the writing up. LV coordinated the project and assisted in the writing up. KM developed the methodology for the literature search and assisted in the writing up. AG participated in the design and coordination of the study. MB assisted in the critical appraisal of the included studies. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Search strategies. The search strategies used to search electronic databases to identify studies relevant to this review.Click here for file


DNA repair genes encode proteins that protect organisms against genetic damage generated by environmental agents and by-products of cell metabolism. The importance of these genes in life maintenance is supported by their high conservation, and the presence of duplications of such genes may be easily traced, especially in prokaryotic genomes.Xanthomonas species were used as the basis for phylogenetic analyses of genes related to DNA repair that were found duplicated. Although 16S rRNA phylogenetic analyses confirm their classification at the basis of the gamma proteobacteria subdivision, differences were found in the origin of the various genes investigated. Except for lexA, detected as a recent duplication, most of the genes in more than one copy are represented by two highly divergent orthologs. Basically, one of such duplications is frequently positioned close to other gamma proteobacteria, but the second is often positioned close to unrelated bacteria. These orthologs may have occurred from old duplication events, followed by extensive gene loss, or were originated from lateral gene transfer (LGT), as is the case of the uvrD homolog.The genome sequences of two Xanthomonas, seem to have arisen from old events and possibly enlarge both functional and evolutionary genome potentiality.Duplications of DNA repair related genes may result in redundancy and also improve the organisms' responses to environmental challenges. Most of such duplications, in  The availability of complete genome sequences from different organisms makes it possible to identify, by similarity, potential homologs of genes that have been experimentally tested in other living beings, resulting in the recognition of putative functions for the proteins encoded by them. This research concept represents a revolutionary tool in modern biology, as the data generated allow for recognition of the presence or absence of genes, giving indications of the metabolic pathways present in such organisms and revealing possible particularities of the individuals in their natural habitat. Moreover, the possibility of obtaining gene sequences from organisms that have long diverged makes it feasible to use these data to trace their evolutionary origin . ComputeAll known forms of life present efficient systems to maintain the integrity of their genetic material. As DNA is under constant attack by different environmental agents and metabolic by-products, evolution has provided organisms with several DNA repair pathways to remove or to tolerate lesions in their genetic material. In fact, these pathways have at least two important contrasting roles in evolution, safeguarding the genome, and allowing for a certain level of mutations in the course of evolution. The critical balance of these two activities is probably the best reason for the high levels of conservation observed in DNA repair related proteins, even across the three kingdoms, Bacteria, Archaea and Eukarya. Detailed studies of many of the different DNA repair genes and protein domains have been described previously ,7, confiXanthomonas axonopodis, X. campestris and Xylella fastidiosa, we found that certain DNA repair genes present duplications in both Xanthomonas species. The great relevance of gene duplication for expanding gene families, as well as for gene innovation, is a consensus among researchers. New genes can facilitate survival in new environments or make possible the use of new metabolites. We investigated the phylogeny of such duplicated genes, in search of evidence on the mechanisms by which cells evolve their DNA repair machinery. Although most DNA repair genes follow the conserved vertical transmission found for 16S rRNA phylogeny, the data clearly show that duplications may have arisen by different means. Examples of a recent duplication, as well as of other old or LGT events, generating paralogs, are presented. These results also help in tracing the origins of LGT events that lead to redundancy and also to functional diversification, especially of Xanthomonas bacteria.In this work, we investigated the DNA repair genes in the recently described genomes from the Xanthomonadales group ,9 followX. fastidiosa, when compared to both Xanthomonas species: ada-alkA (fusion), tag, phr, dinB and ligB. For evolution purposes, however, the presence of several duplications, especially in the Xanthomonas genomes, will be focused here.DNA repair-related genes are normally highly conserved, and grant good genetic information for investigating the evolution of organisms. Orthologs of known bacterial DNA repair proteins were identified in the plant pathogen bacteria of the Xanthomonadales group and are listed in Table Neisseria and Ralstonia).The RecA protein is almost universal among bacteria and is a clear example of phylogeny that follows 16S rRNA gene evolution. In fact, this gene is always unique in all genomes analyzed and has been proposed as an alternative molecule to be used in systematic studies of Bacteria . Figure E. coli, the RecA protein has been shown to participate in recombinational repair, as well as in the control of a set of physiological changes to DNA damage, known as SOS response. The induction of this regulon stems from the co-protease activity of the RecA protein, which cleaves a repressor protein, denominated LexA, allowing the expression of the set of SOS genes. The presence of lexA and recA homologs in the Xanthomonadales indicates that these bacteria also present a SOS regulon. However, in Xanthomonas genomes, there are two copies of the lexA gene. The phylogeny of LexA proteins is presented in Figure lexA gene is not found in all groups of bacteria. The products of the two copies of the Xanthomonas lexA paralogs are positioned close together in the same branch of Xanthomonadales, indicating a recent duplication of such a gene. The fact that they had branched before Xylella's single lexA divergence indicates that this bacterium may have lost the duplication. Gene losses are expected to be extensive in Xylella, in agreement with this bacterium having a more specialized parasitic way of life [lexA in Xanthomonas points to a highly controlled SOS regulon, that may be important for fine-tuning the bacterial responses to stress induced in environmental changes. Indeed, functional characterization of lexA1 in X. campestris has been performed by gene disruption, and the data indicate this protein controls the expression of the recA gene, as expected for the SOS regulatory circuit [lexA gene in Deinococcus radiodurans does not change the level of RecA expression, suggesting that LexA protein may be related to functions other than controlling the SOS regulon in that organism [In  of life . The dup circuit . The funorganism .uvrB gene from X. campestris has been cloned and it was shown to participate in the resistance of these bacteria after UV irradiation [Methanothermobacter thermautotrophicus, Methanosarcina sp and Halobacterium sp. NRC-1 [The nucleotide excision repair (NER) pathway is one of the most important, versatile and conserved systems of DNA damage removal in bacteria. It recognizes damages, which cause significant helix distortions in the DNA molecule, these being excised as an oligonucleotide by several enzymes that act as helicases and endonucleases. The main proteins that participate in such a pathway are known as UvrA, UvrB and UvrC, which, in sequential steps, interact with one another, recognize the damaged strand (UvrA), open the double helix (UvrB), and cleave DNA (UvrC) at both sides, few nucleotides away from the lesion. Subsequently, the oligonucleotide containing the damage is removed by a DNA helicase known as UvrD. The adiation . The NERp. NRC-1 . Other ap. NRC-1 .uvrA, uvrC and uvrD are duplicated in Xanthomonas and the phylogenies of the proteins encoded by these orthologs, together with the single uvrB, are presented in figure Xanthomonas being positioned in the same branch of the Xylella's ortholog, close to gamma proteobacteria as expected for a vertical descent. However, for the duplications, the patterns are different and more complex. The second ortholog of uvrA in X. axonopodis is phylogenetically closer to the also duplicated genes found in several other bacteria (figure D. radiodurans) points to an origin due to horizontal gene transfer from other bacteria. The functions of both uvrA orthologs are not necessarily related to DNA repair, since this activity can be provided by either one of them. It is relevant to mention that for the duplication found in D. radiodurans, White et al [The genes a figure , thus inte et al  have prote et al .X. axonopodis is close to the uvrC orthologs from other gamma proteobacteria, following a vertical transmission. However, a protein with considerable similarity to the N-terminal of UvrC is found in both Xanthomonas species. These orthologs form an independent branch of the phylogenetic tree of UvrC, close to a heterogeneous group of gram-positive bacteria. The role of this second UvrC homolog of E. coli in DNA repair  has recently been investigated in detail, and was shown to have an endonuclease activity in damaged DNA [in vitro [Azotobacter vinelandii, Escherichia coli, Salmonella typhimurium, Shiguella flexneri, and Xanthomonas. Curiously, in Beta proteobacteria, Ralstonia metallidurans and Chromobacterium violaceum, an endonuclease domain similar to Cho appears as a C-terminal fusion with a putative 3'-exonuclease, corresponding to the epsilon subunit of DNA polymerase III. A similar fusion protein was found in Mycobacterium tuberculosis, and an interesting coordinated action of these two activities (endonuclease and exonuclease) was proposed as a new mechanism of DNA repair [cho in these bacteria, but, again, one would have to concede extensive gene loss in other species. Once more, horizontal transfer events, involving part of the uvrC gene  to some gamma proteobacteria, could explain the limited occurrence of cho only in these bacteria. Moreover, some organisms present a duplication of the complete uvrC gene (Clostridium acetobutylicum and Listeria). The proteins encoded by these paralogs are closely positioned in the tree, thus indicating a recent origin for them, and, possibly, functional redundancy.A similar pattern of evolution was observed for the phylogeny of UvrC protein figure . One of aged DNA . This prin vitro . Cho mayin vitro . The oriA repair . A formeuvrD gene is part of a DNA helicase family, which includes the pcrA and rep genes [uvrD gene participates in the removal of damaged DNA strand, after the incision steps of NER or DNA mismatch repair have occurred. The evolution tree of these proteins is presented in figure uvrA and uvrC, there are two orthologs for these genes in the X. axonopodis genome. One of the orthologs encoded by the uvrD gene is closer to the gamma proteobacteria, branching with X. fastidiosa, following a typical vertical inheritance, while the second is similar to proteins from other prokaryotes, including Archaea and Bacteria, mainly from the firmicutes and alpha proteobacteria groups. The second ortholog of this gene from Sinorhizobium meliloti is found in plasmid DNA for which an alien origin is suggested, due to its lower GC content [uvrD duplication in the X. axonopodis genome indicates that it is located close to transposon-related genes  in both Xanthomonas is another remarkable feature of BER in this group. Phylogenetic trees generated for this protein family show these orthologs located in different branches, indicating that the duplication is not a recent event. However, a miscellaneous branching pattern of the tree obtained for the different bacterial groups, results difficult to track the evolution of these genes (data not shown).Base excision repair (BER) protects genetic material from a wide range of DNA damaging agents . The plaNA genes . This duet al [ligA gene, encoding the NAD-dependent DNA ligase, is found in the Xanthomonas and Xylella genomes. The phylogeny for these proteins is presented in figure Xanthomonas genomes present extra copies of putative ATP-dependent DNA ligases (two in X. axonopodis and three in X. campestris). The phylogenetic tree of ATP-dependent DNA ligases is shown in figure Mycobacterium tuberculosis and Streptomyces coelicolor. The other bacterial orthologs branch independently, wherein alpha proteobacteria predominate. A curious observation is the high and variable number of ATP DNA-ligases within the genomes of plant symbiontes, especially in the alpha proteobacteria . It is possible that these X. axonopodis genes may have arisen from recent duplications, soon after gene introduction in alpha proteobacteria, probably by means of LGT from Archaea. The fact that these bacteria inhabit a common niche in plants could facilitate mutual gene exchange. However, the function of such ligases in these bacteria is unknown. As the NAD (+)-dependent ligase is present in all bacterial genomes, these Archaea-related ligase orthologs, present in certain bacteria, seem to be redundant, their roles in cell metabolism remaining a puzzle. Since NAD+-dependent DNA ligases are typically eubacterial, and cannot be replaced [+-dependent DNA ligases appear to perform more accurate ligation reactions, with few mispaired nucleotides being allowed in the DNA extremities [+-dependent DNA ligase from Thermus species exhibit enhanced mismatched ligations under certain conditions but catalyze reactions with 1–2 orders of magnitude more discriminative towards correct nucleotide matches than the ATP-dependent DNA ligase from T4-phage [Thermococcus kodakaraensis, Nakatani et al [The DNA ligases catalyze the joining of breaks in the phosphodiester backbone of the DNA molecule and, thus, play an essential role in several processes of DNA repair, replication and recombination. These enzymes are evolutionary related, although two distinct families of DNA ligases are found: one that is typical for Bacteria, using NAD+ as cofactor, and a second that is typical of Eukaryotes and Archaea, but using ATP as cofactor . As forreplaced , the preremities . NAD+-deT4-phage . Workingni et al  found thAgrobacterium sp T-DNA [Rhizobium species amplicons present in pNGR234 plasmid [Xanthomonas the presence of the ATP-dependent DNA ligase could also be linked to the elevated number of transposable elements [Therefore, the presence of ATP-dependent DNA ligases in certain bacteria could be connected to the ligation of DNA breaks under several contexts that would generate genetic variability, with possible evolutionary advantages. In the alpha-proteobacteria group, a non homologous end joining appears to be important for the integration of inserting elements in the genome of host plants, as occurs with sp T-DNA  or for t plasmid . In Xantelements . Howeverphr) in X. fastidiosa, while present in both Xanthomonas, may be related to its limited habitat within the plant xylem or inside the insect vector [X. campestris bears the umuC and umuD genes, which encode the DNA polymerase V (UmuD'2C), related to translesion synthesis [X. axonopodis uvrD2 gene.Some other features of the DNA repair genes in Xanthomonadales are interesting to mention Table . The abst vector . It is aynthesis . In factynthesis , suggestXanthomonas (5.1 Mbp) are much larger than that of X. fastidiosa (2.7 Mbp), it was not a surprise to find more duplicated DNA repair genes in the former bacteria. Mechanisms for the protection of genetic information may reflect how the organism deals with stress and a hostile environment. Thus, the increased number of DNA repair genes in Xanthomonas may be due to the fact that these bacteria have a more variable habitat when compared to Xylella, which lives most of the time inside its hosts as a parasite. In this work, DNA repair genes, which appeared duplicated in the genomes, were analyzed focusing on their evolution, although it should be pointed out that their functions in vivo remain to be investigated. The duplicated genes found in Xanthomonas have close orthologs in other bacteria. As Xanthomonas are very closely related to Xylella, it is thus possible that, in these cases, duplications arose before the split Xanthomonas-Xylella, and were lost in the latter.Since the genomes of lexA paralogs of Xanthomonas, but this seems to be an unusual example. The possibility that gene duplications would have occurred in an early common ancestor, followed by gene loss in most other bacteria, cannot be the only explanation for many of the genes analyzed. Therefore, horizontal gene transfer among different bacteria possibly originated some of these paralogs. A clear example of recent LGT is the uvrD duplication, which is often found associated with DNA mobile elements. An origin from other life kingdoms may also have occurred, as for the ATP dependent DNA ligase, a common ligase in Archaea, which may have been acquired and were established in certain bacterial genomes, including Xanthomonas. The LGT, more than any other genetic process, makes possible faster ecological changes with the immediate incorporation of a gene or group of genes [For the genes investigated, evolution patterns indicate that duplicated genes may result mainly from relatively ancient origins. Phylogenetic errors of construction, such as long branch attraction effect, cannot be completely excluded, although the presence of close orthologs reinforce the trees generated. Moreover, the duplications are normally positioned near to orthologs also found in the genomes of some distantly related bacteria. A clear exception is the most likely recent duplication found for the of genes . Eventuaof genes ,33. In fof genes .uvrC homolog (cho), which has been found to have a complementary function in E. coli [The new genes acquired by lateral gene exchange are expected to be maintained when they provide a selective advantage to the recipient cell . For the E. coli -20. The Mycobacterium tuberculosis some DNA repair genes are induced by DNA damage, independently of the RecA protein [uvrA, uvrD and ligB, which are duplicated in Xanthomonas. This novel mechanism present in M. tuberculosis may also occur in Xanthomonas and support the idea that these duplications are also required protecting the genome against damage. Curiously, the duplicated genes found in Xanthomonas do not replace the orthologs that present vertical inheritance, similar to 16S rRNA. This reinforces the idea that they may complement the known DNA repair mechanisms with other different functions. The search for such novel functions for these genes may not only improve our knowledge on how cells protect their genomes against DNA damage, but also about how DNA repair processes evolve in bacteria.In  protein . Among tXanthomonas was performed comparing the two different genomes of the species X. axonopodis and X. campestris. As most of their genes are very similar, only X. axonopodis homologs are shown, differences being described in the text. Protein sequences from complete genomes (Bacteria and Archaea) were aligned using the ClustalX multiple sequence alignment program [Sequences of DNA repair-related proteins were obtained at the National Center of Biotechnology Information GenBank database (35). The list of organisms, with abbreviations used and proteins analyzed, is shown in Table  program  with man program . Parsimo program . Bootstr program . The samXanthomonas sp. All authors read and approved the final manuscript.MM-P carried out the phylogenetic analyses and, together with RSG and CFMM, designed and conceived the ideas and the writing of the manuscript. CL gave substantial contribution on the possible involvement of ATP-dependent DNA ligases, when present in bacterial genomes. KML-B and KAA participated in the sequencing and DNA repair genes annotation in Accession numbers for the genes indicated in the figures. Contains all accession numbers of the genes used in the phylogenetic analyses.Click here for file


We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s-1. This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s-1) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s-1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood.Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an  Platelet activation and aggregation play an important role in the development of cardiovascular disease, which is the leading cause of death in the United States. Over the last several years, enormous advances have been made in understanding the molecular mechanisms that regulate platelet function. A general paradigm has emerged that endothelial cells synthesize and release substances that inhibit platelet activation (e.g. nitric oxide and prostacyclin) except near a site of vascular injury, while platelets synthesize and release substances that promote further platelet activation. In contrast to this paradigm, several recent studies have suggested that, in the presence of certain agonists , platelets will produce nitric oxide (NO), a potent inhibitor of platelet activation . Althoug2+ mobilization [Platelet-derived NO is synthesized by membrane-bound endothelial-type nitric oxide synthase (eNOS). A common signaling mechanism shared by many eNOS agonists is binding to a surface receptor, followed by activation of phosphatidylinositol-3-kinase (PI3K), which results in phosphorylation of eNOS at serine 1179 through Akt -8. The alization . The con-17 mole NO/platelet (determined by microelectrode in PRP for 2 minutes following addition of 5 μM ADP), is sufficient to alter platelet function [As a small, hydrophilic free radical NO is highly diffusible in the aqueous environment of the blood. However, it is also highly reactive with a very short half-life estimated to be only on the order of a few seconds  in the bThe purpose of our study was to determine if platelet-derived NO plays a role in thrombus formation in the presence of shear stress. We examined the effect of several external factors on platelet thrombus formation, including insulin, the β-adrenoceptor agonist isoproterenol, and shear stress. Previous studies showed that insulin and isoproterenol both induced NO formation by platelets ,4. Shear-1 are shown in Figure Platelets from whole blood adhered avidly to the collagen-coated surface in the presence of physiologically relevant levels of fluid mechanical shear stress. Very little, if any, platelets were detected on the albumin-coated portion of the slide. Detectable levels of platelet adhesion were evident within 20–30 seconds of the initiation of blood flow over the surface. Platelet adherence occurred predominantly at the interface between albumin and collagen and increased as a function of time similar to results that have been obtained by others using a similar system -16 Repre-1 resulted in increasing inhibition of platelet accumulation on the surface. Higher concentrations of either insulin or isoproterenol had no additional affect on the extent of platelet accumulation on the surface (data not shown). Inhibition of thrombus formation was significant (p < 0.05) up to 6 minutes with 1000 pM insulin and up to 3 minutes with 1000 μM isoproterenol. Significance was determined with SPSS using the Post Hoc test as described in Methods.The effect of insulin and isoproterenol on mural thrombus formation was studied and the results are presented in Figures -1. In the presence of L-NAME as well as either insulin or isoproterenol, thrombus formation is increased, returning to levels seen in the absence of added agonist. This suggests a role for nitric oxide in mediating the agonist induced reduction in platelet accumulation on the surface. Platelet thrombus formation on the surface at lower (100 s-1) and higher (2000 s-1) levels of shear rate are shown in Figures -1) platelet accumulation on the surface is not significantly altered by either insulin (500 pM) or isoproterenol (100 μM). Higher concentrations of either agonist had no affect as well (data not shown). In addition, L-NAME by itself or in combination with either agonist did not significantly affect platelet deposition on the surface  as well as L-NAME, a specific inhibitor of nitric oxide production. The results are presented in Figures  Figures . At the  Figures , platele-1 we observed a curious result when the blood was treated with L-NAME but in the absence of any agonist. The results are presented in Figure -1 even in the absence of insulin or isoproterenol.At the high shear rate of 2000 s-1 and in the presence of insulin was slightly increased by addition of ODQ. No significant effect of ODQ on thrombus formation was observed with isoproterenol  that we tested, platelet derived NO did not appear to play a role in modulating platelet accumulation on the surface. This result was not surprising due to the many types of adhesion receptors on platelets that function well in a low shear environment. The primary platelet receptors for collagen III on a surface are GPVI and α2β1 [2β1 and αIIbβ3 into a high affinity state through a process termed inside-out signaling. In this high affinity state, these integrins are capable of supporting platelet-platelet interactions and promoting thrombus growth. Therefore, platelet adhesion at low levels of shear rate can be mediated by a number of different adhesion molecules, some of which do not require platelet activation. Our results are consistent with this model since we found that platelet derived nitric oxide did not inhibit the rate of thrombus growth at these low, venous levels of shear rate.At the low, venous level of shear , integrins α2β1 and αIIbβ3 play a more significant role in platelet adhesion and recruitment. These platelet integrins are essential for thrombus formation at intermediate to high shear [At intermediate shear  and in the presence of whole blood. Platelet-derived NO did not affect platelet adhesion at low shear but had a significant effect at intermediate and high shear. Production of NO in platelets was dominated by receptor-mediated interactions at intermediate shear and mechano-transduction at high shear. This study and future work may lead to a better understanding of platelet-derived NO and its role in healthy and diabetic vascular wound healing.Low molecular weight heparin, HEPES, NaCl, collagen III from calf skin, bovine serum albumin (BSA), L-nitro-amine-methyl-ester (L-NAME), insulin from porcine pancreas, and isoproterenol were obtained from Sigma. Recombinant hirudin (r-hirudin) was obtained from Pentapharm and 1H-,4oxadiaz2O and 10 mL 95% ethanol. After washing was complete, the cover slips were then placed in a 95% ethanol bath for at least 12 hours. Before use, each cover slip was rinsed with an additional 20 mL 95% ethanol and allowed to air-dry for 15 minutes.Each cover slip was washed with 20 mL nanopure HCollagen III solution was prepared at 0.8 mg/mL in 17 mM acetic acid (pH 2.6) at least 24 hours prior to use. BSA solution was prepared at 0.1% in 10 mM HEPES/115 mM NaCl buffer (pH 7.4). Half of each cover slip was coated with collagen and allowed to incubate for 4 hours in a humidified environment (80–90%) at room temperature (~24°C). After incubation, each slide was rinsed with 10–15 mL of HEPES/NaCl buffer solution to remove excess collagen and coated with 0.1% BSA solution for at least 1 hour.Venous blood collected from healthy donors  was anticoagulated with low molecular weight heparin to a final concentration of 20 U/mL or r-hirudin to a final concentration of 40 anti-thrombin units/mL (ATU/mL). The fluorescent dye mepacrine was then added to a final concentration of 10 μM and allowed to incubate for 10–15 minutes. All donors gave informed consent to participate in our study according to methods approved by the University of Oklahoma Institutional Review Board.in vitro parallel plate flow chamber similar to those previously characterized [-1, 0.013 × .8 cm for shear rate of 500 s-1, and 0.013 × .25 cm for shear rates of 1,000 and 2,000 s-1.The arterial flow environment was modeled with an cterized ,33. The In some studies, 500 pM insulin or 100 μM isoproterenol was added to the anti-coagulated blood 10 minutes before the start of an experiment. In other studies, L-NAME, an inhibitor of platelet NO synthesis was added at a concentration of 200 μM for 20 minutes before the start of an experiment. In additional studies, ODQ, an inhibitor of guanylate cyclase was added at a concentration of 100 μM for 20 minutes before the start of an experiment ,19,34. IIn all studies, blood was used in experiments within two hours of collection. The order of experiments was varied randomly to insure that time-dependent platelet phenotype was not skewing the results. Additionally, flow cytometric studies of platelets showed no significant change in platelet activation, as measured by P-selectin expression, over the course of 4 hours (data not shown).® image analysis software by Inovision. A background image was acquired after blood began flowing over the surface but before adhesion of any platelets. This image was subtracted from subsequent images. Platelets were identified based on size and intensity using adjustable parameters. Images were acquired and analyzed every 10–30 seconds over the course of each experiment, which lasted from 5–10 minutes.A syringe pump provided the flow through the flow chamber. The flow chamber was placed on the stage of a Nikon Diaphot 300 inverted microscope. The illumination was provided by a 75W Xenon light source passing through a 480 nm excitation filter and a 40 × fluorite objective lens. The fluorescent emission from adherent platelets was passed through a 514 nm filter and converted to analog signal by an image intensifier and CCD camera. The image was recorded on VHS tape for subsequent analysis. The extent of mural thrombus formation on the surface was quantified in terms of the percent coverage of a representative area of the collagen-coated surface at the interface between the collagen and albumin. The analyzed area was approximately 100 μm wide by 150 μm in the direction of flow. Digital image analysis was performed on an SGI Indy workstation running the ISEEStatistical significance of the results was assessed with the aid of the software package SPSS (version 11.5 for Windows). The statistical tests used were the Post Hoc test for multiple comparisons and the student's t-test. Results were deemed significant if p < 0.05.BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; eNOS, endothelial nitric oxide synthase, FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; L-NAME, L-nitro-amine-methyl-ester; NO, nitric oxide; ODQ, ,2,4oxadiThe authors declare that they have no competing interests.RW performed all of the experiments and MN conceived of the project and coordinated the data analysis. All authors read and approved the final manuscript.


Human endogenous retroviruses (HERVs) comprise a large class of repetitive retroelements. Most HERVs are ancient and invaded our genome at least 25 million years ago, except for the evolutionary young HERV-K group. The far majority of the encoded genes are degenerate due to mutational decay and only a few non-HERV-K loci are known to retain intact reading frames. Additional intact HERV genes may exist, since retroviral reading frames have not been systematically annotated on a genome-wide scale.gammaretroviral types. One encodes a protein similar to a recently discovered zebrafish retrovirus (ZFERV) while another shows partial, C-terminal, homology to Syncytin (HERV-W/FRD).By clustering of hits from multiple BLAST searches using known retroviral sequences we have mapped 1.1% of the human genome as retrovirus related. The coding potential of all identified HERV regions were analyzed by annotating viral open reading frames (vORFs) and we report 7836 loci as verified by protein homology criteria. Among 59 intact or almost-intact viral polyproteins scattered around the human genome we have found 29 envelope genes including two novel .This compilation of HERV sequences and their coding potential provide a useful tool for pursuing functional analysis such as RNA expression profiling and effects of viral proteins, which may, in turn, reveal a role for HERVs in human health and disease. All data are publicly available through a database at  This stands in stark contrast to various types of repetitive DNA, and it has been estimated that transposable elements alone take up almost half of our genome [pol) [env) [Retroviridae most of the seven genera are represented by endogenous members, and HERVs are divided into class I, II and III depending on sequence relatedness to gammaretroviruses, betaretroviruses or spumaviruses, respectively. Many HERVs are named according to tRNA usage (i.e. HERV-K has a primer binding site that matches a lysine tRNA), while others have been more or less provisionally named by their discoverer. It seems increasingly clear that the nomenclature for endogenous retroviruses (ERVs) needs to be revised to accommodate such wide diversity. Furthermore, it is evident that many more ERVs are yet to be discovered as retroviral elements are present in most, if not all, vertebrates and even in some invertebrates [Phylogenetic analysis of the retroviral polymerase gene (pol)  and envel) [env)  have idel) [env) ,5. Withitebrates ,7.With a single exception (HERV-K) all HERV groups are ancient (i.e. entered the genome prior to human speciation) and entered our genome at least 25 million years ago ,8,9 presi) replication and (random) insertion of new proviral structures, ii) effect on adjacent cellular genes, iii) long range genomic effects and iv) expression of viral proteins (or RNA). Since the majority of HERVs are highly defective no de novo insertions have been observed and presumably HERV mobilization very rarely results in spontaneous genetic disorders or gene knock-outs as seen with other active retrotransposons such as L1 elements [gag, (pro), pol and env lead to expression of 3 viral polyproteins  that are processed by a viral or host protease into the active structural and enzymatic subunits. Although most HERV genes are no longer intact, a small fraction has escaped mutational decay. For a subgroup of HERV-K (HDTV) all proteins can apparently be expressed and particle formation has been detected in teratocarcinoma cell lines [env genes from HERV-H [The presence of endogenous retroviral sequences in our genome has several possible implications: elements . Howeverelements . Moreoveelements . Such efelements . The frell lines . Furtherll lines ,22. Locim HERV-H , HERV-W m HERV-H  and HERVm HERV-H . Conservm HERV-H ,27. Recem HERV-H  was idenm HERV-H .i) the possible association of Rec (HERV-K) with germ-cell tumors [ii) the immunosuppressive abilities of HERV-H Env in a murine cancer model resulting in disturbed tumor clearance [iii) the possible superantigenic (SAg) properties of envelopes from HERV-K and HERV-W [In the literature, expression of HERVs has frequently been linked with human disease including various cancers and a number of autoimmune disorders . While cl tumors , ii) thelearance  and iii)d HERV-W ,34 and td HERV-W , schizopd HERV-W  and typed HERV-W . SAg expd HERV-W ,38. One Among Env-associated effects the mechanism of SAg-like activity is believed to involve true epitope-independent stimulation of T-cells, while the mechanism of action of the immunosuppressive CKS-17-like domain is still unknown. This immunosuppressive peptide region maps to the envelope gene  and may pol gene [env gene [env genes by nucleotide similarity [et al. [env genes in a recent survey by de Parseval et al [Computer-assisted identification of HERV loci has previously been reported. These include searching conserved amino-acid motifs within the pol gene ,40 and eenv gene , detectimilarity  and compmilarity ,42. Curr [et al. ,42 provi [et al. . Howeveral et al  and a deIn order to screen the human genome for HERV-related sequences we have performed multiple nucleotide BLAST searches and subsequently clustered neighboring hits into larger regions up to about 10 kb in size Figure . The que Our data include i) chromosomal coordinates and sequence information of the 7836 HERV regions, ii) annotation of ~38000 retroviral ORFs within these regions and iii) graphical visualization of individual HERV regions . Table gag genes (HERV ID 26658–9. W). We also find other examples of closely situated HERV loci as for instance HERV ID 44313 that is composed of two proviruses of distinct origin (HERV-K and a γ-retrovirus-like sequence) both severely degenerated.The 7836 HERV regions (~2.7 per Mb) are not uniformly distributed among the 22+2 chromosomes , and only 13% of our 7836 HERV regions are situated inside a gene, despite that 33% of the genome is spanned by genes  for intragenic HERVs to be orientated antisense relative to the gene  genes are significantly reduced as compared to a random distribution  are short . Our method only detects a single non-betaretroviral Gag ORF above 500 codons , while two long Pol ORFs are both present in full-length HERV-Fc and HERV-H elements (HERV ID 1178 and 10816) that also harbour intact env genes [Of the ~38000 retroviral ORFs 25% are classified as Gag, 7% as Pro, 55% as Pol and 13% as Env proteins. This correlates well with the expected size of the nv genes ,45.If one extends the search criteria and scans the human genome for retroviral genes where a single mutation  either removes premature termination or restores the correct reading frame, the number of long Gag, Pol and Env proteins increases two-fold to 27, 23 and 43, respectively  shows similarity to a recently reported full-length endogenous retrovirus from Zebrafish (Danio rerio), dubbed ZFERV [gammaretroviruses [gammaretroviruses (weak similarity to Feline leukemia virus). Also, two loci (HERV ID 44200–1 and 44204–5) harbor novel Env-like ORFs that C-terminally show homology to Env from HERV-W/syncytin-1 [pol and env reading frames. The same deletion is present in the HERV-K18 Env locus that has been reported to have SAg-like activity [Our method detects 29 Env ORFs (stop to stop) above 500 codons Table , which ced ZFERV . A phylooviruses . An analncytin-1 ,27 and Hncytin-1 , while tactivity .env genes four of 6 ESTs are also derived from placental tissues.We mapped 265 ESTs to one of the 42 HERV regions that encode a long Gag, Pol or Env ORF Figure . The ESTWe report a mapping of 7836 loci in the human genome that show nucleotide sequence similarity to retroviral genomes and importantly, we provide a detailed analysis of their coding potential by annotation of all viral ORFs (stop-codon to stop-codon fragments longer than 62). This compilation of HERV regions and their corresponding viral ORFs is available as a searchable database . A graphgag, pol or env genes can be restored by a single point mutation or a single insertion-deletion event.The vast majority of the mapped HERV regions contain several frame-shift mutations or in-frame stop codons that truncate the viral ORFs and thus testify to their old association with the human genome. In fact, we detect only 42 proviruses that have retained Gag, Pol or Env ORFs in the size range that approach full-length proteins  in several of these provirus loci strongly suggests that selection has preserved the env genes. In case of syncytin-1 and -2  evolutionary conservation can be understood in functional terms, since the encoded envelope proteins have been suggested to play an essential role in placental development by causing trophoblast syncytia formation [Within our list of intact or almost-intact viral ORFs in the human genome, we detect only a single al Table . The fraormation ,48. Compormation ,49,50.env gene. Potential cellular roles for envelope genes that may drive purifying selection include i) protection from infection by related retroviruses by receptor interference as demonstrated for the murine fv4 locus [ii) mediator of organized cell-cell fusion like the syncytin genes [iii) a hypothesized role in preventing the immune response against the developing embryo by means of the immunosuppressive domain [Concerning other ancient loci such as HERV-R (erv3) no evidence for a physiological role has yet been established despite a remarkable conservation and expression of the v4 locus , ii) medin genes -28 and ie domain .env genes not detected in the recent survey of intact human envelope genes [gammaretroviral genera. Whether this gene is still active or whether the encoded protein still maintains function and/or plays a cellular role is yet to be established. Although we were unable to detect any unambiguous EST matches to this gene . BLAST ne Table , RT-PCR ta Table . We havebetaretroviral env genes five turned out to carry a specific 292 bp deletion that fuses the pol and env reading frames. This deletion variant of the HERV-K (HML-2) group is indicative of the type 1 genomes [Among the long  genomes  that des genomes ,54. It i genomes , and a senv gene reported by us was only detected due to inclusion of the ZFERV sequence [Although our analysis is extensive it is most likely not exhaustive. The sensitivity is obviously limited by our query sequences, and some ancient HERVs may have suffered from the mutational decay to a degree which makes is impossible to detect them by homology. For instance, the ZFERV-related sequence , and altsequence  also poisequence . Thus, isequence . Finallysequence ), and lii) there is an unequal distribution of HERVs between chromosomes and along the genome. In particular the Y chromosome stands out with a five-fold excess of our vORF positive  HERV sequences (Table ii) HERVs are underrepresented within genes and iii) HERVs found in introns are predominantly orientated in the antisense direction  and the presence of flanking direct repeat sequences (putative LTRs). Subsequently, ORFs were categorized based on a library of known retroviral proteins and non-retroviral proteins.), with default parameters except for W = 8, E = 0.001, V = 1000000, B = 1000000. Search results were stored in a MySql database and mapped to chromosomal positions using Ensembl Bioperl packages [In order to cover as many different HERV families as possible we compiled a query set of 237 publicly available sequences from Genbank, published papers and Repbase sequences . These spackages .Overlapping BLAST hits were clustered into putative HERV regions allowing a gap of 500 nucleotides between hits. A region-score was calculated based on the sum of e-value weighted hitlengths divided by region length. Only regions longer than 300 nucleotides and a region-score > 3.0  were kept, resulting in 45658 putative HERV region.Detection of direct flanking repeats (putative LTRs) were done by comparing a window before and after the HERV region.pro and pol genes . Therefore the predicted proteins in particular for gag and env genes may contain incorrect N-terminal regions that must be removed by looking for appropriate start codons. ORF lengths below 63 aa were discarded as the probability of finding ORFs less than 63 aa in a random sequence increases to more than 0.05 .For the 45658 putative HERV loci, we scanned the DNA sequence (including 1000 bases flanking the locus) for forward open reading frames (stop-codon to stop-codon) of lengths > 62 aminoacids (aa). Stop-codon-to-stop-codon fragments were chosen to accommodate the use of non-conventional translational initiation by retroviruses at the internal All ORFs were then assigned to a category by FASTA searching against a library of known retroviral proteins (RV) and known non-retroviral proteins (NON_RV). RV proteins were downloaded from NCBI and categorized into either GAG, POL, PRO, ENV, ACC (accessory protein) or UNWANTED (for unwanted or unknown proteins). NON_RV proteins consists of all human SwissProt proteins of length 400–700 aa not including the words "endogenous, virus, envelope, env-, env, gag-, gag, pol-, pol, reverse". The final library consisted of 6260 records (3454 RV proteins + 2806 NON_RV proteins). ORF was assigned to the same category as the highest scoring hit. All loci with a significant RV ORF (vORF) were flagged as HERVs (E < 0.0005) – this data set consists of 7836 loci. Manual inspection of long ORF above 400 codons revealed that two envelope ORFs (ORF ID 86185 and 312172) were (mis)categorized as non-significant (NonS) due to low sequence similarity to our retroviral protein library.In order to match the human ESTs to the vORF positive HERV regions we first performed an all against all search using NCBI MegaBLAST . The outERV Endogenous retrovirusEST Expressed sequence tagHERV Human endogenous retrovirusLTR Long terminal repeatvORF Viral open reading frameThe authors declare that they have no competing interests., while LAA prepared query sequences, detailed analysis of the results and drafted the manuscript and PV and CW performed the statistical analysis. All authors read and approved the final manuscript.The study was conceived by LAA and FSP; PV and LAA participated in designing and coordinating the study; PV carried out all programming and compilation of Table 1. Genes with one or more vORF HERVs inside. Genes were selected from Ensembl (Current Release 21.34d.1) and compared with all HERV regions containing a retroviral ORF. 813 genes (642 with descriptions) contained 1182 HERVs (969) overlapping the gene chromosomal coordinates (exons + introns). The HERV score is a measure of the density of retroviral blast hits in the region.Click here for fileTable 2. ESTs matching HERVs containing a long viral ORF. ESTs were compared to HERVs using megaBLAST. Only ESTs that best matched the target HERV were kept. Finally, ESTs mapping conclusively to the same genomic regions as the target HERV were kept. EST library information (organ and tissue) was parsed from Genbank. The positions are in NCBI35 coordinates due to overly stringent settings of EST mappings in the NCBI34 mapping at UCSC. HERV positions were lifted to NCBI35 coordinates using the "lift genome annotations" tool at Click here for file