The objective of this paper is to describe the numbers, characteristics, and trends in the migration to the United States of physicians trained in sub-Saharan Africa.We used the American Medical Association 2002 Masterfile to identify and describe physicians who received their medical training in sub-Saharan Africa and are currently practicing in the USA.More than 23% of America's 771 491 physicians received their medical training outside the USA, the majority (64%) in low-income or lower middle-income countries. A total of 5334 physicians from sub-Saharan Africa are in that group, a number that represents more than 6% of the physicians practicing in sub-Saharan Africa now. Nearly 86% of these Africans practicing in the USA originate from only three countries: Nigeria, South Africa and Ghana. Furthermore, 79% were trained at only 10 medical schools.Physician migration from poor countries to rich ones contributes to worldwide health workforce imbalances that may be detrimental to the health systems of source countries. The migration of over 5000 doctors from sub-Saharan Africa to the USA has had a significantly negative effect on the doctor-to-population ratio of Africa. The finding that the bulk of migration occurs from only a few countries and medical schools suggests policy interventions in only a few locations could be effective in stemming the brain drain. Doctors migrate from developing countries to wealthier countries in order to further their careers, or improve their economic or social situation. The World Health Organization (WHO) has long recognized that migration of health personnel from developing to developed countries creates unfortunate imbalances in the global health workforce .America's physician workforce has been significantly infused with foreign-trained international medical graduates (IMGs) since World War II. The purpose of this paper is to describe a sub-population of IMGs in the USA, those who have trained in one of the 47 African subcontinent nations.African governments have been very clear about their objections to the wholesale migration of their physicians to rich countries. In 1996, South Africa's then-Deputy President Thabo Mbeki implored the World Health Assembly to take measures to stop the flow of physicians from poor countries to rich ones. In 1995, South Africa itself banned the recruitment of doctors from other Organization of African Unity countries .Nonetheless, large numbers of African-trained physicians leave home upon completion of their medical school training in search of careers in higher-income countries. They leave behind health systems in sub-Saharan Africa that are severely stressed: life expectancy is only 50 years, 162 children in 1000 die before reaching their fifth birthdays, and only half have access to clean water sources . FurtherAfrican country health systems and workforce data are poor, making it difficult to estimate the effects of physician migration on sending countries. The World Bank has documented this data gap, noting "Quantitative data on the health workforce is notoriously unreliable in most countries...In poor countries, government and professional information systems are weak, when they exist at all, and are rarely comprehensive (often there is no information on the private sector) and up-to-date" . Indeed,The 47 nations of sub-Saharan Africa have a total of 87 medical schools, although 11 countries have no medical school at all and 24 have only one each .One of the most common initial points of entry for IMG physicians into the USA medical workforce is residency training program enrollment, even if physicians have already completed postgraduate training in their home countries. The reliance of many inner-city hospitals on IMGs has thwarted calls by medical policy organizations, such as the Council on Graduate Medical Education, to reduce the number of IMGs admitted to residency programs as a means of narrowing the IMG pipeline to the USAThere is little debate within the USA government or other institutions about the social justice implications of obtaining health professionals from poor countries . TypicalThe ethics of health professional migration from poor countries to rich ones is complicated by the competition of legitimate interests – each country's need for an adequate health workforce as opposed to each individual's human right to travel. When health professionals travel to receive training and then return to apply their skills, there are advantages to the home country. Additionally, emigrants of all social classes from poor countries typically send funds home to relatives, although sub-Saharan African remittances, at less than USD 5 billion, comprise the lowest dollar amounts of any other poor world region . FurtherImmigration theory informs us that "push factors" prompt professionals to leave poor countries in favor of settling in higher income countries . NegativThe "world systems framework theory" stresses the more permeable barriers between and among countries created by the standardized curriculum and English language used in world medical schools, the use of common research methods and shared scientific knowledge, the easy articulation of requirements of practice across countries, and the weakened nationalism that occurs as a result of professional training . Other tGiven the enduring migration from poor countries to rich ones, only likely to increase with the international liberalization of trade in health services , concernTo describe the numbers and types of physicians practicing in the USA who earned medical degrees in Africa, we performed a cross-sectional study using the 2002 American Medical Association Physician Masterfile . This daWe reviewed these data for all physicians in the USA who received their training in sub-Saharan Africa (those 47 countries south of the Saharan desert on the African continent). These data included year of birth, gender, year of medical school graduation, name of medical school, current practice location, specialty of practice, and practice activity . Birth country information is missing for 68% of those who graduated from a sub-Saharan African medical school, so we did not analyze birth country data. To detect changes in migrant waves over time, we analyzed the data by cohorts, categorizing physicians who had graduated from medical school during four periods: before 1970, during the 1970s, during the 1980s, and 1990 and beyond.We linked geographic data about practice locations to a four-category, rural-to-urban status and taxonomy, a condensed version of the Rural-Urban Commuting Area (RUCA) codes, to determine whether these physicians are practicing in rural or urban areas. RUCAs are a census tract-based classification scheme, that have also been adapted for zip codes, combining USA Census population data with work commuting information to characterize the types of rural and urban status .Research colleagues in Canada and the UK provided some data on sub-Saharan African physicians in their countries, as well.A total of 179 978 (23.3%) of the 771 491 active non-federal physicians in the USA in the year 2002 received their medical qualification in another country. The largest portion of these, or 115 835 physicians, originate from low and lower-middle income nations, as defined by the World Bank. Indeed, the most frequent countries of origin of IMGs in the USA include India (36 634), the Philippines (17 755), Mexico (10 404), and Pakistan (8563). Canadian physicians conventionally are not included in the IMG count because the body that accredits USA medical schools ) offers reciprocal accreditation to Canadian medical schools . Canadians are, however, still subject to relevant immigration requirements.Sub-Saharan African medical schools in 22 countries have trained approximately 5334 physicians currently practicing in the USA. Only nine nations, however, have lost more than 40 physicians each , the University of Cape Town (South Africa, 655), the University of Ibadan (Nigeria, 643), the University of Lagos (Nigeria, 429), the University of Nigeria (Nigeria, 394), the University of Ghana (Ghana, 389), Addis Ababa University (Ethiopia, 200), the University of Benin (Nigeria, 183), the University of Ife (Nigeria, 156), and the University of Pretoria (South Africa, 132), for a total of 4234 physicians.An analysis of the numbers of sub-Saharan African IMGs coming to the USA in each of the last decades illustrates that it takes some time between graduation and emigration. The number of recent graduates currently in a USA residency program is higher than those in previous decades because of the obvious correlation between age and career stage.Among sub-Saharan physicians in the USA, 78.3% are male. The picture is changing over time, however. Of the cohort who were trained in 1969 or earlier, 90% were male, but now only 66.3% of those who graduated from medical school in 1990 or later are male , and South Africans are the oldest (only 20% are under 40).A higher proportion of sub-Saharan physicians were in residency training programs (21.2%) than were USA physicians (14.1%), because many emigrate specifically for that reason. While 41.9% are in generalist specialty areas, compared to 34.7% of USA-trained physicians, the number has been rising with each new cohort. Table While 31.6% of all sub-Saharan African physicians in the USA are identified as family practitioners or general internists, it may be that this ratio of generalists will increase, as 45.4% of those in residency programs are in those two specialties. The next largest specialty groups are pediatrics (9.7%), psychiatry (5.5%), anesthesiology (5.4%), obstetrics and gynecology (3.3%) and general surgery (3.0%).Urban areas attracted 93% of sub-Saharan African physicians (compared to 90.9% of other IMGs), even after excluding residents, who are typically based in urban teaching hospitals. Graduates of USA medical schools distribute themselves similarly, with 87% of USA-trained physicians in urban areas, even though a smaller 81% of the population lives in urban areas . The staThe 1943 physicians trained in South African medical schools are somewhat different from their fellow African trainees from the subcontinent. They are older, more of them are male, they are typically white (94%) and they are more often in a subspecialty practice. This may reflect a particular wave of physicians seeking subspecialty training and practice opportunities in the USA during the political turmoil South Africa experienced in the 1970s and 1980s. It is unlikely they are seeking training abroad that is unavailable in their home country, as the medical training opportunities in South Africa are quite comprehensive.The 5000-plus physicians trained in sub-Saharan Africa who have migrated to the USA comprise only a small proportion of the total number of IMGs practicing in the USA. However, their relatively small numeric addition to the USA medical workforce contrasts markedly with the impact of their migration on the medical workforce in sub-Saharan Africa. Moreover, in absolute terms, the USA has drawn more of the medical workforce of Africa than either Canada, with 2151 African graduates , or the UK, with 3451 , largely because of the relative size of the health care system in the USA.Including the USA, the UK, and Canada, then, 10 936 physicians trained in sub-Saharan Africa are practicing in the three countries, a number that represents 12% of all African physicians. We expect there are many more African physicians who are in the UK, as well, as our figures from there include only those who arrived post-1992. While some of the physicians in residency training will return home, there are unknown others who could be practicing medicine at home but were not able to get licenses abroad and therefore are engaged in other occupations. Almost all the medical schools sending graduates to the USA provide their instruction in English.Our study provides some measures of sub-Saharan Africa physician migration to the USA. A next step is to collect data from other developed countries and begin to create a physician migration data set from multiple countries. Migration from country to country within the subcontinent is also worthy of further examination, and it would be useful to identify the relationship between country of birth and country of training. There are likely to be some medical schools that draw students from several African countries. It should be noted, as well, that there are physicians practicing in Africa who did not train there, most prominently Cubans.Some sub-Saharan countries lose a larger proportion of their physicians to the USA than others. For example, while Ghana has a reported 1210 practicing physicians in its country, 478 graduates of Ghanaian medical schools are practicing in the USA. Without even considering those who have migrated to other countries, these 478 Ghanaian graduates in the USA represent 30% of Ghana's potential medical workforce (see Table The flight of physicians from West Africa: views of African physicians and implications for policy, unpublished 2003).The migration of physicians from sub-Saharan Africa represents a lost investment of significant training costs, since graduates of medical schools in Africa are likely to have contributed financially to only a small portion of the costs of their medical education . MedicalThe United Nations Commission for Trade and Development has estimated that each professional leaving Africa costs the continent USD 184 000, or USD 4 billion a year – one third of official development funds to Africa . The losOstensibly, the USA welcomes IMGs for two purposes. First, as a form of foreign aid, it provides specialty training that physicians can take back to their home countries for the benefit of residents of those nations. Second, IMGs fill positions in specialties and locations that are less attractive to their USA counterparts, and may help to correct physician maldistribution in some rural or underserved areas of the USA. Longitudinal tracking of physicians entering the USA has indicated, however, that few IMGs ever leave the USA after arriving for residency training , and theAttracting physicians to rural practice in most countries is difficult, and is accomplished only through a careful set of policies designed to provide incentives for rural service. In most poor as well as rich countries, physicians are concentrated around urban hospitals that offer tertiary care, even though more rational service delivery systems might focus on a geographically decentralized system of primary and preventive care. Poor countries that offer medical training to produce too many physicians with highly technical skills, some of whom who cannot find satisfying jobs, may further contribute to physician migration . India aOur findings show African physicians are unlikely to select small or remote rural practice opportunities in either their home countries or in the USA, but the preponderance of African physicians in American inner-city underserved areas may to some extent be helpful to USA needs by boosting the number of minority physicians in the urban health workforce. The growing number of African immigrant female physicians follows the trend for increasing numbers of female physicians trained in the USA, and probably has similar implications. Some researchers have found, for instance, that female physicians are less likely to practice in rural areas .While the sub-Saharan Africa region as a whole loses many of its physicians, it is apparent that a small handful of medical schools is the sources of the majority of this migration. Ten medical schools in four countries – South Africa, Nigeria, Ghana and Ethiopia – produce 79.4% of the émigré physicians to the USA, out of a total of 87 medical schools in the region. This suggests policy approaches to reducing the "brain drain" from Africa could be targeted at only these few countries or medical schools, a less daunting task than addressing the problem in 47 different countries.Medical migration results from the complex interaction of myriad social, legal and economic forces. Single country policies are unlikely to alter the flows significantly. Even if the USA acknowledges that that it benefits from luring medical professionals here for whose medical school training we do not pay, solutions that would be compatible with social justice principles are not clear. Furthermore, if all African doctors returned to their home countries today, they would not necessarily find satisfactory employment opportunities in cash-strapped health systems.The 57th World Health Assembly, in 2004, adopted a resolution to urge member countries to develop strategies to mitigate the adverse effects of migration of health workers; to develop policies that could provide incentive for health workers to remain in their countries; and, among other issues, requests WHO to help countries set up information systems to monitor the movement of health resources for health, and to include human resources for health development as a top-priority program at WHO from 2006 to 2015 .In an ideal world, freedom of movement is a universal right for individuals, as there is ostensibly no rational reason why anyone would have a stronger right to be in any place more than anyone else . Today, Importing health professionals from poor countries to provide care in rich countries is not consistent with a rational workforce policy rooted in social justice principles. In the short run, Mullan  and otheThe New York Times editorialized that the "obvious long-term solution to the medical brain drain is for wealthier countries to reimburse Africa's health and educational systems for the cost of poaching their professionals, and to greatly increase the financing and technical help for Africa's health systems" [In response to a recent report by the USA Physicians for Human Rights , The Newsystems" . This unThe New York Times response made a strong recommendation that the International Monetary Fund, World Bank, and other donors refrain from withholding loans or grants from countries that increase their spending on "health, education and other sectors and activities needed to promote human development, including to enhance salaries to health staff or to hire new health personnel."The same Physicians for Human Rights report that prompted One of the major limitations African nations face in addressing their health workforce problems is the lack of reliable data on how many health workers have graduated from their schools, how many are working in the country and in what locations, and how many have emigrated. It is urgent that poor countries put together the information systems required to track these data, as a basis for workforce policy and investment decisions.And, finally, the fact that so few medical schools generate the vast majority of emigrants creates an opportunity to focus attention in a strategic way. These schools might be enticed to redirect their missions towards producing graduates who intend to serve their own countries. This would likely require curriculum changes, admissions policy changes, and a change in faculty culture to ensure that emigration is not promoted as a mark of prestige.The author(s) declare that they have no competing interests.AH conceived the project, designed the research and wrote the paper. MJT and KEJ actively participated in the conceptualization and re-writing of the paper. MF conducted the data management and analysis. LGH provided guidance, advice and editing assistance. All authors read and approved the final manuscript.


Every spring and fall, billions of songbirds fly thousands of miles between their summer breeding grounds in North America and their wintering grounds in the more hospitable climes of southern California, Mexico, and Central and South America. While some birds fly during the day, most, including the white-crowned sparrow, fly under cover of night. Many aspects of this remarkable voyage remain obscure, especially if, and how, nocturnal migrators get any sleep at night.A tracking study of the Swainson's thrush found that the roughly seven-inch birds flew up to seven hours straight on six of seven nights, racking up over 930 miles. While the study didn't track their daytime behavior, the birds' migratory pace—as well as the increased activity required to sustain migrations—suggests little time for sleep. Yet field observations indicate that presumably sleep-deprived fliers appear no worse for wear, foraging, navigating, and avoiding predators with aplomb. Researchers are left trying to reconcile this observation with the vast body of evidence linking sleep deprivation to impaired neurobehavioral and physiological function. How do songbirds cope with so little sleep? Do they take power naps? Have they taken “sleep walking” to new heights? Or have they managed to selectively short-circuit the adverse effects of sleep deprivation during migratory stints?To investigate these questions, Ruth Benca and colleagues studied cognitive and sleep behaviors in captive white-crowned sparrows over the course of a year. The sparrows fly nearly 2,700 miles twice a year between their Alaska and southern California homes. In laboratory cages, the birds' migratory instincts manifest as increased restlessness at night during the migratory season, with lots of hopping around and wing flapping.Niels Rattenborg et al. characterized the birds' activity levels with motion-detection measurements and video recordings, and placed sensors on their brains to monitor their seasonal sleep patterns. The brain recordings showed a marked seasonal difference in both the amount and type of sleep during a 24-hour period: migrating birds spent roughly two-thirds less time sleeping than nonmigratory birds and fell into REM sleep (the dream stage of sleep, marked by rapid eye movements) much sooner. Birds displaying active migratory behavior appeared completely awake during such activity. Cognitive tests—birds performed a task that involved pecking a key in exchange for seed—revealed that birds in the nonmigrating state suffered cognitive deficits when sleep-deprived but displayed an “unprecedented” ability to maintain cognitive function in the face of ongoing sleep loss in the migratory state.These results suggest that wild songbirds drastically reduce sleep time during migration, though Benca and colleagues concede it's impossible to know for sure without recording the birds in action. And it is unclear what molecular mechanisms jumpstart the migratory mindset. Such an ability to temporarily circumvent the need for sleep, however, could prove useful for humans in situations that demand continuous performance. Some studies link migration with increased neuroendocrine activity, which is in turn associated with sleep disruption, accelerated timing of REM cycles, and mood disorders in humans. “Like migrating sparrows,” the authors note, “both depressed and manic patients show reduced latency to REM sleep, loss of slow-wave sleep, and reduced amounts of total sleep.” Given the parallels between migratory behaviors and bipolar illness, it's possible that similar mechanisms may be involved in both.Whatever the mechanism, the unprecedented imperviousness of migrating songbirds to sleep deprivation, the authors conclude, clearly warrants further testing. But it also raises interesting questions about the role of sleep, which recent studies suggest is required to incorporate novel perceptions into the brain's memory banks. If this is true, how do songbirds consolidate memories of migratory events with so little sleep? Understanding the mechanisms that power the sleepless flight of songbirds promises to unravel one of the longstanding mysteries of their improbable journey. It may also shed light on the origins of sleep-related seasonal disorders and the much-debated role of sleep itself.


Synovial sarcoma is a high-grade malignant tumor of soft tissue, characterized by the specific chromosomal translocation t, and its resultant SYT-SSX fusion gene. Despite intensive multimodality therapy, the majority of metastatic or relapsed diseases still remain incurable, thus suggesting a need for new therapeutic options. We previously demonstrated the antigenicity of SYT-SSX gene-derived peptides by in vitro analyses. The present study was designed to evaluate in vivo immunological property of a SYT-SSX junction peptide in selected patients with synovial sarcoma.A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Vaccinations with SYT-SSX B peptide (0.1 mg or 1.0 mg) were given subcutaneously six times at 14-day intervals. These patients were evaluated for DTH skin test, adverse events, tumor size, tetramer staining, and peptide-specific CTL induction.A total of 16 vaccinations were carried out in six patients. The results were (i) no serious adverse effects or DTH reactions, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients.Our findings indicate the safety of the SYT-SSX junction peptide in the use of vaccination and also give support to the property of the peptide to evoke in vivo immunological responses. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy. Synovial sarcoma is a relatively rare, high-grade malignant tumor of soft tissue, characterized by biphasic or monophasic histology, specific chromosomal translocation tX;18), and its resultant SYT-SSX fusion gene8, and it-5. HowevVaccination of tumor antigenic peptide serves as a commonly accepted method in anti-cancer immunotherapy,7. This Currently tumor specific chromosomal translocations are defined in leukemias, lymphomas, and sarcomas,10. The A 9-mer peptide (SYT-SSX B: GYDQIMPKK) spanning the SYT-SSX fusion region was synthesized under good manufacturing practice (GMP) conditions by Multiple Peptide Systems (San Diego, CA). The identity of the peptide was confirmed by mass spectral analysis, and was shown to have more than 98% purity when assessed by high pressure liquid chromatography analysis. The peptide was delivered us in the form of a freeze-dried, sterile white powder. It was dissolved in 1.0 ml of physiological saline  and stored at -80°C until just before usage. The affinity of the peptide to HLA-A24 molecules and its antigenicity were determined in previous studies,14.The study protocol was approved by the Clinical Institutional Ethical Review Board of the Medical Institute of Bioregulation, Sapporo Medical University, Japan. Eligible patients were those (i) who have histologically and genetically confirmed, unresectable synovial sarcoma (SYT-SSX1 or SYT-SSX2 positive), (ii) HLA-A*2402 positive, (iii) between 20 and 70 years old, (iv) ECOG performance status between 0 and 3, and (v) who gave informed consent. Exclusion criteria included (i) prior chemotherapy, steroid therapy, or other immunotherapy within the past 4 weeks, (ii) presence of other cancers that might influence the prognosis, (iii) immunodeficiency or a history of splenectomy, (iv) severe cardiac insufficiency, acute infection, or hematopoietic failure, (v) ongoing breast-feeding, (vi) unsuitability for the trial based on the clinical judgment of the doctors involved. This study was carried out at the Department of Orthopaedic Surgery, Sapporo Medical University Hospital from June 2003 until the end of September 2004.Vaccinations with SYT-SSX B peptide were administered subcutaneously into the upper arm six times at 14-day intervals. In order to set up a dose-escalation trial, the patients were separated into the two groups. Each group included three patients. Those from group 1 received 0.1 mg and group 2 participants received 1.0 mg.Delayed-type hypersensitivity (DTH) skin test was performed at each vaccination. The peptide (10 μg) solution in physiological saline (0.1 ml) or physiological saline alone (0.1 ml) were separately injected intradermally into the forearm. A positive reaction was defined as a diameter of erythema of more than 4 mm, 48 hr after the injection.Patients were examined closely for signs of toxicity during and after vaccination. Adverse events were recorded using the National Cancer Institute Common Toxicity Criteria (NCI-CTC).Physical examinations and hematological examinations were monitored before and after each vaccination. Tumor size was evaluated by computed tomography (CT) scans before treatment, and again after three vaccinations, and then at the end of the study period. A complete response (CR) was defined as complete disappearance of all measurable diseases. A partial response (PR) was defined as a >= 50% decrease from the baseline in size of all measurable lesions  lasting for a period of at least 4 weeks. Progressive disease (PD) was defined as an increase in the sum of the bi-dimensional measurements of all known disease sites by at least 25% or by the appearance of new lesions. No change (NC) was defined as the absence of matched criteria for CR, PR, or PD.st, 3rd, and 6th vaccination. Cells were stained with PE-labeled tetramers at 37°C for 20 min and a FITC-conjugated anti-CD8 mAb (Becton Dickinson) at 4°C for 30 min. Analysis of stained PBMCs was performed using FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson). The frequency of CTL precursors was calculated as the number of tetramer positive cells / the number of CD8+ cells.HLA-A24/peptide tetramers (HLA-A24/B, HLA-A24/R49.2, and HLA-A24/HIV) were previously constructed -15. Flow51Cr release assay[+ T cells were mixed with the labeled target cells. After a 6-h incubation period at 37°C, the release of the 51Cr label was measured by collecting the supernatant, followed by quantification in an automated gamma counter. The percentage of specific cytotoxicity was calculated as the percentage of specific 51Cr release: [ / (maximum 51Cr release - spontaneous 51Cr release)] × 100. Maximum 51Cr release was measured by incubating the labelled target cells with 2% NP-40, instead of the stimulated CD8+ T cells. CTL induction was determined as successful when specific cyotoxicity of 10% or more was achieved on Fuji, HS-SY-II, and SYT-SSX B peptide-pulsed T2-A*2402 cells.Cytotoxic T lymphocytes (CTLs) were induced from the PBMCs of patients using SYT-SSX B peptides according to the method described before,14. The ase assay. As targSix patients were enrolled in the study Table . There wOne patient (case 5) experienced slight fever (grade 1) after the first vaccination. No other adverse events were observed during vaccination. DTH skin test was performed at each vaccination and assessed 48 hr later. Reactions were determined as negative in all patients.Recognized disease progression occurred in five out of six patients during the vaccination period Table , Fig. 1.Peptide-specific immunological responses were evaluated in five patients by using HLA-A24/peptide tetramer analysis and in vitro CTL induction. As determined by flowcytometric analysis using HLA/peptide tetramers Table . In the Table The present study was designed to evaluate the in vivo immunological property of a 9-mer SYT-SSX junction peptide in patients with disseminated synovial sarcoma. A total of 16 vaccinations of the peptide in six patients revealed (i) no serious adverse effects in any case, (ii) suppression of tumor progression in one patient, (iii) increases in the frequency of peptide-specific CTLs in three patients and a decrease in one patient, and (iv) successful induction of peptide-specific CTLs from four patients. These findings suggest that the SYT-SSX junction peptide is safe to use as a vaccine and also has the property to evoke in vivo immunological responses.With respect to the clinical efficacy, none of the enrolled patients showed signs of tumor remission. However, in the case 5 patient, tumors showed some dormancy during the vaccination period, in comparison to the other five patients in whom tumors grew rapidly from the early phase of the regimen. The case 5 patient had received one cycle of systemic chemotherapy four months before enrollment, making the possibility of residual chemotherapeutic efficacy unlikely. The other five patients, except in case 1, had received systemic chemotherapy one to two months before perticipation. Notably, frequency of circulating peptide-specific CTLs decreased in the case 5 patient, while CTL frequencies remained unchanged or increased significantly in the other four patients examined. Decrease in circulating CTLs in the case 5 patient may have resulted from accumulation of CTLs at the tumor sites, although biopsy of the tumors was not performed.Vaccination trials of fusion gene-derived peptides have been reported with BCR-ABL in 12 patients with chronic myelogenous leukemia, EWS-FLITo monitor the immunological responses, we used HLA/peptide tetramer and in vitro CTL induction. Other monitoring procedures such as ELISPOT assay should provide further information. Nevertheless, the use of an internal control tetramer with HIV peptide added strength in this present comparative analysis. As shown in Table Due to the rarity and highly malignant nature of synovial sarcoma, three out of six patients failed to complete the six-time vaccination regimen. Such difficulty in continuation of vaccination has also been found in a trial of patients with Ewing's sarcoma and alveolar rhabdomyosarcoma. AnotherThis is the first clinical trial of SYT-SSX fusion gene-derived peptide in patients with synovial sarcoma. The present trial demonstrated the safety and immunogenic property of the peptide. Modification of both the peptide itself and the related protocol is required to further improve the therapeutic efficacy.CTL; cytotoxic T lymphocyte, HLA; human leukocyte antigen, MHC; major histocompatibility complex, PBMC; peripheral blood mononuclear cellThe author(s) declare that they have no competing interests.SK1 and YS carried out vaccinations and clinical evaluations. KI, TT, SK1,2, HA and KS carried out monitoring procedures. TW, SN, HS, HI, TT, HH, TI, ST, NS, and TY made substantial contributions to the design of the study.


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders In the very thoughtful essay “Reason as Our Guide” by Drs. Blackburn and Rowley point out many areas of the report “Monitoring Stem Cell Research” that needed correction from a scientific standpoint. While it is impossible to include every suggestion in a report that seeks to draw consensus from a large panel of members, in a heated, political debate like that surrounding embryonic stem cell research and therapeutic cloning, providing the most accurate and complete scientific information to policy makers is crucial. Unfortunately, with the recent dismissal of Dr. Blackburn from the Council, there will now be one less voice for scientific research and for the potential the research holds for curing disease and alleviating the suffering of millions.Speaking for the Coalition for the Advancement of Medical Research, our concern is not only the small number of researchers on the Council and lack of complete scientific data being shared with policy makers, but the absence of patient representation on the Council itself. With the exception of public comment periods, patient organizations have no voice in the work of the Council as it discusses issues that profoundly impact them. Now, with one less member standing up for research and thus patients, our concern grows even stronger.The Blackburn and Rowley essay also correctly points out that there is more published work on adult stem cell research because of a “paucity of funding for research using embryonic stem cells.” Despite this lack of federal and private funding, advances continue to be made—but just think of the advances we could have had if only there were a supportive federal policy that encouraged embryonic stem cell research instead of stifling it. We hope—in light of scientific advances made over the past several years and the strong support of the scientific community, including the National Institutes of Health, the Health and Human Services Department, and the National Academy of Sciences—that the President will reevaluate the current federal policy for stem cell research and consider easing the restrictions.We commend Drs. Blackburn and Rowley for trying to set the record straight in their essay, and applaud their efforts to stand up for medical research, which has the potential to benefit us all.


All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used.We developed a novel configuration of a cooled charge-coupled device (CCD) for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures.The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves. This is in contrast to the more widely known bioluminescence from animals that visibly glow, such as certain species of jellyfish, fireflies and beetles, which employ fluorescent proteins and/or luciferase enzymes to catalyse reactions that result in chemiluminescence. Ultra-weak bioluminescence is generally considered to result from oxidative chemistry occurring within cells [e.g. [It is well documented that essentially all living systems spontaneously generate and emit very low levels of light (reviewed in ). This uin cells , plants ls [e.g. ) and anils [e.g. ,8. For tIn plants, increases in spontaneous low-level luminescence have been observed in response to pathogen infection , salt stet al., [Quantitative measurements of such ultra-weak photon emissions are normally obtained using sensitive photomultiplier tubes as photon counting devices. However, more recently, 2-dimensional photomultiplier tubes and cooled charge-coupled device (CCD) cameras have also been employed as a means of imaging the spatial distribution of light emission from diseased and damaged plant tissues ,9. The cHere, we present a novel configuration of a cooled CCD that enables high sensitivity, high resolution 2-dimensional imaging with short integration times. We demonstrate the utility of the system to image and quantify delayed chlorophyll fluorescence and wound-induced luminescence from plant leaves.2 from wounded leaves (see below). In addition to its high sensitivity, the imager is significantly less expensive than equipment based on photomultiplier tubes.The experimental imaging set-up, shown in Figure Arabidopsis thaliana and Tradescantia albiflora . To test whether chlorophyll might be the source of wound-induced photon emission, we used the carotenoid biosynthesis inhibitor, norflurazon, to generate photobleached leaves lacking chlorophyll [In order to understand more about the origin of the luminescence emitted by wounded leaves, we used coloured filters to investigate its spectral characteristics. The results are shown in Figure orophyll . Compareorophyll .That delayed chlorophyll fluorescence exhibits temperature-dependence has been shown previously in several systems . Since wFurthermore, we found that heating could activate luminescence in leaves wounded up to 3 hours before imaging. For example, Figure The novel configuration of cooled CCD that we have used in this study to capture 2-dimensional images of plant leaves provides excellent temporal and spatial resolution of bioluminescence/chemiluminescence processes in biological materials, which are important markers of various forms of stress and disease. Using this system, we are able to define the origin of wound-induced luminescence from plant leaves as chlorophyll, and suggest that this arises via the temperature-dependent release of energy from excited triplet carbonyls produced by oxidative stress.Arabidopsis thaliana, Columbia ecotype, were grown in soil in a glasshouse at 22°C with a 16-hour photoperiod. Wounding was performed by crushing leaves with a haemostat. To generate leaves lacking chlorophyll, Arabidopsis plants grown for 3 weeks under 16 h light/8 h dark, were watered with 0.1 mM norflurazon (Sigma Aldrich PS1044) and grown under constant illumination at 120 μmol/m2/sec. Under these conditions, newly emerging leaf tissue was white. Leaves with white regions were taken 15 days after the start of norflurazon treatment, and used for luminescence imaging.Plants of i.e. at approx 1100 nm; however, for ultimate sensitivity the detector output is monochrome, i.e. there is no wavelength selectivity in the present instrument. Further information is available on request from Michel Flor-Henry, Biolumonics Ltd.The imager used is a two-dimensional imaging system based on a slow-scan 'scientific grade' Silicon Photo Area-Detector [SPAD] (Integrated SPAD Imaging System – PiXx*ell 1A, Biolumonics Ltd), which consists of a cooled ultra-sensitive area photo-detector within a stainless steel vacuum vessel and associated image acquisition electronics. The input image is coupled to the detector with optical fibres . The detector contains 222,336 pixels, and in the experiments presented here, ran at an operating temperature of -58°C. The combination of the proprietary low read-out noise electronics, cooling of the detector and the optical fibre coupling results in a sensitive imaging system capable of recording the faintest luminescences at high resolution, (40 μm to 200 μm depending on geometry of sample and integration time). The spectral absorption response of the SPAD ranges from the near UV to IR, thus including the entire visible spectrum up to the band-gap energy barrier of Silicon (1.1 eV) , though readers should note that the transmittance spectra provided with the filters covers the range 300–800 nm, rather than the more limited 400–700 nm spectra shown on the web site. Filters were placed between the leaf and the sample stage prior to imaging.Coloured filters were obtained from Lee Filters (Andover, UK). We used lighting effect filters LEE 182 – "light red," LEE 118 – "light blue," LEE 183 – "moonlight blue" and LEE 735 – "velvet green." Spectral characteristics of the filters can be viewed at MF-H designed the PiXx*ell 1A luminescence imager. MF-H and MRR conceived of the current study, participated in its design and coordination, carried out experiments and wrote the manuscript. MF-H performed data analysis. TCM performed the norflurazon experiment, and both TCM and GLdB participated in some experimental design and execution. All authors read and approved the final manuscript.Arabidopsis leaves.Animated gif file showing images from five sequential 5-minute exposures of one control leaf (bottom) and four wounded Click here for fileArabidopsis leaves. Images were initially captured from samples on a cold imaging stage, but the heater in the lid of the sample stage was switched on when indicated.Animated gif file showing images from seven sequential 5-minute exposures of two control leaves (bottom) and four wounded Click here for file


Endometriosis is a condition that affects women of reproductive age, where the glandular and/or stromal tissues from the eutopic endometrium implant in ectopic locations. It is well established that the survival of ectopic implants is due to lower levels of apoptosis, but no consensus exists as to which pathway/s this is mediated by. The ubiquitin protein shares a similar sequence homology to an anti-apoptotic protein called BAG-1 and is expressed in the normal endometrium. Currently, no studies have been conducted to determine ubiquitin expression and its possible anti-apoptotic effects in endometriosis.Archived endometrial tissues from endometriosis patients and women undergoing laparoscopic diagnosis (controls) from January 2000 to July 2003 at Westmead Hospital were examined, where 14 cases of endometriosis and 55 controls were included in the study.Both the ubiquitin protein and apoptosis were expressed in both glandular and stromal cells throughout the menstrual cycle of the eutopic endometrium, in which ubiquitin exhibited a cyclic expression, reaching a peak in late proliferative phase. In contrast, ubiquitin was predominantly expressed in cells of stromal origin in endometriosis, was no longer regulated by a cyclic pattern and was associated with an aberrant level of cell survival.For the first time, this study shows that ubiquitin is expressed in endometriotic cells and may contribute to a reduced sensitivity of ectopic endometrial tissue to apoptosis. These findings also suggest that stromal cells contribute differentially to the development of ectopic endometrial tissue. Endometriosis is a condition that affects 10% of women of reproductive age . HoweverUbiquitin is a 76 amino acid protein  that is Accumulating evidence suggests that dysregulated apoptotic machinery in endometriosis has a role in its pathogenesis, but no consensus exists as to which pathway/s this is mediated by . TransfoTherefore, this study was undertaken to determine the expression of ubiquitin within endometriosis during the menstrual cycle and to establish whether it is related to a decrease in the incidence of apoptosis in this tissue, thus potentially promoting ectopic endometrial cell survival.Approval to conduct this study was granted by the Human Research Ethics Committee of the Western Sydney Area Health Service. Archived formalin fixed, paraffin embedded tissues from 14 women with endometriosis and 55 women undergoing laparoscopy for non-endometrial pathologies such as leiomyomata and benign ovarian cyst were studied. Endometriotic implants (n = 20) and endometrial tissues (n = 59) were obtained from the women above, respectively. All tissues were observed from the Department of Pathology at Westmead Hospital between January 2000 and July 2003. Mean ages for endometriosis and control groups were 42.5 ± 2.69 and 36.4 ± 1.17 years, respectively. Women with histological evidence of malignancy, necrosis, active inflammation or hormonal treatment were excluded from the study. Control subjects had normal, regular menstrual cycles and endometrial samples were collected from the proliferative and secretory phases of the cycle.Sections (4 μm) of endometriosis and control endometrial tissue blocks were cut on a microtome (Microtome, Stauffenberg, Germany), placed on superfrost slides (Menzel-Glaser, Braunscheig, Germany) and air-dried at room temperature for 24 hours.De-waxing was performed with two changes of histolene and absolute ethanol through a single change of 95% (v/v) and 70% (v/v) ethanol and a final change of tap water. All incubations were of 5 min duration. Following the suppression of endogenous peroxidase activity for 10 min with a blocking agent  sections were incubated with normal goat serum for 60 min to prevent non-specific binding. A polyclonal rabbit anti-ubiquitin primary antibody  prepared at a titre of 1:100 was added for a further 60 min at 37°C. Slides were then washed three times in tris-buffered saline  before the biotinylated goat anti-rabbit IgG secondary antibody  prepared at 1:200 was applied (30 min). A horseradish peroxidase avidin biotinylated complex kit (HRP-ABC) and diaminobenzidine (DAB) were used according to the manufacturer's instructions  to detect the secondary antibody. All washes used TBS except after DAB administration, where water was used. Sections were counterstained with haematoxylin and eosin (H&E) and cover slips were attached using a DAKO aqueous based mounting medium. Negative controls were obtained through the omission of the primary antibody to ubiquitin (that showed the absence of specific staining). Ubiquitin has increased expression in the normal endometrium in both the late proliferative and late secretory phase. In this study, late secretory phase tissue was used as a positive control .Currently, no single assay exists that detects apoptosis with high specificity and sensitivity . Thus thSections were deparaffinized and rehydrated with histolene and a graded series of alcohols (absolute, 95%, 90%, 80% and 70%) for 5 min each. This was followed by a 20 min incubation of sections with proteinase K [15 μg/ml in 10 mM Tris/HCl, pH 7.5] at room temperature. Sections were then rinsed twice in phosphate-buffered saline (PBS) and reacted with 50 μl of the TUNEL reaction mixture  for 60 min in a dark, humidified chamber at room temperature. The sections were then rinsed three times in PBS and incubated for a further 30 min with 50 μl of the Converter-POD  followed by 10 min with DAB. This procedure ensures the detection of TUNEL labelled cells. Finally, sections are counterstained and mounted as described for ubiquitin staining. For positive controls, sections were treated with DNAse 1 to induce DNA strand breaks or peroxidase blocking solution was excluded. Negative controls were achieved by omitting terminal deoxynucleotidyl transferase .The Department of Pathology, Westmead Hospital, kindly provided the H&E slides used for this study. The basis for this procedure is to identify cells with cell blebbing, nuclear condensation and cell shrinkage, which are characteristic features of apoptosis.The ubiquitin staining in the normal and endometriotic tissue was calculated using a semi-quantitative method to determine the average intensity scores of the protein in the nucleus of glands and the stroma using an Optimas Image Analysis program (Silver Spring, USA). Five randomly selected fields were viewed and evaluated with a grading of 0, 1, 2 or 3 (negative, weak, moderate, strong) according to a scale created by Watanabe and colleagues .TUNEL apoptotic cell numbers were determined by counting darkly labelled cells in five randomly selected fields at X400 and expressed as the apoptotic cell mean/field according to Meresman and colleagues .Ten randomly chosen fields at X600 magnification were used to determine the number of apoptotic stromal and glandular cells according to a modified grading scale used by Meresman and colleagues : (-) < 3This modified grading system was used to make statistical analysis more accurate, as there were only a few samples that exhibited > 8 apoptotic cells/field. Also, apoptotic cells were identified by their characteristic morphological features in H&E-stained late secretory endometrial sections. These included cell shrinkage and chromatin margination or chromatin condensation with formation of apoptotic bodies .Apoptotic bodies were identified by the presence of one, or several of the following features ,14:1) Single round mass of condensed strongly eosinophilic cytoplasm with a clump of strongly basophilic, homogenous chromatin.2) Cytoplasm with more than one piece of condensed chromatin.3) Condensed chromatin fragments without cytoplasm.All data is expressed as mean ± SEM. Comparisons between experimental groups were performed using the independent t-test and Mann-Whitney U test for parametric and non-parametric analysis, respectively. A P-value less than 0.05 was considered a significant difference between groups.Analysis of the effect of the menstrual cycle phase on glandular epithelial cell ubiquitin expression showed a greater level of ubiquitin during the proliferative phase than the secretory phase in controls  in contrast to ectopic stromal cells of endometriosis tissues where ubiquitin is higher during the secretory phase (P = 0.020; Figure A significantly higher level of apoptosis was observed using H&E in ectopic glandular cells of patients with endometriosis in comparison to controls in the proliferative phase (2.79 ± 0.43 vs 1.90 ± 0.15 respectively; P = 0.04; Figure Similar levels of apoptosis were observed within proliferative phase ectopic stromal cells of patients with endometriosis and controls using the H&E method (4.57 ± 0.85 vs 5.08 ± 0.50 respectively; P > 0.05; Figure Apoptotic cells are seen using the TUNEL technique Figure  and 4F. Ubiquitin is implicated in the removal of short lived, regulatory or misfolded proteins but is also widely known to play a part in DNA repair and removal of virus budding . The expIn this study, apoptosis was found to be present in endometriotic tissue with varying intensity. A previous study of endometriosis has also shown this result  but the Our data shows that the expression of ubiquitin is increased during the secretory phase of the menstrual cycle in both glands Figure  and stroThis study suggests that increased levels of ubiquitin within ectopic endometrial cells may allow their continued survival, through a yet to be established pathway. The up-regulation of ubiquitin in human endometriotic tissue may facilitate ectopic endometrial cell survival, particularly allowing those of stromal origin to grow, survive and evade T-cell mediated disposal. This finding is of particular interest because ubiquitin has primarily been attributed to the removal of aberrant proteins but seems, from our data, to be also associated with cell survival.This duality in ubiquitin's role may be attributed to the type of lysine residue linkage that occurs during polyubiquitination. Studies by Deng and colleagues  have shoOur data is consistent with the hypothesis that ubiquitin has a protective effect on ectopic endometrial cells, particularly those of stromal origin, as shown by the increased level of ubiquitin with an associated decrease in apoptosis during the secretory phase. However, a different mechanism may apply to glandular endometriosis, where despite a greater ubiquitin expression during the secretory phase, no significant association was found with ectopic glandular cell survival. This suggests that an insufficient ubiquitin tagging during the proliferative phase may cause ectopic glandular cells to undergo apoptosis.Other factors that may explain ectopic endometrial cell survival include the down-regulation of apoptotic receptors , failureThe significant increase in age of patients with endometriosis may have an unknown effect on the level of apoptosis as older women may have increased incidence of endometrial cell death due to increased longevity. However, this age difference is consistent with another study that also shows a higher age range in women with the condition .Our results suggest that ubiquitin is important for endometrial function throughout the menstrual cycle where it may play an important role in the regeneration of the endometrium . Also, aIn conclusion, this study demonstrates for the first time that ubiquitin is expressed in endometriotic cells. Furthermore, the up-regulation of ubiquitin expression may contribute preferentially to the development of these ectopic endometrial lesions due to their reduced sensitivity to apoptosis.RI was responsible for carrying out this study as part of her honours degree. CB provided RI with training in the techniques used. SF and CM were responsible for the conception and design of the study, research funding, and for supervision of RI's work. SF, CB and CM read and approved the final manuscript.


RescueMu system. The study provides a large resource of tagged and sequenced maize alleles, as well as some insights into the biology of RescueMu.The authors describe a large-scale transposon-tagging study in maize using the  Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.Derived from the maize  MuDR/Mu transposable elements are widely used for mutagenesis and as tags for gene cloning in maize [Mu insertional mutagenesis regulated by MuDR in highly active Mutator lines reflects four features of this transposon family. First, a plant typically has 10-50 copies of the mobile Mu elements [Mu insertion alleles in targeted screens [Mu-induced mutations for a chosen target gene is 10-3-10-5 [bronze1 exon [glossy8 [Mu insertion in specific regions, which may explain the higher frequency of mutable allele recovery for these genes.in maize ,2. The helements , althoug screens -6. In di0-3-10-5 . Interesze1 exon  and the [glossy8  contain Mu elements and the regulatory MuDR element become hypermethylated [Mu element mobility.Somatic mutability, visualized as revertant sectors on a mutant background, is indicative of transposon mobility. By monitoring maintenance of a mutable phenotype, it was established that the Mutator transposon system is subject to abrupt epigenetic silencing, which affects some individuals in most families ,11. A mothylated ,13. WithMu mutagenesis has been exploited in several reverse genetics strategies. The first protocol described used PCR to screen plant DNA samples to find Mu insertions into specific genes using one primer reading out from the conserved Mu terminal inverted repeats (TIRs) and a gene-specific primer [Mu insertions yields a list of tagged genes in each plant [RescueMu, a Mu1 element containing a pBluescript plasmid, to conduct plasmid rescue by transformation of Escherichia coli with total maize DNA samples. To identify insertions in genes of interest, RescueMu plasmids can be screened or the contiguous host genomic DNA can be sequenced using primers permitting selective sequencing from the right or left TIRs of Mu1 [The high efficiency of c primer -17. Altech plant ,19. A ths of Mu1 .RescueMu tagging effort conducted by the Maize Gene Discovery Project. The tagging strategy employed grids of up to 2,304 plants organized into 48 rows and 48 columns. Plasmid rescue was undertaken from individual pools of up to 48 plants per row or column. Genomic sequences next to RescueMu insertion sites were obtained for all the rows and for a subset of columns of six grids. Maize genomic sequences were subsequently assembled into 14,887 unique genomic loci using computational approaches. These loci were analyzed for gene content, the presence of repetitive DNA and correspondence to mapped maize genes and ESTs. Gene models were built by co-assembling the genomic sequence with ESTs and cDNAs by spliced alignment and by ab initio gene prediction. Identified gene models were tentatively classified using gene ontology terms of potential homologs [Here we describe the initial results of a large scale homologs .Mu element behavior have been examined previously using hundreds of tagged alleles or by analyzing the population of Mu elements in particular plants and a few descendants. With single founder individuals for the analyzed tagging grids, we could examine the distribution of new insertion sites of RescueMu in large progeny sets. The contiguous genomic sequences were analyzed to determine if there were insertion hotspots, preferential insertion site motifs, routine generation of the expected 9-base-pair (bp) direct target sequence duplication (TSD) and evidence of pre-meiotic insertion events.Many features of Mu elements, RescueMu exhibits a strong bias for insertion into or near genes, as few insertions were recovered in retrotransposons or other repetitive DNA. In addition, for the set of RescueMu insertions into confirmed genes, a bias for insertions into exons (rather than introns) was observed, consistent with the well-established use of Mutator as a mutagen. The gene-enrichment exhibited by RescueMu was compared against two physical methods of gene enrichment, methyl filtration [0t Cgenome fractionation [Like other ltration  and highionation .Mu1 elements maintain copy number through successive outcrosses, indicating that some type of duplicative transposition occurs [Mu insertion events .In standard Mutator lines, n occurs  in the an occurs . Most nen occurs ,27. Consd callus . Using etrRescueMu. By DNA blot hybridization of individuals within grids B through E, the RescueMu transposition frequencies ranged from 0.1 to 0.26 (Table trRescueMu were identified that had inserted into likely maize genes and generated the diagnostic 9-bp TSD characteristic of Mu transposition (data not shown). There were also events initially scored as transposition by blot hybridization that represented RescueMu rearrangements within the transgene array, and deleted forms of RescueMu were detected by blot hybridization and gel electrophoretic sizing of rescued plasmids (data not shown). Although RescueMu insertion frequency was low, overall Mu movement was very high in these grids; visible, independent seedling mutations were identified in 10.1-28.3% of the selfed progeny . Four plants each with a single trRescueMu were identified by these criteria and crossed to r-g. A DNA blot hybridization screen was conducted on 393 progeny of these four individuals. Seven progeny were identified with two new trRescueMu, seven plants were identified with three events, and 33 plants had a single trRescueMu; the original, parental trRescueMu elements were shown to segregate as Mendelian factors in the populations screened (data not shown). The 14 plants with two or three new trRescueMu were each crossed by an anthocyanin tester and also crossed multiple times as pollen parents to tester lines to generate sufficient progeny to construct one grid from each founder plant. Inexplicably, in sampling seedling progeny from each outcross ear, some lineages had very few new trRescueMu. The lines with the highest transposition frequencies had two trRescueMu and were used in grids G through J; DNA blot hybridization analysis of 30-200 grid plants was used to estimate transposition frequencies within each grid, which ranged from 0.38 to 0.66 . The resulting library plates contain 56-96 individual row and column libraries representing the diversity of germinal trRescueMu and a sampling of somatic events present in the harvested leaf tissue (each well in a library plate is a pool of 20-48 plants from a row or column). The parental RescueMu insertion sites inherited from the grid founder(s) are present in every library.As shown schematically in Figure RescueMu shows a strong bias for insertion into genes [trRescueMu insertion sites , with a range of 0.4-15 kb (data not shown). To accommodate the large size of some plasmids, a PCR template preparation protocol was devised to amplify genomic inserts of up to 16 kb for high-throughput sequencing [RescueMu insertion sites. Plasmids from all rows plus several columns of a grid were sequenced, with a routine yield of 80-92% success. A subset of plasmids could not be bidirectionally sequenced, because they lacked the TIRs at one or both ends. Deleted forms of trRescueMu were detected in several percent of the individuals surveyed by DNA blot hybridization  for each grid, and 59% (17,239) were recovered multiple times . In addition, a total of 24,875 parental plasmids were transmitted from the founder plants. The percentage of parental plasmids within each grid varied from 17% for grid G to 61% for grid P. Some grids had more parentals than other grids and some parental plasmids were preferentially sequenced for unknown reasons. The parental insertion sites include the two or three known parental sites that each segregated into 50% of the progeny. Somatic sectors in the tassel or ear of the parental plant that generated plasmids found in multiple individuals within the grid are analyzed in a later section.Previous analysis of ecovered . Out of trRescueMu, equivalent to a frequency of 0.68 new insertions per plant. Using a Poisson model based on this transposition frequency for an individual grid ). By PCR screening of library plates containing the immortalized row and column plasmids, plants containing a specific insertion event can be verified .RescueMu insertions occur in diverse gene types. Illustrating the utility of Mu tagging, insertions are found in housekeeping genes, such as actin, as well as in regulatory genes such as those for transcription factors and protein kinases. Using the database of mapped maize genes and expressed sequence tags (ESTs) [RescueMu insertions are identified in genes on all 10 maize chromosomes [Mu insertions using DNA blot hybridization that established that these elements insert throughout the genome and do not show a measurable bias for insertion locally [RescueMu insertion sites that match maize ESTs correspond to genes of unknown function, suggesting the discovery of novel genes.As shown in Figure s (ESTs) , RescueMomosomes . These dRescueMu insertion sites identified in six grids (multiple insertions into a gene from the same grid being counted only once because the majority are the same insertion event), 88% represent single instances of transposon insertion locations. There were 596 instances of a specific genomic sequence having two or more RescueMu insertion events. If the maize genome contains 50,000 distinct genes that are targets of Mu insertional mutagenesis, then far fewer cases of duplicate recovery would be expected by chance alone, given the number of events analyzed (p < 0.001); therefore, RescueMu exhibits some preference for particular genes.Of the 14,887 RescueMu insertion within particular genes, data were compared between grids with independent founder individuals. As summarized in Table RescueMu insertion sites were found in just one grid. This was true for both probable germinal insertion events (plasmids found two or more times within a grid) as well as for singlet sites . The 10% of insertion sites found in two or more grids represent independent recovery of a RescueMu insertion into the same locus.To determine if there were 'hotspots' for et al. [Mu insertions at glossy8 were in the 5' untranslated region, with 15 insertions at the same base; similarly, the beginning of exon 2 within bronze1 is the most frequent site of Mu insertion in that gene [In addition to the computational comparison in which an overlap of 50 bases (95% identity) was scored as insertion into the same gene, over 730 insertion sites were examined manually for 250 cases of genes with insertions from more than one grid. Of these insertion sites, 80% were at different locations within the same locus; we found 85 cases of insertions within a 1-10 bp region and 67 cases of insertions at the same base. Previously, Dietrich RescueMu contig from the Genomic Survey Sequencing (GSS) section of GenBank, ZM_RM_GSStuc03-10-31.4765 [RescueMu insertion, with six plasmids sequenced from row 42 of grid G and one each from grids H, I, and M. Insertion sites were identical across the grids. Sequences generated to both the left and right of the RescueMu element were aligned as demonstrated in Figure RescueMu insertion site was recovered multiple times in grid G, a heritable insertion may exist. After PCR screening of grid G plasmid libraries, summarized in Figure RescueMu insertion site, total leaf DNA was extracted from selfed seed of this plant, namely G 42-22, obtained from the Maize Genetics Cooperation Stock Center. PCR screening of the DNA  or a single missing or additional base. Given that all sequences were single pass but of high average quality , we consider that 9-bp TSDs exist in virtually all trRescueMu insertion sites. A few cases showed anomalies in the TSDs, which probably reflect rearrangements near RescueMu.Because a 9-bp TSD is characteristic of  Figures , 3; notesequence ,35; thesMu based on smaller data sets [RescueMu insertion regions [et al. [RescueMu targeting is at least partially dependent on sequence features. In addition, we have compared the profiles derived independently from insertion sites within confirmed exons, introns and uncharacterized regions, respectively, and found the same base preferences in all three sets (data not shown).Several groups have reported weak consensus insertion site preferences for ata sets ,18,20. W regions . The pro [et al. , showingRescueMu insertion sites that do not match an EST or gene in another species represent introns or other non-coding genic regions. On the basis of the gene structure annotated by maize EST matching, we have located 968 TSD sites within genes. Of these, 849 are inside exons. To check if RescueMu has a preference for insertion into exons , a standard binomial test with normal approximation was performed. On the basis of the matching to ESTs, the lengths of all exons and introns observed from all RescueMu contigs were counted as 2,182,954 bp and 439,403 bp, respectively. Assuming that RescueMu does not have a preference to insert into exons (null hypothesis), the probability of observing an exon insertion event is proportional to the length of exons . The probability of observing at least 849 exon insertion events was calculated . This result suggests that RescueMu has some preference to target exon regions within genes.Of 14,887 genomic loci, 62% matched maize or other plant EST/cDNAs. As more genomic sequence becomes available that can be assembled with ESTs to annotate the non-coding portions of maize genes, it will be interesting to determine if the RescueMu GSS sequences were scanned and masked for repetitive elements as collected in The Institute for Genomic Research (TIGR) Cereal Repeat Database [0t Cselection (HC) using the same repeat-masking criteria [RescueMu, MF and HC, respectively.As outlined in Materials and methods, the Database . The repcriteria . The perRescueMu shows similar repeat content as the physical enrichment methods. However, after we assembled the RescueMu GSS sequences to remove redundancy, only about 3% of the RescueMu loci are composed of repetitive DNA  from TIGR [RescueMu and HC have similar bias against repetitive DNA, superior to the MF bias. It should be noted, however, that the MF and HC GSS sequencing has generated, on average, much longer contigs than RescueMu .Therefore, on the nucleotide level, rom TIGR  and applRescueMu insertions were found in either the approximately 10,000 copies of the 9.1 kb 28S + 18S rRNA genes [In addition, only 0.4% of the NA genes  comprisiRescueMu loci match strongly to maize or other plant ESTs or appear to encode proteins with high similarity to known proteins. In addition, about another 5% of the loci were predicted to be genic regions with high stringency by ab initio gene prediction programs. As a control, we matched ESTs to contigs assembled from unfiltered (random) maize GSS sequences [RescueMu contigs contain more than threefold enrichment of genic regions than random sequencing. This is consistent with our expectation that RescueMu preferentially inserts into genes. It is worth pointing out that plant EST collections contain ESTs from repetitive elements. Although we masked contigs using the annotated TIGR repeat database [RescueMu results, because these authors used much higher stringency for the EST spliced alignments with the BLAT program [RescueMu data, which tolerates less sequence matching without compromising gene structure prediction accuracy [RescueMu, MF, and HC are very similar (data not shown).Also shown in Table equences . From abdatabase , it is pdatabase . However program , requiriaccuracy . The reset al. [RescueMu by comparing the respective sequence sets to rice gene models. They concluded that unique gene discovery is most efficient with MF at a sequencing depth when EST sampling saturates. However, their reported low gene discovery rate for RescueMu does not reflect the RescueMu insertion bias, because their dataset included all sequences deposited in GenBank. That is, they did not remove the redundancy resulting from multiple sequencing of parental insertions.Palmer et al.  evaluateRescueMu insertion sites were found in two or more row libraries  from female transmission. Given that the founder plants produced copious pollen, there is a low probability that two grains carrying the same meiotic insertion will both result in seed; therefore, the same RescueMu insertion site found in two rows should usually be from a pre-meiotic transposition event. For all events found in three or more rows, the insertion event must be pre-meiotic.To calculate the extent and timing of pre-meiotic sectors, the sequenced plasmids from grids G, H, I, K, M and P were classified as occurring in a single row or in multiple rows. The development of the tassel and ear must be considered when evaluating these data. An insertion event that occurs during meiosis can be represented in two haploid cells. During microgametophyte (haploid plant) ontogeny, both of these cells survive, resulting in two pollen grains with the same event. In contrast, only one megagametophyte develops after megaspore meiosis; therefore, female meiotic and subsequent events in the haploid megagametophyte are always represented in just one progeny plant. Most grid plants resulted from male transmission of RescueMu insertion event very early in the somatic development of the inflorescence or in the apical meristems. The majority of trRescueMu insertion sites are found in only one row . The nuRescueMu insertion sites. Only about 8% of the RescueMu insertion sites match repetitive elements and few insertions (0.4%) were recovered in genes transcribed by RNA polymerase I or III. These results suggest that a chromatin component associated with polymerase II transcription units or the absence of a structure in other classes of genes is important in targeting RescueMu and other Mu elements to maize genes. Similarly, recombination during meiosis and transcription per se is targeted to genes. It is likely that the parasitic Mu elements exploit an element of host gene packaging that evolved for other reasons to facilitate transposition into genes.Given that the maize genome comprises approximately 70% retrotransposons and other highly repetitive sequences, including around 10,000 copies of the rRNA genes , these cRescueMu is close to methyl filtration and high 0t Cfractionation. The probable germinal insertion class defines a collection of mutations of enormous potential for the phenotypic characterization of maize with specifically disrupted functions. However, the low cost of template production is a distinct advantage of both physical enrichment methods compared to the high cost of designing, sampling and self-pollinating tagging grids. Current levels of sample sequencing from the physical enrichment templates highlight the desired redundancy of the RescueMu method, which is important for distinguishing somatic from germinal insertions at individual loci. The physical enrichment methods are considerably below one times coverage of the transcriptome of around 250 Mb; hence the current efficiency of generating novel sequence (the likelihood that the next clone sequenced is new) is much higher with these methods than with RescueMu.The biological specificity for maize genes exhibited by RescueMu insertion site data, several parameters of Mu transposition behavior were investigated. We confirm that a 9-bp TSD is characteristic of virtually all Mu insertion sites. We confirm that a small percentage of trRescueMu suffer deletions, including loss of a TIR, as noted in previous studies of Mu1 [Mu insertion sites [RescueMu insertion sites is consistent with the previously proposed motifs. A bias exists for G+C-rich sequence, reflecting the composition of maize exons. We confirm that there are hotspots for Mu insertion, identified by finding identical trRescueMu insertion sites in independent grids. A few loci were recovered in four or more of the six grids analyzed, and many more in two (1,295 genes) or three (233 genes) grids. There is no strong DNA consensus motif at these hotspots, and we consider it more likely that a specific DNA structure or a protein associated with genes establishes conditions for efficient Mu insertion at particular sites. It is important to note that active transcription is not a requirement for Mu element insertion; otherwise Mu would preferentially insert into genes active late in floral development and in gametophytes.Using the s of Mu1 . Throughon sites , consenstrRescueMu insertion sites represent a mixture of non-heritable somatic insertions present in leaves, germinal insertions in single grid individuals, insertion events in pre-germinal sectors within flowers, and parental elements. Parental elements identified in a grid founder plant segregated 1:1 in the progeny as expected. In addition, some insertion events were found in three or more grid rows, and hence in three or more individuals, and must be pre-meiotic transposition events in the founder. This class represented 10.2% (321/3,138) of all the likely germinal insertions identified  defining predictable insertion locations. RescueMu properties confirm attributes established with smaller populations of standard Mu elements.A public resource of transposon-tagged maize alleles was constructed and evaluated. RescueMu contains all of Mu1 plus a 400-bp segment of Sinorhizobium meliloti and pBluescript (Stratagene), as described previously by Raizada et al. [RescueMu was obtained in this study using PCR primers to amplify overlapping sections of the element [RescueMu transposon was placed in the 5' untranslated region of a 35S:Lc expression plasmid where it blocked expression [Lc is a member of the R family of transcriptional regulators of the anthocyanin pathway [r-r/r-g, A1, Bz1, Bz2) hybrid background were crossed to r-g testers and subsequently with r-g Mutator lines containing multiple copies of MuDR to visualize RescueMu somatic excision as red anthocyanin sectors in an otherwise white aleurone. The tagging populations used here were developed by screening for transposition of RescueMu from the original, complex transgene arrays to diverse genomic locations. Using DNA blot hybridization, these once-transposed RescueMu (trRescueMu) were closely monitored for subsequent transposition, and lines were monitored for Mu1 and/or MuDR methylation in the TIRs, a sign of incipient Mutator silencing. Details of line development and evaluation, including DNA blot hybridization methods, will be presented elsewhere. The anthocyanin tester lines (recessive for r-g, a1, bz1 or bz2) were in inbreds W23, K55, A188, or hybrid combinations of these lines. Some RescueMu lines used in tagging grids were crossed to inbreds A619 or B73, which are both r-g, A1, Bz1, Bz2. Grid backgrounds are presented in detail at [a et al. . The com element  for bidipression . Lc is a pathway . Transgeetail at .BamHI plus BglII digestion of the genomic DNA preparations. These libraries were immortalized in library plates available from the project [RescueMu; although the primers were selective for one TIR, there was some cross-priming. All grid rows plus several columns were sequenced. Three 96-well plates were normally sequenced for each row or column to obtain sequence information for a desired minimum of 200 plasmids; additional sequencing reactions were conducted if necessary. Matches of row and column sequences are designated as probable germinal insertions, because they represent an insertion site present in two leaves of that plant (designated by its row and column address); when only row sequences were available from a particular plasmid, probable germinal insertions were designated after recovery of the same sequence two or more times. Plasmid types recovered just once are a mixture of heritable and strictly somatic insertions. Parental RescueMu insertion sites present in a grid founder plant segregated in the grid progeny, and these insertion sites were expected to be found in all rows and columns. In some cases, particular parental plasmids were over-represented in the sequenced plasmid population. To reduce their contribution and increase recovery of new insertion sites, a rare-cutting restriction enzyme site was identified in the parental plasmid and the corresponding enzyme was included in the genomic DNA preparation to bias against recovery of that parental plasmid.Detailed protocols are presented at , and a s project . Plated RescueMu insertion hotspot Six gene primers plus one RescueMu left readout primer were used in this study:PCR screening of a library plate to quantify a 1. 5'-TTGGGAGGTTGAAGGTAAAGACAT-3'2. 5'-GTGCTG GATTGGTTACTCCG-3'3. 5'-CGATGATTCTAGTTGAGCGTCTG-3'4. 5'-ACTCGCACCAACATGAATACC-3'5. 5'-GTTTCCGAGGACGCAGAGG-3'6. 5'-AGCGCCAGGGCCAGGGGATTC-3'RescueMu)L. 5'-CAT TTC GTC GAA TCC CCT TCC-3' , AGATCT (BglII), or a GGATCT (BglII/BamHI) or AGATCC (BamHI/BglII) motif. The two restriction digestion sites represent a true ligation site of sequence that was non-contiguous in the maize genome, but the post-restriction site sequences can unambiguously be assigned to the right or the left of RescueMu. On the other hand, the GGATCT or AGATCC motif could be contiguous genomic sequence or could have been generated during the ligation step of the plasmid rescue. Consequently, assignment of the position of the sequence beyond the GGATCT or AGATCC motif is ambiguous. If the RescueMu insertion site matched EST sequence across and beyond the GGATCT or AGATCC motif, the post-ligation sequence could be properly assigned  per plasmid.As shown in Figure nsertion , the seq GenBank , for com program  to deterRescueMu GSS sequences deposited at GenBank were screened for vector sequences against the UniVec database at the National Center for Biotechnology Information (NCBI) [RescueMu-transformed plant contains the parental RescueMu elements that were recovered at a high frequency during sequencing (from every sequenced row or column). Because our goal is to analyze the gene discovery by newly inserted RescueMu , we decided to filter out the parental sequences as much as possible. We used Vmatch to cluster near-identical left and right sequences for each grid. A parental cluster contains sequences from nearly all the row or column sequences. A total of 59,069 parental sequences were identified and were excluded from the subsequent assembly. All the non-parental sequences were first preassembled for each plasmid using the left and right 9-bp TSD overlap. The merged GSSs were first clustered by PaCE [RescueMu insertion sites. For the particular case where TSDs were not recovered during sequencing, the left and right sequences could not be assembled together, even though they were from the same plasmid. Therefore, a Perl script was developed to conduct single-linkage clustering based on clone-pair constraints to assemble the GSS to the same 'genomic loci' if they were derived from the same plasmid clone.The 131,364 n (NCBI)  using thn (NCBI)  using thn (NCBI)  with the by PaCE   was used in combination with the TIGR cereal repeat database (version 2 consisting of maize, rice, barley, sorghum and wheat repeats). The contigs were also scanned from tRNA genes by tRANscan-SE program [To be successful as a gene-discovery tool, the transposon insertions must be predominantly into the genic regions of the maize genome. To quantify the potential enrichment of the  program  with itsab initio approaches have been used to detect gene structures of the GSS contigs. For the similarity-based approach, GeneSeqer [ab initio prediction, GENSCAN [Both similarity-based and eneSeqer  programseneSeqer . For the GENSCAN   was derived from the sequences and their reverse-complement orientation determined using the Expectation Maximization Algorithm [For the analysis of lgorithm .The GSS seqeunces present in each tentative unique contig (TUCs) were extracted from  and assiRescueMu flanking sequences where established. Instructions on how to obtain seed of grid plants is also provided.Grid plants were self-pollinated unless male or female-sterile. The resulting F1 families were evaluated by inspection of ears and kernels, at weekly intervals for five weeks after germination in a sand bench in a greenhouse, and at weekly intervals throughout the life cycle in the field. Phenotypes observed were recorded and are assembled into a searchable database at . Unique RescueMu (Additional data file The following additional data are available with the online version of this article: a table listing the internal primers used in sequencing RescueMuA table listing the internal primers used in sequencing Click here for additional data fileSupplementary material for this paper, including details of methodsClick here for additional data file


Longitudinal assessments of quality of life are needed to measure changes over the course of a disease and treatment. Computer versions of quality of life instruments have increased the feasibility of obtaining longitudinal measurements. However, there remain occasions when patients are not able to complete these questionnaires. This study examined whether changes measured using a computer version of the Functional Assessment of Cancer Therapy – General (FACT-G) on two occasions would be obtained if patients completed a paper version on one of the two occasions.Gynecologic oncology patients completed a computer version of the FACT-G pre-operatively and at six months. Patients were given the option of using the paper version instead of the computer at either time point. Repeated measures analysis of variance was used.One hundred nineteen patients completed the FACT-G at both time points. Seventy-one (60%) patients used the computer at both visits, 26 (21.8%) used the computer followed by the paper version, 17 (14.3%) used the paper version followed by the computer version, and five patients (4.2%) used the paper version at both visits. Significant effects over time were obtained in the physical, functional, and emotional well-being domains, and in total scores, but there were no effects of method of administration of the questionnaires and no interaction between method of administration and changes over time.These data indicate that women are responding to the content of the questionnaire and not method of data collection. Although using the same method of administration of instruments over time is desirable, using alternate methods is preferable to forgoing data collection entirely. Large scale studies should be conducted to determine if the multiple methods of data collection that are becoming increasingly available are producing interchangeable information. Measurement of changes in quality of life (QoL) has become a standard outcome variable in evaluating different therapeutic regimes in cancer -3. StandComputer versions of these questionnaires have become available and can be used for longitudinal assessments -17. ThesA potential barrier to longitudinal measurements is that compliance may decrease over time. Patients may be initially willing to answer questions on the computer, but be less willing to do so on subsequent visits ,17. ReasWomen attending a gynecologic oncology practice were enrolled in a longitudinal study of QoL. Women completed a computer version of a QoL questionnaire pre-operatively and again at six months. They were given the option of using the paper version at either time point, and the effect of this choice was examined. An additional issue examined was whether use of the paper version was widespread or sporadic. The goal of the study was to compare changes over time obtained when women used a touch-screen computer on two occasions with changes obtained when women used a paper version of the questionnaire on one of the occasions.Patients who were scheduled to undergo surgery for endometrial cancer, ovarian cancer or an adnexal mass were invited to participate in a long term study of QoL, complementary medicine use and diet. Women at two gynecologic oncology offices in Northeast Ohio were recruited from 2001 – 2003. Informed consent was obtained for participation in this IRB approved study. Private office records and hospital discharge records were reviewed to abstract demographics and final pathology diagnosis. Baseline demographics were ascertained by interview with a research assistant pre-operatively. Patients completed the questionnaire pre-operatively and again at six months.Computer kiosks with a 15 inch monitor were programmed with the Functional Assessment of Cancer Therapy-General questionnaire (FACT-G) along with an additional fatigue module . The FACPatients were categorized into four groups; those who completed the FACT-G on the computer on both occasions (CC), those who completed the initial assessment by computer and used paper format at six months (CP), patients who completed the initial assessment on paper and the six-month via computer (PC) and patients who completed both assessments on paper (PP). Analysis of variance or chi-square statistic was used to compare baseline demographic variables between patients who always used the computer and those who utilized the paper version at either time point.Repeated measures analysis-of-variance was used to analyze change in the domain score from baseline to six months (time effect), whether there was an effect of group (CC, CP, PC and PP) and whether there was an interaction between group and time. Significance was set at p < 0.01 due to multiple comparisons. SPSS version 10.0 was used for analysis (Chicago, IL).A total of 187 patients were asked to participate in this longitudinal study and 151 agreed (81%). Following completion of the initial assessment, 32 patients were lost to follow-up, moved, missed the second appointment entirely or refused to complete the questionnaire the second time . A total of 119 patients (79% of patients who agreed to participate in the study) completed the FACT-G assessments at both time points. Forty patients had endometrial cancer, 40 had ovarian cancer and 39 had a benign adnexal mass. Twenty of the cancer patients had Stage III or IV disease. Virtually all of the patients were Caucasian (96.6%).Patients returned the questionnaire by mail within a few days of their scheduled visit. Seventy-one (60%) patients used the computer at both visits (CC), 26 (21.8%) used the computer initially followed by the paper version at six months (CP), 17 (14.3%) used the paper version initially followed by the computer version (PC), and five patients (4.2%) used the paper version at both visits (PP). Patients in the PP group were excluded from statistical analyses as the numbers in that group were small (n = 5).2 = 2.75, p = 0.60) between the CC, PC and CP groups (Table 2 = 11.07, p = 0.026) as they were more likely to decline to come in for an office visit and request the FACT-G be sent home than were the patients with a cancer diagnosis (Table There were no differences in the age (F = 0.225, p = 0.80) or level of education . The one significant interaction effect was observed with the social well-being domain, which appeared due to a high baseline score in the CP group. At baseline, the CP group was the same as the CC group  so it is not clear why there would be a high baseline score in the group that would use a paper version six months later. It is possible that with the number of tests conducted, one spurious finding would be obtained. The trend across all the tests is very strong, however. There are clear and significant changes with time but not with the method of obtaining the data.Given the choice between using the computer version and the paper version, a small number of women chose the paper version. Of the 238 total measurements, the paper version was used a total of 53 times (22%). Reasons for not using the computer included not wanting to come in to the physician's office at all and patient preference but also instances beyond the patients' control such as scheduling complications and researcher unavailability on a small number of occasions. Designing strategies to increase computer availability may result in further reductions in patient use of the paper versions. If patients can log onto the computer using a unique identifier and complete the questionnaires on their own in the waiting room, the number of women who have to take questionnaires home or forgo completing them should decrease even further.The second assessment occurred six months following major surgery for all women. The majority of women with ovarian cancer received chemotherapy, but were not receiving it at six months. This time point therefore allows a relatively stable point to assess changes in QoL relative to pre-operative scores in these groups of women. It is possible that differences in method of data collection would be obtained if women were acutely ill at the time of measurement, however the time frame of seven days used in the FACT-G reduces the likelihood that a separation in time of a day or two between using the computer in the office or the paper version at home will result in different responses. The time frame used in the FACT-G, and the relatively stable time point chosen may therefore contribute to the lack of measurement effect obtained in these groups of women.A limitation of this study is the lack of minority representation which may reduce the generalizability of these results. Additionally, 19% of patients refused to participate in the study. Of the patients who did participate, 21% did not complete the second assessment, although this figure includes 16 women with a benign adnexal mass who may have returned to their referring physician, and women with cancer who moved or transferred their care. Nonetheless, the women who remained on study may differ from those who did not agree to participate or who did not complete the second assessment. They may, for example, have a greater degree of commitment to the research process.A second limitation is that women were not randomly assigned to use either the computer or the paper versions. This is a preliminary examination of existing data to determine whether there appeared to be a selection bias, or major effect, of using the paper version. Women with QoL scores that differed markedly from the norm, for example, might have chosen to take the paper version home. This did not appear to be the case, however, as highly significant effects of time were observed, but group and interaction effects were markedly non-significant. Related limitations include the remote, but possible, explanation that the first method of administration had an effect on participants at the second time point. Additionally, patient choice itself may have had an unmeasured effect. For example, women with benign adnexal mass were more likely to forego the second office visit and complete the questionnaire at home. Disease and questionnaire administration are therefore confounded. These limitations may have influenced group choice, as well as responses on the second measurement.These exploratory data suggest that women are responding to questionnaires presented on a computer in the same manner as questionnaires on paper. This study therefore differed somewhat from studies that found differences in method of administration ,20. An iLongitudinal measurements of health- related QoL are increasingly used in cancer patients. This study examined whether two different methods of measuring QoL (computer and paper) would provide interchangeable data. It appears that patients are dealing with issues of significant concern and they are responding to the content of the questions and not the method of data collection. It is clearly desirable to standardize the method of data collection and have conditions remain constant across time. The results of this study, however, demonstrate that valid data are obtained with alternate methods of data collection and this is preferable to foregoing data collection entirely.KG and VVG conceived of the study, and participated in its design and coordination. HF, MH, EJ and VVG implemented the study and were responsible for day to day conduct of the study. KG and HF analyzed the data. KG, HF, VVG drafted the manuscript; JE and MH provided critical review. All authors read and approved the final manuscript.