In this study we evaluated if salivary flow and composition as well as dental caries are oral markers of disease severity in pSS.in 20 patients fulfilling the American-European Consensus criteria for pSS and 20 age-matched healthy controls whole and parotid saliva flow rates and composition, measures of oral dryness, scores of decayed, missing and filled tooth surfaces (DMFS), periodontal indices, oral hygiene, and dietary habits were examined.in pSS, salivary flow rates, pH, and buffer capacities were lower, and DMFS, salivary sodium and chloride concentrations higher than in the healthy controls. DMFS also correlated inversely to salivary flow rates and positively to oral dryness. Apart from slightly increased gingival index, and more frequent dental visits in pSS, the periodontal condition, oral hygiene or sugar intake did not differ between these two groups. In pSS, findings were correlated to labial salivary gland focus score (FS) and presence of serum-autoantibodies to SSA/SSB (AB). The patients having both presence of AB and the highest FS (>2) also had the highest salivary sodium and chloride concentrations, the lowest salivary phosphate concentrations, lowest salivary flow rates, and highest DMFS compared to those with normal salivary concentrations of sodium and chloride at a given flow rate.the salivary changes observed in some pSS patients reflect impaired ductal salt reabsorption, but unaffected acinar transport mechanisms, despite low salivary secretion. Our results suggest that changes in salivary flow and composition as well as dental caries may serve as potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS. The study also indicates that the ductal epithelium is functionally affected in some pSS patients, which calls for future pathophysiological studies on the mechanisms underlying this impaired salt reabsorption. Primary Sjögren's syndrome (pSS) is a common systemic autoimmune disease that mainly affects middle-aged women. The exact aetiology remains elusive, and appears multifactorial. The disease is characterised by a chronic periductal lymphocytic infiltration of the exocrine glands, predominantly of the lacrimal and salivary glands. The chronic inflammation is assumed to lead to glandular tissue destruction, resulting in keratoconjunctivitis sicca and hyposalivation and associated symptoms of ocular and oral dryness ,2.The salivary dysfunction in pSS is of significant clinical importance and may cause chronic oral discomfort as well as compromised oropharyngeal functions -5. SubseThe American-European Consensus Classification Criteria for pSS include evaluation of salivary gland dysfunction by means of parotid sialography, salivary gland scintigraphy, whole saliva sialometry and labial salivary gland biopsy as well as questionnaire concerning symptoms of oral dryness . The purThe study included 20 randomly selected patients with pSS, who attended the Department of Oral Medicine, University of Copenhagen, for routine follow-up examinations. They were diagnosed according to the Copenhagen criteria, but also met the American-European Consensus Classification Criteria for pSS ,14. All In order to understand the underlying salivary pathophysiology in pSS, the study also included 20 healthy controls selected among persons attending the Institute of Odontology for dental treatment to obtain reference values of normalcy. The selection was based on the requirements that controls had no present or past medical history of systemic disease, did not take any medication including contraceptives and hormonal replacement, had no current use of tobacco products, and matched the pSS patients with regard to age.One examiner (AMLP) conducted the whole saliva collections, interviews, and oral clinical examinations. About 2 hours after collection of whole saliva, another examiner (AB) conducted collections of parotid saliva. In order to minimize the influence of circadian cycle on salivary secretion and composition all procedures were carried out in the same order and at a fixed time of the day, i.e. between 9.00 and 11.45 a.m. The local Danish Ethical Committees approved the study protocol, and all participants completed an informed consent form according to the Declaration of Helsinki. The number of decayed, missing and filled surfaces (DMFS) was recorded excluding third molars as previously described in details . Each su2 was used /[C]) * (1-([CB]/[C]))2.3 [C] * ([CB]) states the concentration of the base in the buffer system. The concentrations of the bases was calculated as previously described ) states the total concentration of the buffer system and  of saliva with respect to hydroxyapatite, i.e. the calcium phosphate salt and main mineral of the tooth tissues, was calculated according to the method described by Schmidt-Nielsen [HAP) was calculated from the saliva p [Ca2+], p [HPO42-], and p [H+]. The negative logarithm of the solubility product of hydroxyapatite (pSPHAP) was calculated by the constant given by Schmidt-Nielsen [HAP acceptable. Finally, the critical pH of parotid saliva, i.e. the pH value at which saliva is saturated  with respect to hydroxyapatite  was calculated.The degree of saturation . Fisher's test (less than ten in one category) and the chi-squared test (more than ten) were used for analysis of distributions between the two groups. Associations between variables were analysed by the Spearman rank order correlation analysis (rs). In order to find the best predictor for a given outcome, multiple regression analysis was used with stepwise backward elimination with the adjusted R-squared values given. Significance was selected at a level of P ≤ 0.05.Differences in the salivary and clinical parameters between the patient and control groups were analysed by Wilcoxon rank-sum test (categorised variables) and two-sampled rs = -0.50, P = 0.025), but only a tendency toward a relationship between the oral dryness and UWS, UPS and SPS. On the other hand, in both groups as a whole, displaying large variations in oral dryness and salivary flow rates, the subjective measures were significantly inversely correlated to UWS , SWS , UPS , and SPS .Table rs = 0.33, P = 0.04).The results of the clinical examination are shown in Table rs = 0.79, P < 0.001 and rs = 0.45, P < 0.05, respectively), but only in pSS a mutual correlation was obtained between UPS and SPS .As shown in Tables In 10, 9, 13 and 7 cases, respectively, volume of UWS, SWS, UPS and SPS samples from pSS patients were insufficient for full sialochemical assessment. In pSS, the concentrations of sodium were significantly higher in both whole and parotid saliva as compared to the healthy controls. Similar results were obtained for chloride concentrations (except for the UPS) Tables . RegardiHAP) in their UPS than the controls. In pSS, the pH in UPS was in fact only 0.1 pH units from their critical pH. Overall, the salivary β and DSHAP were reduced due to low pH, bicarbonate and phosphate concentrations. In pSS, PCO2 of SPS was lower than in that of the healthy controls (P = 0.02). Nevertheless, at corresponding parotid flow rates, be it stimulated or not, the relations between the flow rates, the saliva pH and the bicarbonate concentrations were similar to those of the healthy controls . Also the prevalence of dental treatment, i.e. the number of filled surfaces relative to the total number of present tooth surfaces, was significantly higher in the patients (55 ± 27%) than in the controls (25 ± 14%) (P < 0.001). However, no significant differences were obtained for the number of decayed surfaces between the patients and controls.As shown in Table rs = 0.63, P < 0.001). As shown DMFS increases with increasing age (per year) and increasing complaints of oral dryness, but decreases with increasing salivary flow rates.Table rs = 0.53, P < 0.05) and the prevalence of dental treatment . In addition, the prevalence of dental treatment increased with the duration of pSS , independently of the patient's age.In the pSS patients, regardless of their age, the complaint of oral dryness, but not the actual salivary flow rate, was associated with dental status (DMFS). The lack of significant correlation between flow rate and DMFS may be ascribed to the narrow window of flow rates in pSS. Scores of oral dryness correlated significantly with the total DMFS , SWS , SPS , but not to UPS. Focus scores were not correlated to oral dryness, time since pSS diagnosis, and duration of disease symptoms. It is noteworthy, however, that the patient group exhibited large variability with regard to time since diagnosis and age . Although highly increased sodium and chloride concentrations were found in UPS as well, the number of observations was too small to generate statistical significance due to the sample volumes that did not allow for sialochemistry , presence of serum autoantibodies (n = 14), highest concentrations of salivary sodium and chloride, but lowest salivary concentrations of total phosphate compared to patients with UWS ≥ 0.05 ml/min. Pooling data of UPS and SPS for the pSS group allowed us to compare profiles of the patients with high sodium and chloride concentrations to those with low ones. The results of this analysis revealed that pSS with more than 40 mM sodium in their parotid saliva also had significantly higher focus scores as well as presence of serum autoantibodies, than pSS patients below this value (P < 0.01). The former also tended to have lower parotid flow rates, more decayed tooth surfaces and to be younger than the other pSS patients.In the pSS group, the focus scores (which ranged from 0 to 4) were significantly inversely correlated to UWS , thereby protecting the teeth against demineralisation. The buffer systems responsible for the human saliva buffer capacity include the bicarbonate, phosphate and protein systems ,36-38. IIn this study, the parotid saliva buffer capacity was calculated individually for each pSS patient and healthy control based on the saliva bicarbonate and phosphate concentrations at the respective saliva pH values. This calculation gives an estimate of the buffer capacity of the saliva at the time it is secreted from the parotid gland. In the pSS patients, the buffer capacity of UPS was significantly lower than in the healthy controls, mainly due to the low pH value and bicarbonate concentration in saliva caused by the low flow rates. Moreover, in pSS most of the pH values of UPS were below the relevant pK values of both the bicarbonate and phosphate buffer systems. As compared to healthy controls, the pSS patients will therefore experience far more abundant pH drops in their saliva if exposed to acidic challenges leading to a higher risk of tooth demineralisation. Apart from a low buffer capacity, and thereby impaired ability to maintain a non-acidic saliva pH, the pSS patients also had a significantly lower degree of saturation with respect to HAP in their saliva Table . Thus, i3- concentration and saliva pH did not differ between the two groups  and secrete (at high secretion rates due to increased metabolic turnover of the gland).Despite the differences between pSS patients and healthy controls in sodium and chloride concentrations at comparable low flow rates, stimulated or not, the HCOups Fig. . The traThis study demonstrated a tendency towards lower total phosphate concentrations in pSS patients than in healthy controls. Previous studies have found significantly reduced phosphate concentrations in stimulated parotid and SM/SL saliva of patients with SS compared to healthy controls and patients with conditions resembling SS -42. It sOur sialochemical results are interpreted within the frame of the classical two-stage model of saliva formation . Under nDespite the low salivary flow rates seen in the pSS, the acinar transport mechanisms involved in the formation of primary saliva seem to be unaffected by the glandular lymphocytic infiltration. Accordingly, concentrations of potassium, total calcium, total protein and amylase activity in whole and parotid saliva did not differ from those of the healthy controls, which is in agreement with previous reports ,45. FurtThe changes in salivary composition indicate that the duct epithelium, and mainly the striated duct epithelium, cannot effectively reabsorb the high concentrations of sodium and chloride of the primary saliva in some of the pSS patients, despite low salivary flow rates. The pSS patients who exhibited high concentrations of sodium and chloride were also characterised by having the lowest flow rates, the highest focus score (FS) and highest concentrations of serum autoantibodies (AB) in addition to a tendency of being younger than the pSS patients with normal salivary concentrations of sodium and chloride. Overall, the subgroup of pSS patients with the highest concentrations of salivary sodium and chloride and lowest total phosphate concentrations appeared to be more severely affected by pSS having more exocrine and non-exocrine disease manifestations than those without these salivary changes supporting previous observations . It shouin vitro study on isolated human acini and duct segments from pSS patients have shown that these cells possess functional receptor systems and normal response in changes in the intracellular free calcium concentration upon maximal secretagogue stimulation [via amiloride-sensitive epithelial sodium channel (ENaC) is regulated by circulating adrenal mineralocorticoids, e.g. aldosterone [In pSS, the salivary gland histopathology is characterised by periductal lymphocytic infiltration and acinar destruction. The duct epithelium, however, appears relatively unaffected by the lymphocytic infiltration, which contrasts the observed salivary changes in pSS. On the other hand, the discrepancy may be explained by morphological differences between the labial salivary glands, which predominantly consist of mucous acini and have a very short duct system, and the parotid glands, which comprise serous acini and have long duct system. Nevertheless, it has been stated that the histopathological changes of the labial salivary glands mimic those of the parotid glands ,50. It hmulation . The facosterone  could alThe results of this study indicate that specific sialometric and sialochemical glandular changes, particularly changes in sodium and chloride concentrations, may serve as oral markers of disease severity in pSS. The question arises whether pSS represents a continuum of patients with different stages of disease/affection of salivary glands or different diseases affecting the salivary gland tissues. There is great need of disease assessment and outcome markers in pSS. The hypotheses generated from our results on changes in salivary flow and composition as well as high caries experience as potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS therefore need to be tested in a large prospective cohort study including patients with early to long-standing disease. In addition, the mechanisms underlying the impaired ductal salt reabsorption observed in the pSS patients with presence of both labial salivary gland focus score and serum-autoantibodies need to be further elucidated in future pathophysiological studies.SS = Sjögren's syndrome; pSS = primary Sjögren's syndrome; FS = focal lymphocytic infiltration in the labial salivary glands; UWS = unstimulated whole saliva flow rate; SWS = stimulated whole saliva flow rate; UPS = unstimulated parotid saliva flow rate; SPS = stimulated parotid saliva flow rate; AB = presence of serum autoantibodies to Ro (SSA) and/or La (SSB) antigens; DMFS = decayed, missing and filled tooth surfaces; PI and GI = plaque and gingival indices; PPD = periodontal probing pocket depth; HAP = hydroxyapatite.The author(s) declare that they have no competing interests.All three authors participated in the design of the study and the statistical analysis. AMLP attended coordination of the study and conducted the whole saliva collections, interviews, oral clinical examinations and labial salivary gland biopsies. AB performed the parotid saliva collections and sialochemical analyses. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Young women are a group at high risk of weight gain. This study examined a range of perceived personal, social and environmental barriers to physical activity and healthy eating for weight maintenance among young women, and how these varied by socioeconomic status (SES), overweight status and domestic situation.In October-December 2001, a total of 445 women aged 18–32 years, selected randomly from the Australian electoral roll, completed a mailed self-report survey that included questions on 11 barriers to physical activity and 11 barriers to healthy eating . Height, weight and socio-demographic details were also obtained. Statistical analyses were conducted mid-2003.The most common perceived barriers to physical activity and healthy eating encountered by young women were related to motivation, time and cost. Women with children were particularly likely to report a lack of social support as an important barrier to physical activity, and lack of social support and time as important barriers to healthy eating. Perceived barriers did not differ by SES or overweight status.Health promotion strategies aimed at preventing weight gain should take into account the specific perceived barriers to physical activity and healthy eating faced by women in this age group, particularly lack of motivation, lack of time, and cost. Strategies targeting perceived lack of time and lack of social support are particularly required for young women with children. One gn (2.3%) . This rin (2.3%) .In an effort to reverse the current global epidemic of overweight and obesity, strategies to promote increased physical activity and to encourage healthy eating have been promoted in many countries -15. In AMost existing studies examining perceived barriers to physical activity and healthy eating have focused on the general population,,22-25. wAs well as perceiving different barriers to those faced by other groups in the population, the perceived barriers to increasing physical activity and improving diet that young women face may vary according to their social and personal circumstances. For example, having children is likely to impact on a women's ability to adopt healthy habits ,28,29. IIn order to develop appropriate and effective obesity prevention strategies for young women it is important to understand the barriers they perceive in attempting to control their weight. The aim of this study was to examine perceptions of a range of personal, social and environmental barriers to physical activity and healthy eating, specifically related to weight maintenance, among young women, and how these vary by domestic situation, SES and overweight status.A total of 445 women provided data for this study. Initially, a sample of 1200 women aged 18–32 years was selected from the Australian Electoral Roll using a stratified random sampling procedure, with strata based on the number of eligible cases in each of the eight States/Territories of Australia. As voting is compulsory for Australian adults, the electoral roll provides a complete record of population data on Australian residents aged 18 years and over. Excluding those who had moved and left no forwarding address, the study achieved a response rate of 41% (462 women participated), which is comparable to response rates reported in similar postal surveys with this age group ,34. DataThe socio-demographic characteristics of the sample are reported in full elsewhere . BrieflyA questionnaire was developed and pilot-tested with a convenience sample of 10 women in the same age group as participants. The questionnaire, a study description, an invitation to participate, a consent form and a reply-paid envelope for returns were mailed to the study sample of women in October 2001. Non-responders were sent a reminder postcard two weeks later and a second reminder with replacement questionnaire a further three weeks later.The participants completed the following questions.No-one, I live alone; Partner/spouse; Own children; someone else's children; parents; brothers/sisters; Other adult relatives; and Other adults who are not family members. This was subsequently re-categorized as living with parental family; living alone/share 'flatting'; living with partner (no children); or living with children . Education level  was subsequently categorized as tertiary educated or not tertiary educated and used as an indicator of SES.The socio-demographic questions included domestic situation (household composition) and education. Domestic situation was assessed by asking 'Who lives with you?' with response options: 2)). Self-reported height and weight have been shown to provide a reasonably valid measure of actual height and weight for the purpose of investigating relationships in epidemiological studies [Women were asked to self-report their height and weight and this information was used to calculate body mass index (BMI = weight (kg)/height , participants were asked the following two open-ended questions: 'What is the one thing that makes it hardest for you to be physically active?' and 'What is the one thing that makes it hardest for you to eat a healthy diet?'Analyses were conducted mid-2003, using SPSS version 11.0.0 statistical software. . InitialTwo separate exploratory factor analyses using SPSS FACTOR were performed with the 11 barriers to physical activity and the 11 barriers to healthy eating, to identify underlying patterns of relationships among individual items, and to reduce and simplify the items in order to facilitate subsequent analyses. Principal components analysis with varimax rotation (since factors were not correlated) was used. For any cross-loading items (i.e. items that had loadings of greater than 0.4 on more than one factor), only the higher loading was taken into account when calculating final factor scores. Inter-item reliability for each factor was assessed by Cronbach's α coefficients. Kaiser's measure of sampling adequacy was used to confirm the appropriateness of factor analysis . StandarTable Table Consistent with women's responses to the closed-ended questions, the most important perceived barriers to physical activity reported in response to the open-ended questions were lack of time due to work, study or family commitments (78%), lack of motivation (37%) and childcare issues (25%). The most important perceived barriers to healthy eating related to taste (24%); lack of time (21%); lack of motivation (13%); and the perception that healthy foods are inconvenient or expensive (13%).The factor analysis of the perceived barriers to physical activity revealed three interpretable factors Table  with eigThe principal components analysis of the 11 barriers to healthy eating resulted in two distinct interpretable factors with eigenvalues greater than one Table . The CroMean factor scores did not vary according to women's overweight status or SES. Mean factor scores did differ significantly by domestic situation for two factors: social support barriers to physical activity and social and environmental barriers to healthy eating  or overweight/obese (31%), 18% of the women were underweight . It shouThe findings of this study highlight the need for health promotion strategies that provide increased motivation, support and skills to enable young women to shop and prepare healthy, quick and inexpensive meals. Similarly, the findings suggest a need to promote more time-efficient physical activity alternatives. Additional strategies that recognize the perceived barriers to physical activity and healthy eating faced by young women with children are particularly required.The authors declare that they have no competing interests.SA conducted the literature review, final statistical analyses and early drafts of the results and conclusions sections. KB and DC conceived the study, design and measures, collected the data, coordinated the analyses and participated in the write-up of all sections. NW conducted preliminary analyses and drafting of early results. VI contributed to drafting the final manuscript.


High fruit and vegetable intake is known to reduce the risk of colon cancer. To improve understanding of this phenomenon the action of different phytochemicals on colon cells has been examined. One such compound is quercetin that belongs to the group known as flavonoids. The purpose of this study was to determine the influence of quercetin on the proteome of the SW480 human colon adenocarcinoma cell line, specifically to identify proteins that could be the molecular targets of quercetin in its amelioration of the progression of colon cancer. To this end, two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins that underwent a change in expression following treatment of the cells with 20 μM quercetin. This could elucidate how quercetin may reduce the progression of colon cancer.Quercetin treatment of the SW480 human colon cancer cells was found to result in the decreased expression of three proteins and the increased expression of one protein. The identified proteins with decreased expression were type II cytoskeletal 8 keratin and NADH dehydrogenase Fe-S protein 3. The other protein with decreased expression was not identified. The protein with increased expression belonged to the annexin family.Several proteins were determined to have altered expression following treatment with quercetin. Such changes in the levels of these particular proteins could underlie the chemo-protective action of quercetin towards colon cancer. Colorectal cancer is the third most common cancer diagnosed both in men and in women in the United States . The Amein vitro and in vivo have suggested that quercetin may have a protective role against breast -1-propane-sulfonate;TBP, tributylphosphine;BPB, bromophenol blue;DMSO, dimethylsulfoxide;NCBI, National Center for Biotechnology Information.The authors declare that they have no competing interests.MFM treated the cells, performed the two-dimensional electrophoresis and gel analysis, submitted protein spots for mass spectrometry, undertook the literature search on the identified proteins, drafted the manuscript, submitted the manuscript and completed all the necessary revisions to make it acceptable for publication. KK carried out trypsin digestion of the protein spots and undertook the mass spectrometry. RO provided oversight for the mass spectrometry. JLH and AG participated in conception and design of the study. All the authors read and approved the final manuscript.


Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200–300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a “just-in-time” manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic development of P. falciparum and provide a resource for the identification of new chemotherapeutic and vaccine candidates. Plasmodium genesA tight cascade of gene regulation during the lifecycle of the malaria parasite in human blood cells suggests new functions for many  Plasmodium. Of these four species, Plasmodium falciparum is responsible for the vast majority of the 300–500 million episodes of malaria worldwide and accounts for 0.7–2.7 million annual deaths. In many endemic countries, malaria is responsible for economic stagnation, lowering the annual economic growth in some regions by up to 1.5%  lack sequence similarity to genes from any other known organism  oligonucleotides as representative elements for predicted ORFs in the sequenced genome (strain 3D7)  . Using tP. falciparum IDC was generated by measuring relative mRNA abundance levels in samples collected from a highly synchronized in vitro culture of parasites. The strain used was the well-characterized Honduran chloroquine-sensitive HB3 strain, which was used in the only two experimental crosses carried out thus far with P. falciparum , and the trophozoite-specific gene, dihydrofolate reductase–thymidylate synthetase (dhfr-ts) (asl) (r) is greater than 0.90 for 68% (0.75 for 86%) of the transcripts represented by multiple oligonucleotides with detectable expression during the IDC (r) for these features is 0.98±0.02.To measure the relative abundance of mRNAs throughout the IDC, total RNA from each timepoint was compared to an arbitrary reference pool of total RNA from all timepoints in a standard two-color competitive hybridization . The tradhfr-ts) B and 1C.s) (asl) D, displa the IDC . Cases iP. falciparum IDC dataset, a similar computational approach was taken. We performed simple Fourier analysis, which allowed us to calculate both the apparent phase and frequency of expression for each gene during the IDC (see Periodicity in genome-wide gene expression datasets has been used to identify cell-cycle-regulated genes in both yeast and human cells . Owing t IDC see . The faseba175 (peak B), dhfr-ts (peak C), and asl (peak D) are indicated. It is striking that 79.5% of the expression profiles have a very high score (greater than 70%). For comparison, we applied our FFT analysis to the Saccharomyces cerevisiae cell cycle data, yielding only 194 profiles (3.8%) above a 70% score  or that lack differential expression throughout the IDC. Since the majority of the profiles display a single low-frequency peak in the power spectrum, we have taken advantage of this feature to classify profiles, similar to the application of a low-pass filter in signal processing. By measuring the power present in the peak frequency window (the main component plus two adjacent peaks) relative to the power present at all frequencies of the power spectrum, we were able to define a score (percent power) that we have used to stratify the dataset. The resulting distribution of expression profiles, scored in this way, is shown in 0% score . In addi0% score F. The raP. falciparum . In one case, chr5.glm_185 overlaps with the manually annotated ORF PFE0850w, which is found on the opposite strand. Oligonucleotide elements for both of these ORFs are present on the array. The oligonucleotide corresponding to the automated prediction yielded a robust FFT score and a phase consistent with the translation machinery, yet no PFE0850w expression was detected. These results suggest that the automated prediction for chr5.glm_185 most likely represents the correct gene model for this genomic locus and illustrates the use of the IDC expression data for further verification of the P. falciparum genome annotations.Following invasion, approximately 950 ORFs are induced during the ring and early trophozoite stage, including genes associated with the cytoplasmic transcriptional and translational machinery, glycolysis and ribonucleotide biosynthesis B–2E. Repmerase I  and RNA merase I  and RNA merase I . The cytmerase I C consistPlasmoDB.org). In addition, one of these factors, ribosomal protein S9, has been experimentally immunolocalized within the plastid . In another case, the malaria genome sequencing consortium has predicted two homologues of triose phosphate isomerase, PF14_0378 and PFC0381w. The latter is not detected by our analysis, suggesting that this gene is utilized in another developmental stage or may be a nonfunctional, redundant homologue.In addition to transcription and translation, genes involved in several basic metabolic pathways were also induced during the ring and early-trophozoite stage, including glycolysis and ribonucleotide biosynthesis D and 2E.entified . The glyentified D is tighectively . HoweverP. falciparum parasites generate pyrimidines through a de novo synthesis pathway while purines must be acquired by the organism through a salvage pathway  cycle were detected in the m genome , althougm genome . The peam genome H. Consism genome , and sevm genome . Althougm genome , the expP. falciparum IDC.A transition from early to mid-schizont is marked by the maximal induction of 29 ORFs predicted to encode various subunits of the proteasome I. Seven In the schizont stage, one of the first specialized processes induced was expression from the plastid genome J. The esPlasmodium yoelii merozoite antigen 1. As expected, peak expression of these antigens coincides with the maturation of merozoites and development of several apical organelles, including rhoptries, micronemes, and dense granules. Many of these proteins have been considered as vaccine candidates since antibodies against these antigens were readily detected in the immune sera of both convalescent patients as well as individuals with naturally acquired immunity . RESA1 is secreted into the host cell cytoplasm at the final stages of the invasion process, where it binds to erythrocytic spectrin, possibly via its DnaJ-like chaperone domain   . In eighd genes) C. The avThe largest group demonstrating coregulation on the nuclear chromosomes corresponds to seven genes of the SERA family found on Chromosome 2 see B Miller. Besidesvar, rifin, and stevor families, have a high degree of genomic variability and are highly polymorphic between strains and even within a single strain  when hybridized to the 3D7-based microarray. Thus, the underrepresentation of these gene families in the HB3 IDC transcriptome is likely due to the high degree of sequence variation present in these genes. Excluding the three surface antigen families in the subtelomeric regions, 97% of the remaining oligonucleotide microarray elements exhibit an equivalent signal in the CGH analysis. However, 144 of the differences detected by CGH reside in internal chromosomal regions and include several previously identified plasmodial antigens: MSP1, MSP2 , resulting in a list of 124 in-phase apicoplast genes (PlasmoDB.org as apicoplast proteins by inference from direct assay (IDA). In addition, 30% of the nuclear-encoded translational genes that are not coexpressed with the known cytoplasmic machinery are found within this small group of genes. More importantly, 76 ORFs (62%) are of unknown function, with little or no homology to other genes. This limited subgroup of putative plastid-targeted ORFs are likely excellent candidates for further studies in the ongoing search for malaria-specific functions as putative drug targets.Given that over 10% of the ORFs in the ated see C. We reaated see  , and IV , has been identified in the , and IV . Our dat, and IV . The coiP. falciparum contains a variety of proteases involved in cellular processing, including a group of Clps and signal peptidases that are all expressed maximally at the late-trophozoite stage ; PM X parallels the expression of PfSUB1 (PFE0370c), suggesting that aspartyl proteases may also be involved in merozoite invasion. In addition, the phase of the PfSUB1 homologue suggests a concomitant role, with PM IX slightly preceding merozoite invasion. In total, we have detected gene expression for over 80 putative proteases throughout the entire IDC , yet this was not observed; the mRNA transcripts for 75% of proteins determined to be gamete-, gametocyte-, or sporozoite-specific by mass spectrometry are also transcribed in the plasmodial IDC. These findings confirm previous studies demonstrating that not only genes used for generic cellular processes are present in multiple developmental stages, but also factors of highly specialized unctions . This maP. falciparum genome. The Plasmodium parasite devotes 3.9% of its genome to a complex system of antigenic determinants essential for host immune evasion during a single developmental stage  was grown in a standard 4.5 l microbial bioreactor  equipped with a Bio Controller unit ADI 1030 . Cells were initially grown in a 2% suspension of purified human RBCs and RPMI 1640 media supplemented with 0.25% Albumax II , 2 g/l sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/l gentamycin, at 37°C, 5% O2, and 6% CO2. Cells were synchronized by two consecutive sorbitol treatments for three generations, for a total of six treatments. Large-scale cultures contained 32.5 mM HEPES (pH 7.4). The bioreactor culture was initiated by mixing 25.0 ml of parasitized RBCs  with an additional 115.0 ml of purified RBC in a total of 1.0 l of media (14% hematocrit). Invasion of fresh RBCs occurred during the next 2 h, raising the total parasitemia from an initial 5% to 16%. After this period, the volume of the culture was adjusted to 4.5 l, bringing the final RBC concentration to approximately 3.3% to reduce the invasion of remaining cells. Immediately after the invasion period, greater than 80% of the parasites were in the ring stage. Temperature and gas conditions were managed by the Bio Controller unit. Over the course of 48 h, 3–4 ml of parasitized RBCs was collected every hour, washed with prewarmed PBS, and flash-frozen in liquid nitrogen.A large-scale culture of P. falciparum RNA sample isolation, cDNA synthesis, labeling, and DNA microarray hybridizations were performed as described by http://ucsf-nomad.sourceforge.net/). In brief, a scalar normalization factor was calculated for each array using unflagged features with median intensities greater than zero for each channel and a pixel regression correlation coefficient greater than or equal to 0.75. Quality spots were retained based on the following criteria. The log2(Cy5/Cy3) ratio for array features that were unflagged and had a sum of median intensities greater than the local background plus two times the standard deviation of the background were extracted from the database for further analysis. Subsequently, expression profiles consisting of 43 of 46 timepoints (approximately 95%) were selected. For those timepoints that were represented by multiple arrays, the ratio values were averaged.In total, 55 DNA microarray hybridizations covering 46 timepoints were performed. Timepoints 1, 7, 11, 14, 18, 20, 27, and 31 were represented by more than one array hybridization. Data were acquired and analyzed by GenePix Pro 3 . Array data were stored and normalized using the NOMAD microarray database system (http://www.R-project.org). Fourier analysis was performed on each profile using the fft function of R, padded with zeros to 64 measurements. The power spectrum was calculated using the spectrum function of R. The power at each frequency (Power), the total power (Ptot), and the frequency of maximum power (Fmax) were determined. The periodicity score was defined as Power[(Fmax−1) + (Fmax) + (Fmax+1)]/Ptot. The most frequent value of Fmax across all profiles was deemed the major frequency (m) and used in determining phase information. The phase of each profile was calculated as atan2\[−(I (m)],R (m)\, where atan2 is R's arctangent function and I and R are the imaginary and real parts of the FFT. Profiles were then ordered in increasing phase from −π to π. The loess smooth profiles were drawn through the raw expression data using the loess function found in the modern regression library of R (version 1.5.1). The default parameters were used, with the exception that local weighting was reduced to 30%. For the averaged profiles of the functional groups . Profiles were smoothed with missing values imputed using a locally weighted regression algorithm with local weighting restricted to 12% using R  was isolated from a minimum of 500 ml of total culture for each P. falciparum strain, as previously described , resulting in 124 genes represented by 128 oligonucleotides on the microarray. This select group represents the in-phase plastid targeted genes (see The range of FFT-based phases for the expression profiles of the plastid genome is between 0.32 and 1.05 (or roughly π/9 −π/3). Using the list of 551 apicoplast-targeted genes available at enes see .To select the expression profiles most related to the AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1 vaccine candidates, we calculated the similarity of all expression profiles in the dataset to those of these antigens by Euclidian distance. The minimum Euclidian distance calculated for every profile was then binned into 60 bins and plotted as a histogram. A natural break in the histogram was seen that included the set of 262 ORFs see .Dataset S1(29.5 MB ZIP).Click here for additional data file.Dataset S2(3.7 MB TXT).Click here for additional data file.Dataset S3(2.4 MB TXT).Click here for additional data file.Dataset S4(3.1 MB TXT).Click here for additional data file.Figure S1The percent of power in the maximum frequency of the FFT power spectrum was used to determine periodicity of the yeast cell cycle data from (223 KB EPS).Click here for additional data file.Figure S2PlasmoDB.org.A matrix of the pairwise Pearson correlations was calculated for every expression profile along the chromosomes. The analysis included all annotated ORFs. The gray areas correspond to a Pearson correlation d = 0 and indicate ORFs with no detectable IDC expression or ORFs not represented on the microarray. The starting point (left) and the end point (right) of the chromosomes and the ORF order along the chromosomes are identical to the order in (30.9 MB EPS).Click here for additional data file.Figure S3Genomic differences between strain 3D7 and strain HB3 were measured by CGH. The relative hybridization between the gDNA derived from these two strains is shown as a percent reduction of the signal intensity for 3D7 along individual chromosomes. (1.7 MB ZIP).Click here for additional data file.(A)Click here for additional data file.(B)Click here for additional data file.(C)Click here for additional data file.(D)Click here for additional data file.(E)Click here for additional data file.(F)Click here for additional data file.(G)Click here for additional data file.(H)Click here for additional data file.(I)Click here for additional data file.(J)Click here for additional data file.(K)Click here for additional data file.(L)Click here for additional data file.(M)Click here for additional data file.(N)Click here for additional data file.Figure S4i − yi)2. The Euclidian distance value to the closest vaccine homologue was selected for each IDC profile and used to generate this plot. Genes with d < 20 were selected for the phaseogram of putative vaccine targets  = Σ(xgets see .(494.02 KB EPS).Click here for additional data file.Table S1This table contains all of the ORFs in the analyzed dataset that are represented by multiple oligonucleotides on the DNA microarray. The average Pearson correlation value has been calculated for the expression profiles of all oligonucleotides for each given ORF.(44 KB TXT).Click here for additional data file.Table S2P. falciparum groups discussed. The groups include the following: transcription machinery, cytoplasmic translation machinery, the glycolytic pathway, ribonucleotide synthesis, deoxyribonucleotide synthesis, DNA replication machinery, the TCA cycle, the proteaseome, the plastid genome, merozoite invasion, actin–myosin motility, early-ring transcripts, mitochondrial genes, and the organellar translational group.This table contains all of the (291 KB TXT).Click here for additional data file.Table S3P. falciparum determined by calculating the Pearson correlation between expression profiles for contiguous ORFs. The cutoff was 70% pairwise correlation of greater than 0.75 for each group. Only groups of two ORFs or more are listed.This table contains the regions of coregulation found in the chromosomes of (6 KB TXT).Click here for additional data file.Table S4P. falciparum. The averaged intensities from three microarray hybridization experiments are listed.This table contains all of the intensity data from CGH of gDNA derived from the 3D7 and HB3 strains of (414 KB TXT).Click here for additional data file.Table S5PlasmoDB.org. The presence of each ORF on the DNA microarray is tabulated, as well as whether each ORF is present in the overview set. Finally, the plastid ORFs in-phase with plastid genome expression are listed, as well as the corresponding oligonucleotide identifiers.This table contains all of the predicted apicoplast-targeted ORFs from (147 KB TXT).Click here for additional data file.Table S6The table was constructed by searching the database for any putative protease annotations and contains all of the 92 proteases identified by (59 KB TXT).Click here for additional data file.Table S7The similarity of all expression profiles to seven known vaccine candidates was evaluated by a Euclidian distance calculation to all expression profiles measured. These 262 ORFs constitute the top 5% of genes in the IDC with minimum distance to these seven ORFs. The seven candidates used are AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1.(204 KB TXT).Click here for additional data file.


Many professions in the fields of engineering, aviation and medicine employ this form of scheduling. However, on-call work has received significantly less research attention than other work patterns such as shift work and overtime hours. This paper reviews the current body of peer-reviewed, published research conducted on the health effects of on-call work The health effects studies done in the area of on-call work are limited to mental health, job stress, sleep disturbances and personal safety. The reviewed research suggests that on-call work scheduling can pose a risk to health, although there are critical gaps in the literature. The question of whether work hours and schedules affect people's health has been reviewed for a range of work patterns including shift work and overtime. Research in these areas indicates that shift work, and in particular night work can interrupt sleep patterns , aggravaHowever, the health effects of on-call work, where workers are called to work either between regular hours or during set on-call periods, has not merited as much attention. This form of work scheduling occurs in a variety of diverse occupations, for example medical technologists, doctors, ship engineers, utility workers, electrical technicians, tug boat pilots, midwives, information technologists, media personnel and junior airline pilots. For many of these professions being on-call is not an option, but rather a component of the job. This form of scheduling is often used to provide 24 hour coverage, 7 days a week, for facilities such as hospitals and laboratories, where emergencies require personnel to immediately deal with critical situations and where the volume of evening and weekend work does not necessitate full shift coverage. Having employees on-call, even if they are being paid a stipend for their call time, is often seen as less expensive for employers than providing full shift coverage during off-peak hours .While on-call work scheduling may be less expensive, it is not without human costs. On-call employees must plan their lives and the lives of their families around a call schedule. This often means limiting behaviours and obliges employees to restrict their on-call time to activities that would not interfere with their ability to work. The unpredictability of the call scheduling can also generate a great deal of stress, as home life is interrupted and workers are required to "change hats" to shift to their professional roles at any time during call. These limitations and interferences present unique challenges for on-call workers that are not encountered by those working set schedules or even people with rotating shifts. It is thus not surprising that researchers have found that on-call work patterns can have a major influence on employees' lifestyles and their interactions with family members and friends . HoweverWithin the limited literature that has explored on-call work, there exists some pertinent findings concerning the impact of on-call for an employee's physical and psychological health, and social relationships, which this review seeks to bring together. Specific attention has been devoted to the areas of stress, sleep, mental health and personal safety.The implementation of on-call schedules varies. For many occupations, workers leave their place of employment and are placed "on-call" on evenings and weekends, which means they can be called back to work during these periods. For many professions this form of scheduling is a normal component of the occupation, for example, marine pilots can spend up to 60% of their working time on-call. However, for a limited number of occupations such as airline pilots, on-call hours are reduced with seniority. Generally, but not always, employees are compensated monetarily for the period of call, usually with a stipend which is less than their hourly rate. When on-call employees are usually expected to restrict their use of alcohol and limit distance or travel time from the work-site. The on-call experience of these workers includes aspects of interruption, either of sleep or family or social life, and often includes an element of uncertainty as to the time of call or the occurrence of the call.Other forms of on-call include work done by junior doctors during their medical training. Medical residents spend periods of time "on-call" at a hospital, where space may be provided for them to sleep. This form of on-call work is distinct because workers remain at work to undertake their call duty. During these periods, residents often put in 30–36 hour shifts with little to no sleep , resultiThis review focuses on the health effects of on-call work in which an employee spends a period of time on-call outside of their workplace and/or their regular working hours. The research on medical residents has been excluded because the medical resident experience is distinctly different from that of other professions where on-call is utilized. However, research on medical residents is used to illustrate findings from other work-related areas when appropriate.This review explores the published literature referring to on-call work patterns and health. For the purpose of this review, the on-call period may be formal  or informal . Search terms for this review included "on-call" and "work schedule tolerance". The terms "stand-by" and "night call" are used by some professions to describe on-call type work, and were also used as search parameters. This literature review was undertaken on journal articles included in databases up to December 2000. Database searches were performed on the following electronic sources:1) OVID Databases: including Medline (1966–2000) and Current Contents (1996–2000).2) Canadian Centre for Occupational Health and Safety Database3) Cambridge Abstracts  1981–2000.4) PsycInfo (1989–2000).5) Web of Science: including Science Citation Index and Social Science Citation Index (1989–2000).A manual review of the references generated from the computer-search was also done. Articles were excluded from the review if they were not original research, were not written in English or focused on medical residents experiences with on-call work.Two reviewers read through each of the eligible research papers independently.In total, 24 papers met the search criteria. Eight (8) were excluded as they focused on the impact of on-call work patterns on patient's health and not on the health of workers. The remaining sixteen studies were used for this review. The results are divided into four health-related sections; 1) Stress, 2) Sleep, 3) Mental Health and 4) Personal Safety.Of the five studies pertaining to on-call and stress uncovered in this review, all focus on the General Practitioners (GPs) as their subject. In these studies, the relationship between on-call work and stress was measured through self-report and perceived stress.Three of the studies were part of a major UK study carried out from 1989 to 1998 -13. In tth in a ranking of 14 major stressors. The authors believe this reduction in the level of stress from on-call work could be explained by the introduction of GP co-operatives in the mid 1990s for the management of out-of-hours calls. This cooperative system allowed GPs to either do their own calls or share them with a cooperative formed by 10 or more doctors. The cooperatives gave GPs greater flexibility for how and where they saw their patients and how they implemented 24-hour care and appear to have successfully reduced the stress of night visits for GPs. Indeed, night visit stress went from being one of two top stressors for GPs in 1987 and 1990 to being one of the least stressful issues by 1998. The authors also posit that this "success may also explain the reported reduction by 1998 in stress attributable to disturbance of home/family life" The fourth study also dealt with the changes in the British health care system, in particular the introduction of partial shifts to decrease long on-call periods . A smallThe fifth study on GPs and stress was a qualitative analysis of 25 GPs and their spouses in Manchester . This reOther studies have revealed that the amount of time spent on-call varies between male and female doctors, but no clear pattern has emerged ,17. It hResearch conducted in other professions support the idea that work patterns, particularly night shifts, can increase stress in workers and have a negative impact on family life. Working late afternoon and evening shifts has been related to increased stress for both workers and their families . VariablBesides stress, the interruption of sleep is another major component of on-call work, particularly for those who work nights on-call and in professions that deal with emergencies that occur at all hours. Three studies have dealt specifically with the sleeping patterns and problems experienced by train and ship engineers and transplant coordinators, all of whom regularly work on-call.The first study researched the on-call sleep patterns of 198 train engineers using prospective activity logs over a 14-day period in the United States . It was The second study of on-call and sleep explored the sleeping patterns of 53 predominantly female organ transplant coordinators in the UK, using a postal questionnaire . This reThe third study, conducted on a small sample (n = 5) of ship engineers in Sweden, measured sleep during on-call periods using electroencephalogram (EEG) and electrocardiogram (ECG) recordings and subjective ratings. . This reThe impacts of sleep loss on job performance remain unclear and controversial. For example, research on the cognitive performance in sleep deprived medical residents has produced mixed results -27. HoweWork-related fatigue has been related to an increase in car accidents. A review of traffic accidents determined that falling asleep while driving accounted for a major proportion of accidents while driving under monotonous conditions . This fiSix studies were found that examined the impact of on-call work schedules on mental health. All of these studies used self-reported questionnaires and/or mood diaries. Five studies were conducted on GPs in the UK and one examined gas and electrical employees in France.Two surveys were conducted by Chambers et al. ,34 on GPThe second survey conducted by Chambers et al in 1996 (n = 620) employed the Hospital Anxiety and Depression scale to assess the mental health of GPs . It was A third survey done on GPs in Leeds in 1993 was designed to determine the psychological symptoms and sources of stress among 268 GPs . This suThe fourth study in this area surveyed mental health and job stress on 414 GPs in England in 1992 . This reA pilot study of 44 male and female volunteer GPs using cognitive behavioural diaries assessed self-reported emotional states recorded in conjunction with hourly activities over 2 days . DoctorsThe sixth study examined male gas and electrical employees working in France . EmployeThe research conducted on GPs in the UK supports a negative role of on-call work related to mental health. However, the results from the gas and electrical workers do not reflect the same findings from the research on GPs. This may be the result of either a difference in study methodology or a difference that is profession-specific. On-call gas and electrical workers did experience psychological disruption and the lack of significant diagnostic findings may be a function of other factors, such as self-selection, in this profession, where those most affected opt out early on. The on-call gas and electrical workers experience of family and social life disruption does mirror the experiences of doctors and transplant coordinators as discussed previously ,15,22.Working on-call often necessitates leaving home alone, at night, to attend work, conditions that can jeopardize personal safety. Unfortunately, there is only sparse data regarding this issue. A study done in the north west of England, in a hospital where the on-call sleeping quarters were separate from the hospital found that 40% of anaesthetists feared for their safety while walking through hospital grounds at night .In medical professions, patients can also present a danger to those working on-call. Doctors have cited fear of violence from night call visits as a significant stressor . A studyThis issue has only been peripherally studied and further attention needs to be given to personal safety, particularly when being called in at night.What emerges from this review is the limited research that has been done in the area of on-call work. Preliminary work done in the areas of stress and mental health suggests that on-call work may play a role in increasing stress and decreasing mental well-being. The three studies that examined sleep indicate that on-call work does decrease the quality and quantity of sleep for workers and can leave people feeling fatigued for periods after their on-call work.The current body of literature on the health effects of on-call work is limited in part due to the narrow range of professions studied. The majority of research done to date has been on general practitioners. It is reasonable to assume that the effects of on-call will vary across occupations, given the host of other factors that can influence occupational health. However, the degree to which this variation exists might only be determined by examining a wider occupational base. The need to undertake more on-call research across a greater variety of occupational groups is suggested given that this form of work scheduling touches many occupations, and given that on-call work is estimated to continue to increase in many sectors in the future .There is also an obvious lack of research focusing on the impact of on-call shifts on psychosocial factors. Given the very disruptive and limiting nature of on-call schedules, it would not be surprising that workers' family and social life suffer due to this type of scheduling. The results of the research addressing gender (discussed above) do suggest, albeit indirectly, that such social and familial impacts may be significant. However, without more research, it is not possible to determine the magnitude of these effects, nor the relative importance compared to other factors such as physiological responses.More rigorous methodological designs are needed for future research in the area of on-call work and health. The current research is predominantly cross-sectional in nature, a factor that makes it difficult to determine causality. Only two studies employed external comparison groups ,38 and oFuture research on the health effects of on-call work also needs to examine the role of gender, not only from a physiological standpoint, (e.g. reproductive issues), but also from a psychosocial perspective. Many of the articles reviewed above indicate differences in how males and females experience the stress of on-call work ,15,17,36The range of health effects studied in relation to on-call work has to date been inadequate. Health conditions such as cardiovascular disease, reproductive problems, gastrointestinal issues and overall mortality need to be explored as has been done in conjunction with work patterns such as overtime and shift work ,45. FactWhile the results of this review are limited, initial research in this area suggest that being on-call can have negative impacts on workers' sleep patterns, mental health and personal life. Further research in this area is required to provide a clear picture of the risks of this form of work scheduling.UK, United KingdomUS, United States of AmericaThe authors declare that they have no competing interests.AMN designed the research project, carried out the literature search, reviewed articles and drafted the manuscript. JSB reviewed articles and edited the manuscript. Both authors approved the final manuscript.


We aimed to determine optimal strategies for complete mortality ascertainment comparing death certificates and United States (US) Veterans Administration (VA) records.We constructed a cohort of California veterans who died in fiscal year (FY) 2000 and used VA services the year before death. We determined decedent status using California death certificates linked to VA utilization data and the VA Beneficiary Identification and Records Locator System (BIRLS) death file. We compared the characteristics of decedents who would not have been identified by either single source  with the rest of the cohort.A total of 8,813 veteran decedents were identified from both VA decedent files and death certificates. Of all decedents, 5,698 / 8,813 (65%) veterans were identified in both source files, but 2,426 / 8,813 (28%) decedents were not identified in VA BIRLS, and 689 / 8,813 (8%) were not identified in death certificates. Compared to the rest of the cohort, decedents whose mortality status was ascertained through either single source differed by race / ethnicity, marital status, and California residence. Clinically, veterans identified from either single source had less comorbidity and were less likely to have been users of VA inpatient or long term care, but equally or more likely to have been users of VA outpatient services.As single sources, VA decedent files and death certificates each provided an incomplete record, and death ascertainment was improved by using both source files. Potential bias may vary depending on analytic interest. Clinicians, healthcare administrators, researchers, regulators and policymakers are concerned with optimizing mortality ascertainment using administrative data. In addition to its clinical importance, mortality informs program planning, quality assessment and improvement, and public reporting -8. VeterIn order to evaluate the implications for improving veterans' end-of-life care, we constructed a population-based decedent cohort . For sucThe VA Beneficiary Identification and Records Locator System (BIRLS) contains records of all beneficiaries including veterans whose survivors applied for burial benefits. It includes records of discharged military veterans post-1973 and recipients of Medals of Honor and VA education benefits. After submission to the Veterans Benefits Administration (VBA), deaths are recorded in the BIRLS Death File. A submission to the VBA is typically triggered by a family claim for death benefits  -19. The We first identified 345,380 decedent veterans who died during FY2000 (30 September 1999 – 1 October 2000) from the BIRLS Death File. We used SSNs to link cases to VA NPCD outpatient, inpatient, or long term care records restricted to recipients of any VA services in California within 12 months of death. We extracted records including any inpatient or long term care admission, or outpatient encounters. Veterans who entered the cohort on the basis of using outpatient services were required to have at least one clinical encounter .In addition, we used California death certificates as second source to identify decedent veterans by linking SSNs from death certificates directly to VA utilization files. California death certificates contained 462,561 records for calendar years 1999 and 2000, and we primarily matched decedents identified through death certificates to BIRLS by SSN. We manually inspected matches on SSN only and we also examined matches on criteria other than SSN . Additional cases we accepted after manual inspection involved transpositions of one and rarely more than one SSN digit but agreement in other fields. Thus, the cohort included recipients of VA clinical services verified as deceased based on either BIRLS or death certificates, and all cases were linked to VA utilization files by SSN.In the final decedent cohort, we excluded cases of non-veterans receiving care at VA facilities by examining indicators of veteran status associated with visits. The VA assigns specific codes to non-veterans rendered care for various reasons . We also considered the possibility of erroneous decedent status by looking for evidence of healthcare utilization during the 12 months after death. We excluded cases with evidence of utilization more than one month after the date of death.We used VA encounters and ICD-9-CM codes to demographically  and clinically characterize decedents -26. We iTo understand the limitations of single source mortality ascertainment, we described decedents who would not have been identified by a strategy using either death certificates alone or VA decedent files alone. We compared these cases with the rest of the cohort based on their demographic and clinical attributes and the settings in which they received care. Based on distributions, we used Wilcoxon tests for continuous and chi-square tests for categorical variables.From 345,380 deaths during the period 30 September 1999 to 1 October 2000 identified in BIRLS, we distinguished 6,071 decedents who were users of VA inpatient, outpatient, or long term care services in California. California death certificates included 227,308 deaths during the same period, including 3,580 additional users of VA inpatient, outpatient, or long term care services in California. Using SSN and other identifiers to match decedent cases to VA utilization data, we excluded non-veterans (n = 365), users of only non-clinical care such as laboratory tests (n = 251), those possibly alive based on subsequent VA encounter data (n = 229), and 3 cases for other reasons. Of the final cohort of 8,813 veteran decedents, 5,698 (65%) cases were identified in both source files, while 689 (8%) were only identified in VA decedent files, and 2,426 (28%) additional cases were only identified through death certificates . Ninety-nine percent of decedents missed by using VA data alone were California residents ; whereas, 62% of those missed by using death certificates alone were out-of-state residents . Relatively fewer veterans of white or black ethnicity and relatively more veterans of missing ethnicity were represented among decedents missed by either single source strategy. The proportion of married or previously married veterans was higher and single or missing marital status lower among those decedents missed using only BIRLS, and relative proportions were reversed for a strategy using only death certificates.Decedents missed by either single source approach were less likely to have been diagnosed with an advanced chronic illness than the identified cohort. Veteran decedents missed by using only BIRLS were less likely to be diagnosed with any condition except HIV and dementia, and those missed by using death certificates alone were less likely to be diagnosed with any condition except HIV. With a BIRLS only approach, 37% of missing cases vs. 35% of the remainder cohort p < 0.001) had no diagnosed chronic illness . Veteran decedents missed by either single source approach were equally or more likely to have been users of the outpatient setting, but missed cases were less likely to have been users of inpatient healthcare settings  were both significantly lower among veterans missed by either single source approach.Our findings are consistent with Washington State where the deaths of 25% of 533 veterans who only used outpatient services were only identified with death certificates, and 5% were only identified in BIRLS.  Using BIOne limitation of our study is that we did not identify cases that were only decedents by virtue of VA utilization files alone rather than BIRLS, although Dominitz, et. al., identified only 2.7% of deaths this way . We did Researchers, managers, and policy makers should understand the limitations of sources of mortality ascertainment. The relationship of missing data to bias is related somewhat to how "missingness" is distributed by the outcome of interest. Our findings suggest these concerns may be relatively more important for studies involving veterans and racial-ethnic disparities, co-morbidity, certain disease comparisons, or settings of care. Additional study is needed to compare BIRLS, death certificates, and the NDI for mortality ascertainment in veterans. If our findings are confirmed, the VA may need to consider improving its system for mortality ascertainment through routine linkages to national mortality data. Studies of end-of-life care using decedent cohorts need to pay particular attention to the incompleteness of VA data as the sole source of mortality information.VA, Veterans Administration; FY, fiscal year; BIRLS, Beneficiary Identification and Records Locator System; NPCD, National Patient Care Database; SSN, Social Security Number; CHF, congestive heart failure; COPD, chronic obstructive lung disease; ESLD, end-stage liver disease; ESRD, end-stage renal disease.The author(s) declare that they have no competing interests.KL originated and oversaw all aspects of the conception, design, analysis, and publication of the study. SA, LR, and EY contributed to conception, design, and analysis. MW contributed to analysis and is responsible for programming. All authors reviewed and approved of the manuscript.