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 . 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  were previously constructed -15. Flow51Cr release assay × 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  , 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 . 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 , placed on superfrost slides  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  for 5 min each. This was followed by a 20 min incubation of sections with proteinase K  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 . Five randomly selected fields were viewed and evaluated with a grading of 0, 1, 2 or 3  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%  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  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%  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  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 . 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  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 .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  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  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.


Due to the complexity of health system reform in the post-conflict, post-disaster, and development settings, attempts to restructure health services are fraught with pitfalls that are often unanticipated because of inadequate preliminary assessments. Our proposed Integrated Multimodal Assessment – combining quantitative and qualitative methodologies – may provide a more robust mechanism for identifying programmatic priorities and critical barriers for appropriate and sustainable health system interventions. The purpose of this study is to describe this novel multimodal assessment using emergency medicine in post-conflict Serbia as a model.Integrated quantitative and qualitative methodologies – system characterization and observation, focus group discussions, free-response questionnaires, and by-person factor analysis – were used to identify needs, problems, and potential barriers to the development of emergency medicine in Serbia. Participants included emergency and pre-hospital personnel from all emergency medical institutions in Belgrade.Demographic data indicate a loosely ordered network of part-time emergency departments supported by 24-hour pre-hospital services and an academic emergency center. Focus groups and questionnaires reveal significant impediments to delivery of care and suggest development priorities. By-person factor analysis subsequently divides respondents into distinctive attitudinal types, compares participant opinions, and identifies programmatic priorities.By combining quantitative and qualitative methodologies, our Integrated Multimodal Assessment identified critical needs and barriers to emergency medicine development in Serbia and may serve as a model for future health system assessments in post-conflict, post-disaster, and development settings. In June 1991, a series of civil wars began as the Socialist Federal Republic of Yugoslavia dissolved violently into the four independent republics of Slovenia, Croatia, Bosnia-Herzegovina, the Former Yugoslav Republic of Macedonia, and the semi-autonomous nations of Serbia and Montenegro. Kosovo remains a province of Serbia but under the administration of the international community. The events of the last decade in the Balkans have significantly impacted the health care system of Serbia. The nation and its health care system were devastated by international sanctions, soaring unemployment, political instability, near economic collapse, and the North Atlantic Treaty Organization (NATO) air campaign .A country's emergency medical services are a particularly important component of the health care system for they provide an essential front-line resource for trauma, medical emergencies, and those without other access to health care. Unfortunately, the system of emergency services in Serbia did not escape the devastation of the 1990s and continues to suffer from problems common throughout the health care system. Limited health care funding has resulted in a lack of necessary equipment, supplies, medications, personnel wages, and an economic infrastructure unable to support necessary health care reforms in the public and private sectors. Furthermore, the country has received little external support for health system reform, underscoring the need for focused assessments to best determine priorities for health care development.There is a desperate need for reorganizing and restructuring emergency medical services throughout Serbia. To better understand the problems, needs, and obstacles to development, a team of investigators from Johns Hopkins University Schools of Medicine and Public Health collaborated with the Belgrade University Emergency Center to perform a multimodal assessment of emergency medicine in Serbia. Our goal in this study was to identify the most pressing needs and achievable goals, as perceived by emergency health care personnel, in order to ensure that development programs and available funds are appropriately directed .Evaluating health care systems through the use of demographic data collection, surveys, or focus groups alone – as is often done in health system assessments – cannot adequately elucidate the intricacies of a country's health care system. In order to fully appreciate the complex dynamics of the health care system, it is necessary to perform a complete initial assessment; include strong local participation; identify and address significant barriers to change; and identify unique local needs and other cultural dimensions. To do this effectively, it is important to avoid simply introducing a "carbon copy" of a Western health care system that would further burden the system with inappropriate and unsustainable programs. We propose for the rapid assessment of health care services an Integrated Multimodal Assessment that uniquely combines diverse methodologies, whose individual effectiveness has been well described in the literature.This study used a multimodal approach to assess the strengths, needs, problems, and obstacles related to the development of emergency medical services in Belgrade. The four study modalities were 1) demographic information and observational data; 2) focus group discussions with 68 emergency medicine personnel; 3) individual, free-response questionnaires with 39 emergency medicine personnel; and 4) by-person factor analysis (or Q-methodology) of the attitudes and opinions of 36 emergency medicine personnel regarding the development of emergency medical services. Participants in the study were a non-probabilistic sampling of emergency medicine physicians, nurses, and department directors from all pre-hospital and hospital-based emergency medicine institutions in Belgrade. Many participants took part in more than one study modality. Data collection occurred during July-August 2002. The authors received institutional review board approval and the written informed consent of all participants.The capacity of emergency medical services in Serbia to provide for the needs of the country's citizens was characterized through health system data collection. Health system information was obtained through meetings with key officials in established health surveillance institutions such as the Emergency Center of the Clinical Center of Serbia; World Health Organization Belgrade office; Public Health Institute of Belgrade; Republic of Serbia Ministry of Health; European Agency for Reconstruction; Belgrade University, Institute of Social Medicine, Statistics and Health Research; as well as several non-governmental organizations with offices in Belgrade. Key officials were asked a series of quantitative, closed-response questions about the Serbian health care system and emergency medical services. Developed from a pilot health care assessment in Serbia the previous year, the questions focused on health system organization; patient population size and demographics; health care personnel; medical education and training; materials, supplies, and medications; and provider aptitude and morale .The two-fold purpose of the focus group discussions was 1) to better understand the needs, problems, and obstacles to system development as perceived by those involved directly in providing emergency medical services and 2) to collect a wide variety of opinions and attitudes for subsequent by-person factor analysis. The focus group discussions were held with 68 emergency medicine physicians, nurses, and directors through a non-probabilistic sample taken from the institute for pre-hospital emergency services, the Emergency Center of the Clinical Center of Serbia, and four of Belgrade's six community emergency departments. The discussions were conducted by the primary author in the Serbian language. The audio recordings of the 30- to 60-minute discussions were transcribed and translated into English by a native-English speaker fluent in Serbian (BDN) and a native-Serbian speaker fluent in English (MŠ). The translation was later verified by additional native-Serbian speaking co-authors fluent in English .The investigative field team  summarized the final transcripts and extracted common themes and divisive statements for by-person factor analysis.Free-response questionnaires enabled assessment of emergency health care needs and priorities for change as perceived by those most directly involved in patient care: the emergency health care providers. A non-probabilistic sample of 39 participants was obtained by open invitation, without compensation, through the director's office of each institution. As with the focus group discussions, the institute for pre-hospital emergency services, the Emergency Center of the Clinical Center of Serbia, and four of the city's six community emergency departments were represented. All subjects were administered a written Serbian-language questionnaire comprised of 10 free-response items  were designated respondent type "definers" and closely reviewed to assist in characterizing each respondent type.By-person factor analysis, or Q-methodology, avoids many of the limitations of other modalities by allowing the grouping of participants based on their subjective responses to an issue while preventing investigator preconceptions from influencing the grouping structure. In a letter to ectivity ,7. In ouectivity  followedzdravstvene stanice) and primary health centers (domovi zdravlja). In the capital city of Belgrade, there exists a network of 6 part-time, hospital-based (kliničko-bolnički centri) emergency departments that are supported by around-the-clock pre-hospital emergency services (hitna pomoć) and the academic Emergency Center (Urgentni centar) at the Clinical Center of Serbia. Like many European countries, Serbia has adopted a largely Franco-German model of emergency medicine in which pre-hospital emergency services are provided in the field by physician-staffed ambulances [Emergency medicine is not a recognized health care specialty in Serbia. In smaller communities, emergency services are provided by primary health stations , internal medicine (29%), anesthesia (12%), general medicine (7%), emergency medicine (3%), other (15%)). Discussions were held at the institute for pre-hospital emergency services, the Emergency Center, and four hospital emergency departments.The participants' comments from the focus group discussions are collectively summarized in Table In addition, physicians and medical directors report that their emergency medical system is in significant need of improved education and training programs. Suggested improvements include further training in emergency medicine during medical school, bedside training to develop practical skills, development of standardized treatment protocols, and access to continuing professional education.Questionnaires were completed by 39 physicians at the institute for pre-hospital emergency services, the Emergency Center, and four hospital emergency departments , internal medicine (31%), anesthesia (13%), general medicine (10%), emergency medicine (5%), other (10%)).Respondents show several areas of high agreement – particularly remarkable considering the questionnaire's free-response format. The most frequently reported strength of Serbia's current health care system are the health care workers (69%). Worker enthusiasm (33%) and the collaboration of various medical specialties providing emergency services (21%) are most frequently cited as the greatest positive attributes of emergency service providers. In addition, 46% of respondents report that the various emergency medical service providers  are other strengths of emergency medical services in Serbia.Thirty percent of the participants surveyed believe that lack of incentives to enter the specialty, difficulty of the work, and poor financial compensation are important barriers to becoming a physician in emergency medicine in Serbia. The poor reputation of physicians in emergency medicine relative to other physicians (reported by 46% of participants) may contribute to lower interest in specializing in emergency medicine. Poor organization (26%) and insufficient opportunities for professional development (15%) were also reported as major impediments to being an emergency service provider.Participants were asked what they believed were major problems of emergency medical services in Serbia. Inadequate finances, medications, medical supplies, and modern medical equipment were cited by 54% of the emergency medicine personnel as being a major problem , the absence of treatment protocols (27%), insufficient numbers of emergency medical physicians (17%), and the lack of government regulation (13%) were the most frequently cited priorities for improving the organization of emergency medical services. Other concerns include lack of quality control measures, burden of caring for non-emergent cases, inadequate admission and triage areas, and insufficient hours of operation of hospital-based emergency departments.Many emergency medicine personnel are also critical of the education and training of physicians in emergency medicine. Although 58% of those surveyed feel that physicians receive good theoretical training, 77% report that their educational system is in significant need of improved medical school and postgraduate training programs. Of the individuals advocating for improved education and training, 27% feel that further training in emergency medicine should be included in the medical school curriculum, and 53% believe that additional bedside training to develop practical skills is imperative. Furthermore, 56% of participants in this study express a need for better access to continuing professional education, including shared practical experiences and training between health care institutions. A large number of emergency medicine personnel (36%) stress the need for greater access to international journals, conferences, seminars, and the internet. Many (15%) also communicate a need for international collaboration and training programs between Serbia and foreign medical institutions.Survey participants cite numerous obstacles to the development of emergency medical services in Serbia, including insufficient funding and resources (69%), poor organization of emergency services (36%), lack of governmental support (26%), and inadequate education and training (18%). Also vital to development efforts are the perceptions and attitudes of the people impacted by and involved in these efforts – the public, health care community, and government officials. A slight majority (62%) of individuals surveyed believe that the health care community will support further development of emergency medical services. However, only 46% and 41% of participants feel that the public and government, respectively, will respond positively to development.Of the 36 emergency medicine personnel invited to participate in factor analysis, 33 individuals (91.7%) completed the exercise correctly , internal medicine (27%), anesthesia (27%), general medicine (6%), emergency medicine (6%), other (12%)). The institute for pre-hospital emergency services, the Emergency Center, and two hospital emergency departments participated in this component of the study.Factor analysis followed by manual factor rotation determined levels of agreement among the 33 participants and identified three unique types of respondents. Table In addition to assessing levels of agreement among participants, by-person factor analysis allowed us to identify three different respondent types, each involving multiple respondents with diverse demographics but who share common and unique perspectives relative to the remaining participants. To aid discussion of the three respondent types, each was assigned a brief label: "Utilize", "Develop", and "Invest".Respondent type "Utilize" was heavily loaded by a total of 5 respondents (15.2% of participants) and defined by 4 (12.1%). The principal concerns of the "Utilize" respondent type include the poor utilization of emergency medical services. These individuals believe that emergency personnel are well trained, educated, and prepared to address the emergent needs of the community. However, they consider their role ill-defined and misunderstood, leading to the ineffective use of their services by the public.Respondent type "Develop" was characterized by 6 loaders (18.2%) and 3 definers (9.1%). These individuals support the further development of emergency medicine as an independent specialty in Serbia. They do not consider inadequate finances, supplies, or equipment to be the chief concern of the system of emergency medicine. Instead, the "Develop" respondent type encourages the development of standardized treatment guidelines, improved coordination between the specialties providing emergency care, and additional education and training in the specialty of emergency medicine.The third respondent type, "Invest", included 4 loaders (12.1%) and 2 definers (6.1%). These individuals consider the system of emergency medicine appropriately organized but lacking adequate investment. They believe greater resources should be applied towards equipment and professional training and development. They are also concerned with an apparent lack of coordination between institutions providing primary health care and emergency services.Additional respondent types were defined by single respondents, which prevented adequate characterization. However, the demographic data on each of these isolated respondent types were closely examined to assure that the individual was not in a unique position of authority that could disproportionately influence future health system reform. The specific responses of respondent types "Utilize", "Develop", and "Invest" to the 23 Q-statements are listed in Table The purpose of a rapid assessment is to provide organizations with timely and reliable information to aid targeted interventions. Many rapid assessment methodologies have been utilized, each with its own strengths and weaknesses. One of the most commonly used methodologies is the KAP  survey, which is traditionally a standard questionnaire with pre-developed, closed-response questions . BecauseThe use of more detailed assessment tools – employing a combination of qualitative and quantitative methodologies – can more accurately characterize the country's health care needs and the significant political and cultural barriers to sustainable health care reform. Our proposed Integrated Multimodal Assessment utilizes a diverse approach for strong internal validity and by-person factor analysis to uniquely identify critical subpopulations. By understanding the concerns of subpopulations, more targeted interventions can be developed to directly address these concerns. A trial study evaluating primary health care in Serbia has demonstrated the advantages of using a combination of qualitative and quantitative tools for health system assessment . Each ofOut of the near collapse of the Serbian health care system comes the opportunity to establish a health system more effective than ever in meeting the needs of its citizens. Our focus group discussions, questionnaires, individual surveys, and by-person factor analysis reveal that most emergency medical physicians believe the greatest problems in their system are poor organization of emergency medical services; lack of essential funding, medical supplies, medication, and technical equipment; and inadequate education and training. Several respondents also share the idea that poor incentives for specializing in emergency medicine, difficulties of emergency service work, and inadequate compensation are significant barriers to advancing the field of emergency medicine in Serbia. Although many of these are problems ubiquitous to developing health systems, this study illuminates specific opportunities for emergency medicine providers, the Serbian government, and international institutions to work together to address these issues.According to the majority of emergency medicine providers and administrators surveyed, of foremost priority is reorganization of emergency medical services. In order to begin addressing this need, several participants propose the development of triage and treatment protocols. To facilitate improved routing of patients within the hospitals and to the appropriate emergency health care facility, all health care providers, including those within the primary health centers, should receive formal training in triage. Several participants suggest that leadership development and management training should be provided to medical directors and leading health care providers to improve the organization within and between emergency health care centers. Public education programs should also be developed to increase awareness of the level of care that each health care institution provides so that the public can more properly utilize available health care services.Study participants further reported that the attainment of necessary medications, supplies, and equipment should be a top priority in the development of emergency services. The professionals working in the health care system of Serbia are severely limited by a shortage of these resources and, therefore, are unable to apply their knowledge and skills for proper diagnosis and treatment. Obsolete medical equipment further constrain their diagnostic capabilities, and lack of basic medications and supplies impedes their treatment strategies.This multimodal assessment also shows that the education and training of physicians in emergency medicine need further development. Elements of the educational system needing improvement include medical school and postgraduate bedside training programs; training between health care institutions; access to continuing professional education materials including international journals, conferences, seminars, and internet access; and international collaboration between Serbia and foreign medical institutions, including opportunities for Serbian physicians to gain international clinical experience. Several participants also suggest that all health care workers, including physicians working in the primary health centers, receive periodic training in Basic Life Support (BLS), Advanced Life Support (ALS), and emergency triage.Further development of emergency medicine in Serbia will be complex and will face numerous challenges. Reform will require funding, political commitment, supportive legislation, a revised medical curriculum, multi-phasic implementation, and post-interventional evaluation. Respondents believe that the funding needed for development of emergency services represents a significant barrier to change, but that, however, it is not the universal solution.Although a majority of the emergency medicine personnel surveyed feel that the health care community will support further development of emergency services, just under half of the participants expect similar support from the public and government. Additional studies specifically assessing the needs and concerns of the public and government are recommended.The use of multiple assessment modalities and the resulting complexity of this study involve several limitations that require consideration. All participants in this study were invited to participate by the director's office at each hospital or clinic. The study participants were aware that their participation was entirely voluntary and that they would not receive compensation. Although the study did not provide an economic incentive for participation, it did provide the opportunity for those providers with strong opinions to openly voice their concerns and suggestions. As a result, the study population may not represent all emergency health care providers. While every emergency medicine institution in Belgrade participated, this study was limited to the pre-hospital and hospital-based institutions within the capital city (covering 14.9% of the population of Serbia (excluding Kosovo)). Nevertheless, the participants represent the breadth of medical specialties in Serbia, and they constitute a large number of the stakeholders involved in further development of emergency medicine.Despite the challenges, development of emergency services in Serbia can be accomplished through the dedication and commitment of providers. As a result of studies on emergency services in Kosovo, developmental programs similar to those suggested here have been successfully implemented to improve the training in and provision of Kosovo's emergency medical services . VisitinIt is the hope of the authors that similar reforms will also be implemented to address the specific situation in the Republic of Serbia and that this multimodal assessment will assist by identifying the critical needs, barriers, and priorities for sustainable development. By utilizing both quantitative and qualitative methodologies, the authors submit that this Integrated Multimodal Assessment tool offers a more robust alternative to standard surveys, KAP studies, or the use of anecdotal information for quickly identifying priorities in health system reconstruction in the post-conflict, post-disaster, or development setting.KAP: knowledge, attitudes, and practicesRAP: rapid assessment proceduresThe authors declare that they have no competing interests.Each author has participated sufficiently in the work being reported to take public responsibility for the content. BDN, KD, and MVR conceived and designed the study and obtained research funding. MVR and MM supervised the completion of the study. BDN, KD, MS, MV, and LM undertook recruitment of participating centers and physicians. BDN, KD, and MS completed the data collection, statistical analysis, and first draft of the manuscript. All authors reviewed and contributed to the revision of the manuscript.The pre-publication history for this paper can be accessed here: