Periconceptional use of vitamin supplements containing folic acid reduces the risk of a neural tube defect (NTD). In November 1998, food fortification with folic acid was mandated in Canada, as a public health strategy to increase the folic acid intake of all women of childbearing age. We undertook a comprehensive population based study in Newfoundland to assess the benefits and possible adverse effects of this intervention.This study was carried out in women aged 19–44 years and in seniors from November 1997 to March 1998, and from November 2000 to March 2001. The evaluation was comprised of four components: I) Determination of rates of NTDs; II) Dietary assessment; III) Blood analysis; IV) Assessment of knowledge and use of folic acid supplements.12 deficiencies, and no evidence of improved folate status masking haematological manifestations of vitamin B12 deficiency. The proportion of women aged 19–44 years taking a vitamin supplement containing folic acid increased from 17% to 28%.The annual rates of NTDs in Newfoundland varied greatly between 1976 and 1997, with a mean rate of 3.40 per 1,000 births. There was no significant change in the average rates between 1991–93 and 1994–97 . The rates of NTDs fell by 78% (95% CI 65%–86%) after the implementation of folic acid fortification, from an average of 4.36 per 1,000 births during 1991–1997 to 0.96 per 1,000 births during 1998–2001 (RR 0.22, 95% CI 0.14–0.35). The average dietary intake of folic acid due to fortification was 70 μg/day in women aged 19–44 years and 74 μg/day in seniors. There were significant increases in serum and RBC folate levels for women and seniors after mandatory fortification. Among seniors, there were no significant changes in indices typical of vitamin BBased on these findings, mandatory food fortification in Canada should continue at the current levels. Public education regarding folic acid supplement use by women of childbearing age should also continue. Neural tube defects (NTDs) are birth defects resulting from the failure of neural tube closure during early development of the human embryo. The 1997 Canadian national NTD birth prevalence was 0.75 per 1,000 births (live births and stillbirths), down from 1.16 per 1,000 in 1989 -4. HistoEvidence from a number of studies has demonstrated that periconceptional use of vitamin supplements containing folic acid reduces the risk of NTDs -8. AlthoBecause of concern that public education campaigns alone would not be effective in achieving optimal periconceptional folic acid intake for the majority of women, food fortification with folic acid was proposed as a strategy to ensure that all women of childbearing age increase their dietary intake of this vitamin. In November 1998, Health Canada mandated fortification of white flour and enriched pasta and cornmeal with folic acid . Since d12 deficiency, a condition that affects 10–15% of the population over age 60 years [12 deficiency, thus delaying diagnosis and treatment of the condition while its attendant neurologic manifestations progress. Seniors may be at particular risk since the incidence of vitamin B12 deficiency increases with age.The question of whether folic acid fortification of grain products poses any serious health risk has been controversial. The main concern has been the potential masking of vitamin B60 years ,15. IncrWe therefore undertook a comprehensive population based study to evaluate the effectiveness of the public health strategy of food fortification with folic acid and to determine possible adverse effects resulting from fortification.This evaluative study was designed as a population based study and included four components as follows: I) Determination of rates of NTDs; II) Dietary assessment; III) Blood analysis; IV) Assessment of knowledge and use of folic acid supplements. The latter three components of the study were carried out in two phases; the first phase took place prior to the introduction of mandatory fortification, from November 1997 to March 1998 and the second phase occurred from November 2000 to March 2001, after two years of implementation of mandatory fortification.This study was undertaken in Newfoundland because of the historically high rates of NTDs in the province, and because of strong interest in the health community in this initiative. Newfoundland and Labrador, with a population of approximately 500,000, has about 5,000 births annually. An urban (St. John's) and rural (Clarenville, Port Blandford, Random Island area) location in the province were chosen as the sites for this study. Data collected from these sites were compared between Phase I (November 1997 to March 1998) and Phase II (November 2000 to March 2001). Table In order to examine temporal changes in the rates of NTDs in Newfoundland, data were compiled from the Newfoundland and Labrador Medical Genetics Program from 1976 to 2001. This Program ascertains cases of NTDs annually and maintains an NTD database. The database has recorded cases of NTD since 1976. Cases are identified in the following ways: provincial live birth and stillbirth notification forms, maternal-fetal medicine referrals (only one tertiary care unit in the province), and letters sent to all medical records departments of all provincial hospitals requesting data on cases assigned ICD-9/10 codes associated with NTDs or terminations for NTDs. These multiple sources are utilized to ensure complete ascertainment. NTD cases include anencephaly, spina bifida and encephalocele diagnosed in live births, stillbirths  and fetuses from pregnancies terminated  after a prenatal diagnosis of an NTD.For the knowledge assessment component of the study, women between the ages of 19 and 44 years were recruited through a random telephone survey. In the initial telephone survey, women were asked about their use of vitamin supplements and knowledge of the importance of folic acid for reducing the risk of NTDs or for fetal development. Women who completed the initial telephone survey were subsequently screened for their eligibility for dietary and blood assessments. Women who were not taking supplements containing folic acid and not pregnant were eligible to participate. This sampling procedure for Phase I and Phase II resulted in a response rate of 59.7% and 65.4%, respectively, with no difference between urban and rural response rates. A total of 233 women were recruited into Phase I and 204 women were recruited in Phase II, who completed components II, III and IV of the study.12 deficiency or anaemia and not taking vitamin B12 or supplements containing folic acid, were eligible for dietary and blood sample assessments. A total of 202 seniors were recruited in Phase I and 186 were recruited in Phase II (response rate 45.1% and 44.9%, respectively).Seniors were recruited in the same manner as the samples of women, but were drawn only from St. John's, Newfoundland. Seniors aged 65 years or over, not diagnosed with vitamin BIn order to determine intakes of naturally occurring folate  pre and post fortification, and dietary intakes of folic acid (the synthetic form of the vitamin) post fortification, a Willett food frequency dietary questionnaire  was admi12 status in Phase I and Phase II. Laboratory tests for complete blood count (CBC), red blood cell (RBC) folate, serum folate, creatinine, vitamin B12, plasma homocysteine (HCY) and methylmalonic acid (MMA) were conducted at the laboratories of the Health Care Corporation of St. John's.The women and senior participants were also asked to provide a sample of blood in order to determine blood folate and vitamin Bpermitted in Canada as of December 1996 [Rate of NTDs was defined as the number of above described NTD cases, divided by the total number of live births, stillbirths, and pregnancy terminations for an NTD (termed as "births" hereafter). First we examined the temporal trend in annual rates of NTDs from 1976 to 2001 using 3-year moving average rates, then we focused on comparison of the NTD data for the most recent 11 years, identified as pre-supplementation (1991–1993), pre-fortification (1994–1997) and post-fortification (1998–2001). We regard the year 1997 as a transition period, or partial fortification period, since fortification of white flour and enriched pasta and cornmeal was ber 1996 . Thus weMean daily intakes of naturally occurring folate were calculated for women aged 19–44 years and for seniors in Phase I and Phase II. Also, for Phase II, average daily intakes of folic acid from fortified foods were calculated.12were skewed. Values were therefore log transformed to give an approximate normal distribution for estimation of geometric mean and confidence intervals. Unless otherwise stated, all laboratory values presented in this paper are geometric means and 95% confidence intervals (CI). Differences in the frequency of high or low results based on reference values were tested by Pearson chi-square statistics.Data from the blood analyses were tested for normality with the Komogorov-Smirnov test, and differences between groups were tested using the non-parametric Mann-Whitney U test. The distributions of plasma MMA, plasma HCY, serum folate, RBC folate and serum vitamin BAll data for this study were entered into SPSS  Rel. 10.0 after the end of each phase. Data from the dietary interviews were analyzed using Epi-Info (Version 6.04d), while the laboratory data and the data about knowledge and use of supplements were analyzed using SPSS.There were 617 ascertained cases of NTD among live births, stillbirths and pregnancies terminated for an NTD in Newfoundland over the 26 year period. The annual rates of NTDs in the province varied greatly over time, with the lowest rate of 2.18 per 1,000 births in 1989, and the highest rate of 5.92 per 1,000 births in 1995. The average rate of NTDs between 1976 and 1997 was 3.40 per 1,000 births. A dramatic drop is seen in 1997, in which the rate of NTDs was 2.20 per 1,000 births, down from 5.49 per 1,000 births in the previous year. The decreasing trend continued after 1998 . It is worthwhile to note that there has been no significant increase in the proportion of NTDs from terminated pregnancies since 1994.There was no statistically significant change in the average daily intake of naturally occurring folate among either women aged 19–44 years or seniors between Phase I and Phase II (p = 0.19 and p = 0.18, respectively). Seniors generally had dietary folate intake slightly higher than women of childbearing age. In Phase I, the average daily intake of naturally occurring folate was 306 μg/day for seniors and 262 μg/day for women aged 19–44 years, while in Phase II, the average daily intake of naturally occurring folate was 290 μg/day for seniors and 248 μg/day for women aged 19–44 years.The implementation of mandatory fortification resulted in an average additional dietary intake of 70 μg/day of folic acid in women aged 19–44, and 74 μg/day of folic acid among seniors. It is noteworthy that for the women the average daily folic acid intake due to food fortification was less than the approximately 100 μg that was previously predicted for women of childbearing age. The maximum dietary intake of folic acid due to fortification for an individual woman was 235 μg/day, and for an individual senior was 219 μg/day.Serum folate and RBC folate increased significantly from Phase I to Phase II in both women aged 19–44 years and seniors (p < 0.001). For both age groups, there was a corresponding decrease in mean plasma HCY levels Tables  and 4. T12 levels in women aged 19–44 and seniors (p = 0.020 and p < 0.001, respectively, Tables 12 (<133 pmol/L) was 18.8% prior to fortification and following fortification this proportion declined to 11.8% (p = 0.032) (data not shown). A statistically significant increase in mean plasma MMA levels was observed in women subjects (p = 0.008) but not in seniors (p = 0.229) (Tables There was a significant increase in mean vitamin Bles 12 <13 pmol/L There was a significant increase from Phase I to Phase II in the proportion of women aged 19–44 years who knew the importance of folic acid (from 33% to 46%, p < 0.001). The proportion of women taking a vitamin supplement containing folic acid increased substantially between the two time periods (from 17% to 28%, p < 0.003). Information about folic acid dosage was not collected.The results of a number of studies have led to the conclusion that periconceptional folic acid supplementation reduces the risk of NTDs -8. AmongIn March 1996 the US Food and Drug Administration (FDA) announced that it would permit addition of folic acid to enriched flour and other enriched cereal grain products, and that this addition would be mandatory as of January 1998. The level of fortification was set at 0.14 mg folic acid per 100 g of cereal grain product. It was determined that at this level of fortification, the intake of folate  for the target and the general population would be kept below 1,000 μg/day, which was deemed to be the safe upper limit. This level of fortification was estimated to increase the average daily intake of folic acid in women of childbearing age by about 100 μg . SubsequOur results show a highly significant drop in the rate of NTDs in Newfoundland, taking into account all identified affected pregnancies . The 78% (95% CI 65%–86%) reduction in the NTD rate after implementation of fortification is greater than the 18%–22% reduction predicted at current levels of fortification ,28, and The large reduction in the rate of NTDs in Newfoundland may be due, at least in part, to the fact that Newfoundland had higher background rates of NTDs. This population may be more sensitive to the influence of folic acid. In a large-scale public health campaign in northern and southern China, periconceptional use of 400 μg/day folic acid supplements was associated with a reduction in NTD risk of 79% for women in northern China, where the baseline NTD rate was high and similar to that observed in Newfoundland. A lower risk reduction of 41% was observed in the southern region where the pre-campaign NTD rate was much lower .The 65% increase in the proportion of women taking vitamin supplements containing folic acid, from 17% in Phase I to 28% in Phase II, suggests that an increasing trend in folic acid supplementation may have played a role in the declining NTD rate in Newfoundland. In this study it was not possible to determine the individual contribution of supplementation and fortification to the trend in NTDs.The annual rate of NTDs in the pre-fortification period (1994–97) did not differ significantly from that of the pre-supplementation period (1991–93); this is true whether 1997 is excluded or included in the pre-fortification period. The increase in the rates of NTDs in 1995 and 1996 appears random and largely unexplainable. The changes in the NTD rates between 1994–1996 and 1991–1993 and between 1995–1997 and 1991–1993 were not statistically significant. In addition, our data do not show an obvious increase in the proportion of NTDs in terminated pregnancies during 1994 and 1996 (data available upon request).The questionnaire used in this dietary assessment was a modified Willett questionnaire , adminisThe daily intake of naturally occurring folate among women aged 19–44 years in this study (average 248 μg/day in the Phase II sample) was similar to values found in other studies of women's diet ,35. For The results of this study provide strong evidence of improved blood folate status in women aged 19–44 years following mandatory fortification with folic acid. Women showed evidence of increased levels of serum and RBC folate and decreased levels of plasma HCY. These results are consistent with an earlier study examining the effect of fortification in the Framingham offspring study cohort .Mandatory food fortification with folic acid has resulted in improvements in folate indices in seniors. Both mean serum folate and mean RBC folate increased following folic acid fortification  in seniors post fortification in our study. Thus, these results show no evidence of a deterioration in vitamin B12 status among seniors. Furthermore, there is no evidence of improved folate status resulting in masking of the haematological manifestations of vitamin B12 deficiency among seniors as a group. There was no evidence of deteriorating vitamin B12 status among young women participants based on vitamin B12 measurements. The upward trend in plasma MMA levels and higher proportion of abnormal values among young women is being further evaluated. It is unlikely that this is a direct effect of folic acid fortification and this observation is not consistent with any known effects of folic acid on vitamin B12 status.There was a decline in the proportion of seniors with low vitamin BWe have documented the rate of NTDs among live births, stillbirths and terminated pregnancies known to have an NTD. It was not possible to include NTDs that may have occurred in pregnancies that resulted in a spontaneous abortion or a termination that occurred for reasons other than a congenital anomaly.12 status. On the other hand, the fact that we observed such marked improvements in blood folate status leads us to conclude that there was a real increase in exposure to folic acid through fortification, over the study period.This study, and other studies of fortification in Canada, are limited by the fact that there was no precise date when exposure to food fortified with folic acid began. The addition of folic acid to white flour and enriched pasta and cornmeal was permitted as of December 1996. Industry was switching to folic acid-containing enrichment premixes, especially towards the end of 1997, in anticipation of both US requirements for fortification as of January 1, 1998, and Canadian plans to implement mandatory fortification. Although this requirement did not come into force in Canada until late 1998, the Phase I (November 1997 to March 1998) subjects of our study may have consumed at least some food fortified with folic acid. This would result in an underestimate of improvements in blood folate status due to fortification, and might lead us to miss adverse effects on vitamin BAnother limitation of this study is the possible underestimation of folic acid intake due to fortification. Our calculations were based on the assumption that manufacturers are fortifying flour at the required level. It has been suggested that allowance for "overages" is resulting in higher amounts in the affected products . Also, fWe initially selected a random sample of subjects through random digit dialling, and asked eligible respondents for voluntary participation in the study. The reasonable level of response for the dietary questionnaire and blood sampling among rural and urban women aged 19–44 years suggests that with caution, we can generalize the results to all Newfoundland women of childbearing age. However, these findings may not be representative of the rest of Canada because of population differences in factors such as genetic background and dietary behaviour. These differences may also affect the generalizability of the NTD trend.The sample response rate for the dietary questionnaire and blood sampling in seniors was approximately 45% both in Phase I and in Phase II. Many of the refusals to participate were due to illness of the eligible person. Furthermore, seniors residing in long term care settings were not included. Thus our sample population of seniors may be healthier than the general population age 65 and over in the province.12 status based on biochemical and haematological indices.The implementation of food fortification with folic acid has been accompanied by a marked decrease (78%) in the rate of NTDs in Newfoundland. The blood folate status of women aged 19–44 years improved following mandatory fortification. There is no evidence of adverse effects of the current levels of fortification on individuals aged 65 years and older. Specifically, there is no evidence to suggest an adverse effect of folic acid fortification on detection of abnormalities in vitamin BBased on these findings, mandatory food fortification with folic acid should continue in Canada at the current levels. Over the time period of this study, the proportion of women aged 19–44 years taking a vitamin supplement containing folic acid increased. It was not possible to determine the magnitude of the separate contributions of fortification and supplementation to the decline in NTDs. Therefore, we recommend that public health efforts to promote awareness of the importance of folic acid supplementation among women of childbearing age continue. Ongoing surveillance of NTDs in Newfoundland and other parts of Canada is necessary to determine if the decline in NTD rate is maintained, and to enable further evaluation of prevention strategies. National surveillance of congenital anomalies including NTDs is a critical public health function that should be strengthened where necessary.We look forward to the results of a current epidemiologic study, funded by the Canadian Institutes of Health Research (CIHR), of NTDs in 7 Canadian provinces between 1993 and 2002. There is also research into the relationship between increased folic acid consumption and reduced risk of other congenital anomalies, cardiovascular disease and cancer -45. As tThe authors declare that they have no competing interests.CBC, complete blood countCI, confidence intervalCIHR, Canadian Institutes of Health ResearchFDA, Food and Drug AdministrationHCY, homocysteineMCV, mean corpuscular volumeMMA, methylmalonic acidNTD, neural tube defectRBC, red blood cellSPSS, Statistical Package for Social SciencesSL and CM oversaw the whole study and drafted the manuscript. SL carried out the analysis of NTD rate and statistical analysis. RW, LL, KSO and HS designed the study and carried out the data collection and the dietary assessment. ER, AL and VP participated in the design and carried out the blood analysis. MC carried out the collection of NTD data. All authors read, revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:


BRAF although the clinical importance of these mutations is not clear. We previously reported an analysis of BRAF mutations in metastatic melanoma samples from 68 patients. In this study, we correlated patient baseline characteristics, prognostic factors, and/or clinical outcomes with the presence of BRAF mutations. No significant differences were observed in age, gender, location of primary melanoma, stage at the diagnosis, and depth of primary tumor between patients with and without BRAF mutations. Melanomas harboring BRAF mutations were more likely to metastasize to liver (P = 0.02) and to metastasize to multiple organs (P = 0.048). Neither time to progression to stage IV nor overall survival were associated with BRAF mutations. In conclusion, we observed no significant differences in clinical characteristics or outcomes between melanomas with or without BRAF mutations. Although there was an increased frequency of liver metastasis and tendency to metastasize to multiple organs in tumors with BRAF mutations, there was no detectable effect on survival. Future prospective studies should include analysis of whether BRAF mutations in melanoma tumors correlate with an increased tendency to metastasize to liver or to multiple organs.Forty to eighty percent of melanoma tumors have activating mutations in  BRAF mutations were found with high frequency in malignant melanomas, including primary tumors and cell lines [BRAF mutations in cultured human melanoma cells inhibited the MAPK cascade causing growth arrest and promoting apoptosis [BRAF mutations in malignant transformation in melanoma.The mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to growth signals and activation of this pathway has been shown to be critical in tumor formation, particularly in melanoma -3. Recenll lines ,5. Supprpoptosis , furtherBRAF mutations in a cohort of metastatic melanoma patients [BRAFV599E, which was found in 40% of samples. Since little is known about the clinical implications of activating BRAF mutations in melanoma tumors, we examined whether the melanoma tumors harboring BRAF mutations in this cohort showed different clinical or biological features compared to the melanoma tumors without mutations.We have reported the analysis of patients  and noteBRAF mutations [Cryopreserved metastatic melanoma samples from 68 patients were selected from the Memorial Sloan-Kettering Cancer Center Tumor Bank. Patient demographic data were collected on the 68 patients whose tumors we had previously analyzed for utations . Data coBRAF (exons 11 and 15) was sequenced as previously reported [BRAF. In three patients, two to four metastatic sites were available for sequencing. For patients with multiple specimens, we considered only the first acquisition of tissue in assigning patients to mutant or wild type categories.reported . For 65/2 test, t-test or Cochran-Armitage test to trend. Survival distributions were estimated using the Kaplan-Meier method and compared using the log-rank test. Stage IV patients were stratified into two categories: those with stage M1a or M1b (lymph nodes, soft tissues and/or lung metastasis) and those with stage M1c .The patients were first seen at MSKCC between June 1993 and April 2000. Clinical follow up was available through April, 2003. Comparisons between mutated and wild type were made using either the χBRAF, including one with mutations in both BRAF and NRAS, and 38 patients were wild type. Overall, mutations in BRAF exons 11 and 15 were detected in 30 of 68 (44%) patients.We studied 74 cryopreserved metastatic melanoma samples from 68 patients: 42 men and 26 women Table . Thirty-BRAF mutations (p = 0.12). Similarly, there was no difference in the distribution of primary sites and stages at diagnosis between patients with and without BRAF mutations. We noted that among the 7 melanomas arising from the head and neck region, only 1 harbored a BRAF mutation. Although there were too few of these patients for a meaningful statistical analysis, this observation is consistent with a recent report indicating that mucosal melanomas did not harbor BRAF mutations [BRAF mutations, and 4.83 mm (range: 0.4, 35 mm) for patients without (p = 0.29). The effect of BRAF mutation on other known prognostic features of primary tumor such as the presence or absence of ulceration, regression, tumor-infiltrating lymphocytes, lymph-vascular invasion, and mitotic index could not be assessed because this information was available for only a small proportion of patients.Patients' age ranged from 29 to 97 years; there was no statistically significant difference in patients' age with regards to utations ,9. The mBRAF mutations were more likely to have metastasis to liver compared to those without the mutations   (Table BRAF mutations in their melanomas were found to have more than one metastatic site compared to only 11 of the 24 patients (37.5%) with wild type BRAF.Patients with tumors harboring BRAF mutations, 18 patients received systemic therapy of who two patients achieved complete remission (response rate 11.1%). Fifteen patients with wild-type BRAF received systemic therapy of whom three patients achieved complete remission and two achieved partial remission (response rate 33.3%) (p = 0.12).We examined the response to systemic therapy (chemotherapy or biochemotherapy) for the 33 patients who received such treatments. For patients with BRAF mutations (data not shown). As this is a retrospective study, we cannot rule out the possibility that differences in interval assessments affected our ability to detect a difference in time to progression. On the other hand, date of death is an endpoint not affected by interval assessment times. There was no statistically significant difference between patients with BRAF mutations and those without BRAF mutations.There was no statistically significant difference between time to progression to stage IV disease either from the time of diagnosis or from stage III in patients with or without BRAF mutations has been reported in malignant melanoma [High frequency of melanoma ,5,7, howBRAF mutations are common in melanocytic nevi [BRAF occur early in melanocytic transformation and play an important role in the initiation of malignant transformation. Recently, an alternative view has been suggested by Dong et al who confirmed the high frequency of BRAF mutations present both in nevi and later stage melanomas but found few BRAF mutations in early stage radial growth phase melanomas [BRAF mutations are not involved in the initiation of the majority of melanoma, but rather play a role later in progression.The observation that tic nevi  has led elanomas . They inBRAF mutations in melanoma, we analyzed the clinical characteristics of 68 melanoma patients whose tumors we had previously analyzed for BRAF [BRAF mutation were more likely to have metastasis to the liver and tended to have more organs involved with melanoma than patients without mutations. This is consistent with the idea that activating BRAF mutations affect the pattern of metastatic spread in melanoma, although we await confirmation of these findings in a prospective study.Since little information was available on the biological effects of activating for BRAF . We founBRAF mutations, 15 patients without detectable mutations). There was a trend towards lower response rates among patients with mutations, although this trend was not statistically significant and is confounded by the small number of patients, the heterogeneity of treatments these patients received, and the retrospective nature of these analyses. This is a question that deserves to be revisited in a prospective manner.In our cohort of subjects, there were 33 patients who received systemic therapy . They did not analyze the effect of BRAF mutations on the site of metastatic spread or other biological characteristics of the tumor.Kumar and colleagues found that melanoma patients with utations ,13. TheiBRAF mutation in a metastatic melanoma lesion was associated with a poor prognosis as measured by shortened survival [BRAF mutation. The patient characteristics were not reported by Houben and colleagues but they indicate that most patients had soft-tissue metastases (M1a or M1b). In contrast, most of our patients had M1c melanoma and this could account for the different findings.Houben et al. reported that the presence of survival . In our BRAF mutations. In one patient in whom 2 lung metastasis collected over a period of one month were analyzed, one metastasis contained a BRAFV599E mutation; the other metastasis was wild-type for BRAF. In another patient, metastasis from lung, gastrointestinal (GI) tract, lymph node, and soft tissue were collected of a period of 34 months. All tumors harbored the BRAFV599E mutation except for the GI metastasis which was wild-type. It is possible that this discordance represents a problem with assay sensitivity, but we cannot rule out the possibility that there is true heterogenicity among metastasis with regard to BRAF mutations. Although this discordance among metastasis seems to contradict the observation that BRAF mutations are an early event in melanocytic nevi transformation, one possibility is that in melanomas arising from non-nevus melanocytes, BRAF mutation is a late event occurring in individual metastasis. Consistent with this, Shinozaki et al. recently reported that the incidence of BRAF mutation of primary melanoma did not correlate with Breslow thickness, and there was significantly higher frequency of BRAF mutation in metastasis than in primary melanoma, arguing that BRAF mutation maybe acquired during development of metastasis [BRAF mutational status of the primary and metastasis did not correlate [In three patients, multiple metastatic samples were available for analysis; in 2 of these patients, there was discordance in the presence of detectable tastasis . Houben orrelate . This isBRAF mutations and clinical outcomes in metastatic melanoma. Although we observed a statistically significant higher frequency of liver metastasis and tendency to metastasize to multiple organs in patients with BRAF mutations, there was no significant effect on survival or response to systemic therapy detected by this study. Although this analysis is limited by its retrospective nature and the relatively small number of patients, it appears unlikely from these observations that there will be a major qualitative difference in the biological behavior between melanomas with and without BRAF mutations. Larger prospective studies are required to verify these observations and to clarify other biological consequences of BRAF mutations in melanoma.In summary, this analysis represents the largest study to date correlating


Research indicating that people increasingly prefer to die at home suggests that palliative care is likely to play a more prominent role in the future of Canada's health care system. Unfortunately, at a time when research evidence should be informing policy and service delivery, little is known about health service utilization by Canadians at the end of life. One existing mechanism that can help address this gap is provincial administrative health data. The purpose of this study was to explore the potential of administrative health data to identify characteristics of palliative care users, patterns of formal service utilization and predictors of palliative care use.Bivariate and multivariate analyses were used to examine data from the Capital Health Region, British Columbia Linked Health Databases for the period 1992/93 to 1998/99. The databases examined include continuing care, physician claims, hospital separations, and vital statistics. As the name implies, these databases can be linked at the individual level using unique identifiers so that health services utilization can be tracked across sectors.General patterns of service use among palliative care patients suggest that general practitioner and medical specialist visits have decreased over time and the utilization of hospital beds has increased. Utilization of community-based services (i.e. home support and home nursing care) shows an overall pattern of decline. However, when compared to non-palliative care patients, palliative care patients spent fewer nights in hospital, used fewer hours of home support, and had a greater number of home nursing care visits.Administrative health databases can provide valuable information for examining service utilization patterns over time. However, given that decisions surrounding the designation of palliative care include factors beyond the scope of administrative databases , these databases should only be seen as one source of information to inform service delivery and policy decision making. Despite the fact that we all die, the philosophy and practice of palliative and end of life care is relatively new . CertainQuality palliative and end of life care has emerged as a core value of Canada's health system . Indeed,At a time when research evidence should be informing policy and health service delivery, little is known about health service utilization by Canadians at the end of life. Although countries such as Australia and the United Kingdom have established national palliative care (PC) surveillance data and extensive work has been done in the United States to identify key data elements pertaining to the end of life, Canada has been slower to develop in this area. Many Canadian PC programs have developed regional data systems , and a cThe primary purpose of this paper is to explore the usefulness of provincial administrative health databases as a source of information to inform health care decision making and policy development in palliative and end of life care. Data from the Capital Regional District in British Columbia, Canada are examined for the period 1992/93 to 1998/99. Specifically, our analyses identify characteristics of PC patients, patterns of formal service utilization over time, and predictors of PC service utilization. The implications of our findings for both research and policy, along with recommendations for future data collection initiatives are addressed.Research on dying persons has grown in the past decade due, in part, to a rapidly aging population, policy concerns about increasing health expenditures, and a perception that the quality of care at the end of life is inadequate ,12. SeveLevels of PC services utilization tend to be similar for both males and females ,15,16,18Some research has examined the patterns of formal service utilization of PC patients. The more frequently cited measures of service utilization include scope of services used, along with length of stay (on a particular PC service) and location of death ,13,24,25can be done with these data rather than what the data are actually telling us about PC patients and their health care utilization.While administrative health databases have been used for research purposes for decades in Canada, it was the establishment of the Manitoba Centre for Health Policy and Evaluation and the subsequent development of their population health information system, POPULIS , which bData for this paper came from the British Columbia Linked Health Database. Housed at the University of British Columbia (BC), this database contains all provincial administrative health data collected by the BC Ministry of Health including physician claims, hospital separations, long-term care data, pharmacare data and vital statistics. Each of these components can be analyzed separately, as a stand-alone database, or linked through unique identification numbers and examined in combination or as a whole. The data were originally collected as part of a larger project examining the impact of regionalization in BC from 1990/91 to 1998/99 . HoweverGiven that PC patients could not be identified in the data prior to 1992/93, for the purposes of this paper, data from fiscal years 1992/93 to 1998/99 for all databases  were included. Results are based on a 100 percent sample of the population aged 50 and over residing in the Capital Regional District, on Vancouver Island, British Columbia, Canada for each of the years. Both univariate and multivariate analyses were used to determine the usefulness of administrative databases for PC research in British Columbia.The starting point for these analyses was the identification of all patients designated as being in need of PC . This was made possible through the 'service type' variable found in the direct care services database, one of many databases that make up the continuing care component of the administrative databases. Next, using the unique identification numbers that are consistent across the different databases, it is possible to link these different datasets thereby allowing for the examination of individual service utilization across the various segments of the health care system .The continuing care database is a suite of databases, however for the purpose of this paper only the direct care services and home support databases were accessed. The direct care services databases were used in this paper for 2 purposes. First, and most importantly, it allowed us to identify those designated palliative through an individual variable. It should be noted that those never entering the continuing care system but who receive palliative services in another sector  could not be identified as such and thus are not designated palliative for these analyses. Second, the direct care services database also contains information on home nursing care. For this paper, the number of home nursing visits received by each individual in a given year was calculated. Home support utilization is tracked on a monthly basis. From these files it was possible to calculate the number of hours of home support received by each individual in a given year.All claims made by physicians are tracked in the Medical Services Plan database. It is therefore possible to calculate the number of visits to a physician made by individuals over the course of a year. The hospital separations database is so named because a hospital patient is entered into the system only when they leave the hospital (this includes deaths). Since admission and separation dates are included as variables, it is possible to calculate the number of nights each individual spends in the hospital (no overnight stay is assigned a 0). Total nights in a given year for each individual can then be summed. The vital statistics data that was accessed included underlying cause of death. This variable is comprised of ICD-9 codes that can be classified into disease categories. This procedure allowed for the identification of cancer versus non-cancer deaths used in the multivariate analysis.Each of these health databases includes a small number of demographic variables such as age and gender, and geographic identifiers such as health authority and census tract. Marital status is included in one of the continuing care databases. This lack of what are typically referred to as 'control variables' is one of the biggest limitations of administrative databases. To compensate, it is possible to link Census variables at an aggregate level to the administrative data. Of course this procedure only works for variables that lend themselves to averages. For example, it is possible to calculate the average household income for an area but it is not possible to calculate the average gender for an area. Using this premise, average household income was linked to the administrative data at the enumeration area level . In brief, the average household income for an enumeration area is assigned to an individual residing anywhere within the enumeration area. Thus, income is an aggregate measure while the remainder of the variables used in this paper are individual measures.Finally, as mentioned earlier, each of these databases is linkable. The term linkable is used to describe the assignment of the same unique identification number to an individual regardless of database. In other words, Person A will be assigned the same number in the hospital database and the continuing care database. As such, linkable databases allow researchers to examine health service utilization and health status of the same individual across sectors. Without the ability to link, it would be impossible, for example, to examine underlying cause of death  in relation to the designation of palliative care (continuing care database).As described in the preceding paragraphs, 5 health service utilization measures were calculated: number of general practitioner visits per year; number of medical specialist visits per year; number of nights spent in hospital per year; number of hours of home support received per month; and number of home nursing care visits received per month. Next, using these variables, three different analyses were conducted:1. Descriptive statistics (distribution, mean, median) were used to examine PC patient characteristics and health service utilization by gender. All those identified as being in need of PC for each fiscal year were examined by age, gender, income, and the 5 health service utilization measures. The sample size of PC patients ranged from a low of 74 in 1992/93 to a high of 568 in 1997/98.2. Because service use is likely to differ by diagnosis, we compared patients designated as being in need of PC with patients who were not designated as in need of PC in order to control for diagnosis. Previous research indicates that the majority of persons receiving specialized palliative and end of life care have a cancer diagnosis . While i3. To examine the influence of a PC designation on health service utilization, five multiple linear regression models were estimated using the 1998/99 data from the palliative and non-palliative sample (n = 2,734) described above. The following five dependent variables were regressed on age, gender, average household income, and designation of palliative/non-palliative: (1) number of general practitioner visits; (2) number of medical specialist visits; (3) number of nights spent in the hospital; (4) number of hours of home support; and (5) number of home nursing care visits. Prior to conducting the multivariate analyses, assumptions of normality, linearity, and collinearity were tested and adjustments were made where necessary.The linked administrative database allowed for a description of patient characteristics including age, gender, and income. Table The means for the use of the five health services are presented in Table Health service utilization trends observed across the years show that the number of annual general practitioner and specialist visits is fairly steady until 1997/98 when there is a dramatic drop. In contrast, the number of nights spent in hospital steadily increases over the same period until 1998/99 when a sharp reduction in hospital bed utilization is observed. Patterns of home support and home nursing care utilization over time are more variable. Home support hours per month exhibit a pattern similar to number of hospital nights, although the increases and decreases are not as large. Number of home nursing care visits has fluctuated over the years and shows a general pattern of decline from 1992/93 to 1998/99.Results comparing cancer patients designated as being in need of PC and cancer patients who were not designated as in need of PC are presented in Table Regression results for the five health service utilization regression models are presented in Table The primary purpose of this study was to explore the usefulness of provincial administrative health databases as a source of information to inform health care decision making and policy development in palliative and end of life care. Administrative databases represent an existing source of longitudinal information on health system users. The need to track individuals' health service utilization over time has long been recognized by researchers, and this is a key advantage of such databases. At the same time, however, the breadth of information collected is limited. For example, no direct measurements of quality of life, or for that matter, quality of death are compiled. The researcher who makes use of the databases must therefore work within these confines. The findings from the present study provide insights into the precise manner in which these databases can be of value to health services researchers, within these limits. More specifically, the analyses conducted for this paper exhibit that a sample of palliative patients can be identified in one of the many provincial administrative health databases. Since individuals can be linked across sectors, it is possible to examine a number of health status and utilization indicators of these individuals. Finally, linking the health databases with the vital statistics variables gives the researcher insight into the underlying cause of death.The limitations of administrative databases arise directly from their intended purpose. They were created to serve a billing role, and although they are of value as a research tool, their limitations should be noted. First, socioeconomic variables are limited to age, gender and income. The importance of these variables is unquestioned; however, a wider range of such variables is normally a requirement for social science based health services research. Second, the precise date within a year that patients are designated palliative is not available. Since health service resource utilization has been shown to be much greater during the final few months and weeks of life, information on date designated palliative would be informative. Third, the data that would allow for an examination of either quality of care provided or quality of dying – both of which are central to any study seeking to improve service delivery and associated outcomes – is not collected. Finally, given the complexities of data collection within the health care system, these databases are unlikely to capture all individuals in need of PC. For example, given the fluctuation in the number of palliative patients across years, with special emphasis on the precipitous decline observed between 1997/98 and 1998/99, it appears that the definition and coding of the variable designating an individual palliative may be subject to change over time. If these data are to be used for research purposes, this indicates the need for careful monitoring for continuity of both definition and coding procedures.While the primary objective of this paper was not to focus on the actual trends and findings, the results hold potential for future studies. For example, the observed decrease in both GP and specialist visits between 1992/93 and 1998/99 for males and females designated as being in need of PC may be indicative of changing trends. First, persons designated palliative are increasingly signaling their desire to die at home. Second, perhaps GPs are increasingly visiting dying patients at home, with the result that these visits are not captured in the health databases since physicians cannot bill for such visits. As GPs become more aware of the need for effective PC, and as they acquire the requisite skills to do so, the territory once thought to be the exclusive realm of the specialist may be experiencing erosion. In the larger picture, this may be indicative of a shift away from a curative medical model approach to end of life care, to a more appropriate social model.At the same time that GP and specialist visits among persons designated as being in need of PC have declined, mean number of nights spent in hospital annually have shown a general increase. Ostensibly, the increase in hospital nights until the 1997/98 period is a puzzling trend: especially given findings on the general population that show a decrease in nights spent in hospital throughout the 1990's . A potenAlthough it is not monotonic, the general decline in monthly home nursing visits between 1992/93 and 1998/99 is also somewhat puzzling on the surface, given the calls to enhance home nursing services for the dying. A comparison of home nursing visits between palliative and non-palliative cancer patients, however, reveals that those designated palliative receive considerably more home nursing visits. This latter observation is consistent with the increased care requirements at the end of life. In trying to decipher the differences between these two groups it would be important to examine the time of the visits in relation to death. For instance, do more visits occur in the last months or weeks leading up to death?In the multivariate analyses, it is of particular interest that being designated palliative is related only to number of home nursing care visits. In other words, being designated palliative has no bearing on general practitioner visits, medical specialist visits, number of hospital nights and number of hours of home support. This, of course, is contrary to bivariate findings presented in Table These data provide another piece of the overall picture of service utilization at the end of life. Research into palliative and end of life care is in its infancy at a time when effective and immediate information based on sound scientific research is urgently required. To the extent that administrative databases can bridge this gap, they are of immediate use. Their primary usefulness, however, is probably as a means of identifying and sketching a larger picture, and from that picture, facilitating the generation of key questions that may serve as platforms for launching more in-depth studies. For example, the decline over time in GP and specialist visits, combined with the general decline in home nursing visits for palliative persons raises the question of whether resources have met rhetoric, and if not, why not? Given that health care and policy decisions must include information on factors beyond the scope of administrative databases , these databases should only be seen as one piece of information to inform service delivery and policy decision making for patients and families at the end of life.The author(s) declare that they have no competing interests.All authors participated in the conceptualization and design of the study. DEA drafted the initial background, methods and results sections. KIS and DEA conducted all the analyses. KIS and RCR drafted the discussion section and provided additional edits to all sections. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To determine whether this phenomenon is shared by other herpesviruses, we investigated the effects of IFNs on human cytomegalovirus (HCMV) replication.Recent studies have shown that gamma interferon (IFN-γ) synergizes with the innate IFNs (IFN-α and IFN-β) to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. While pre-treatment of human foreskin fibroblasts (HFFs) with IFN-α, IFN-β or IFN-γ alone inhibited HCMV plaque formation by ~30 to 40-fold, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV plaque formation by 163- and 662-fold, respectively. The generation of isobole plots verified that the observed inhibition of HCMV plaque formation and replication in HFFs by IFN-α/β and IFN-γ was a synergistic interaction. Additionally, real-time PCR analyses of the HCMV immediate early (IE) genes (IE1 and IE2) revealed that IE mRNA expression was profoundly decreased in cells stimulated with IFN-α/β and IFN-γ (~5-11-fold) as compared to vehicle-treated cells. Furthermore, decreased IE mRNA expression was accompanied by a decrease in IE protein expression, as demonstrated by western blotting and immunofluorescence.We have found that as with HSV-1, IFN-γ synergizes with the innate IFNs (IFN-α/β) to potently inhibit HCMV replication in vivo.These findings suggest that IFN-α/β and IFN-γ synergistically inhibit HCMV replication through a mechanism that may involve the regulation of IE gene expression. We hypothesize that IFN-γ produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination  Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that affects 60–80% of the human population . The lytLike other herpesviruses, HCMV establishes lifelong latency in its host from which reactivation can occur and cause severe and fatal disease in immunocompromised individuals . Cellulain vitro and in vivo [Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) are important components of the host immune response to viral infections. IFN-α and IFN-β are produced by most cells as a direct response to viral infection -24, whil in vivo . In addi in vivo , HCV [40 in vivo .in vitro.In the present study, we examined the effects of IFN-α, IFN-β and/or IFN-γ on HCMV replication in human foreskin fibroblasts (HFFs). Treatment of HFFs with IFN-α, IFN-β or IFN-γ separately inhibited HCMV replication by ≤ 40-fold in both plaque reduction and viral growth assays. In contrast, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV replication 10–20 times greater than that achieved by each IFN separately. This effect was synergistic in nature and the mechanism of inhibition may involve, at least in part, the regulation of IE gene expression. As with HSV-1 [The abilities of human IFN-α, IFN-β or IFN-γ to inhibit the replication of HCMV were initially compared in a plaque reduction assay on HFFs. Viral plaque formation was reduced by 9-, 37- or 29-fold in fibroblasts treated with 100 IU/ml of IFN-α, IFN-β or IFN-γ, respectively -treated cells, a known DNA synthesis inhibitor of HCMV, the level of inhibition achieved in GCV-treated cultures was comparable to that in IFN-α and IFN-γ- or IFN-β and IFN-γ-treated cultures at 3 and 4 d p.i.  and type II IFN (IFN-γ) treatment, four-day viral growth assays were performed. In cultures treated with IFN-α, IFN-β or IFN-γ, viral replication was undetectable or below the lower limit of detection at 1 and 2 days (d) post-infection (p.i.). At 3 d p.i., however, HCMV replicated to average titers of 8350, 1050 or 985 PFU/ml in IFN-α-, IFN-β- or IFN-γ-treated cultures, respectively  are the first class of viral genes expressed after HCMV entry into the cell . AlthougIE protein expression plays a pivotal role in controlling subsequent viral and cellular gene expression during productive HCMV infection , such thIf IFN-α/β and IFN-γ synergistically inhibit HCMV replication through inhibition of IE gene expression, we hypothesized that this inhibitory effect would be maintained after multiple rounds of viral replication. To address this question, IE protein expression was analyzed by indirect immunofluorescence over a 5-day period. For all treatment groups, IE protein expression was detected as early as 1 h p.i.; however, as viral replication progressed IE protein expression among IFN-treated groups varied (data not shown). Notably, by day 5 p.i., nearly 100% of the cells treated with vehicle, IFN-α or IFN-β alone stained positive for IE72/86, and approximately 87% of the cells treated with IFN-γ alone were expressing the IE proteins Figure . In contin vitro, these studies are limited as they only examine the effect of one type of cytokine on viral replication rather than examining cytokines in combination. In support of the latter, recent studies have shown that type I and type II IFNs function, in synergy, to inhibit both RNA and DNA viruses, including HCV [in vivo inflammatory response that results after viral infection. The results presented herein are consistent with this hypothesis and establish that type I (IFN-α and IFN-β) and type II (IFN-γ) IFNs synergistically inhibit the replication of HCMV.The immune response to viral infection is responsible for preventing viral dissemination and uncontrolled replication within the host. Following viral infection, type I IFNs are secreted by infected cells and function to induce an antiviral state in neighboring uninfected cells. Infiltrating immune cells, such as NK cells and macrophages, secrete numerous chemokines and cytokines that contribute to the overall antiviral response. Upon activation of the adaptive immune response, T-cells can further add to the milieu of immune cytokines present at the site of viral infection by secreting additional cytokines, including IFN-γ. Although several studies have examined the effects of proinflammatory cytokines on HCMV replication ding HCV , SARS-Coding HCV , Lassa vding HCV  and HSV-ding HCV . These sin vitro. The inhibitory effect by IFN-α/β and IFN-γ was synergistic in nature  demonstrated that treatment of cells with both IFN-α and IFN-γ potently inhibits HCMV replication; however, this study neither determined whether the effect was synergistic nor identified the mechanism of inhibition. However, the authors suggested that IFN-mediated inhibition of HCMV might occur at or prior to early gene expression [Previously, Yamamoto, pression . Similarpression  and/or Hpression ,46. Usinpression ,49,50. Iet al. (1998) have identified numerous genes differentially regulated by IFN-α, IFN-β or IFN-γ [Type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ) activate distinct but related Jak/STAT signal cascades resulting in the transcription of several hundred IFN-stimulated genes . Althougor IFN-γ . In partin vitro. We hypothesize that IFN-γ produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination and facilitate the establishment and/or maintenance of latency in the host. Further studies are required to evaluate the role(s) of both type I and type II IFNs in the regulation of HCMV replication.Guidotti and Chisari have reported a model of noncytolytic control of viral infections by the innate and adaptive immune response, in which cytokines are implicated as having a direct role in viral clearance . Here we2. HCMV strain RVdlMwt-GFP was propagated in HFFs as previously described [HFFs (Viromed, Minneapolis, MN) were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin G (100 U/ml), streptomycin (100 mg/ml), 2 mM L-glutamine, 1 mM sodium pyruvate and 100 μM non-essential amino acids at 37°C in 5% COescribed . RVdlMwtescribed .Recombinant human universal IFN-α, IFN-β and IFN-γ  were added to cell cultures 12 h prior to HCMV infection and maintained after viral infection. Concentrations of 100 IU/ml of each IFN were used in all experiments unless stated otherwise.For plaque reduction assays, vehicle- and IFN-treated HFFs were infected with a fixed inoculum of Towne-GFP. After 2 h adsorption, the inoculum was removed and medium containing 1.0% methylcellulose (Fisher Scientific, Houston, TX) and the respective IFN(s) was added to the cells. Plaque numbers were determined 14 d later by fluorescent microscopy (Nikon TE300 inverted epifluorescent microscope, Nikon USA, Lewisville, TX).For viral replication assays, vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 2.5. After 2 h adsorption, the inoculum was removed, monolayers were washed twice with 1X PBS, and fresh IFN-containing medium was returned to each well. For GCV-treated groups, 100 μM GCV (Sigma, St. Louis, MO) was added to culture medium immediately following infection. One, 2, 3 or 4 d p.i. cells and medium were harvested and titers of infectious virus were determined by a microtiter plaque assay on HFFs .a/Da+db/Db > 1 and da/Da+db/Db <1, where da and db are the IFN concentrations needed to jointly produce the effect under consideration, and Da and Db are the IFN concentrations capable of producing the effect on their own, termed isoeffective doses [95 values at various concentrations of IFN-α or IFN-β in the presence of various concentrations of IFN-γ. Concave isoboles are indicative of synergy while convex isoboles are indicative of an antagonistic effect  and 250 ng was reverse transcribed in a total volume of 20 μl using the iScript cDNA Synthesis Kit (Biorad, Hercules, CA) according to the manufacturer's instructions. For real-time PCR, 1 μl of cDNA was amplified in 1X iQ SYBR Green Supermix containing specific primer pairs using the iCycler iQ Real-Time PCR Detection System (Biorad). The optimal primer concentrations and sequences were as follows: 200 nM IE1, sense 5' CAAGTGACCGAGGATTGCAA 3', antisense 5' CACCATGTCCACTCGAACCTT 3' ; 200 nM IE2, sense 5' TGACCGAGGATTGCAACGA 3', antisense 5' CGGCATGATTGACAGCCTG 3' [target)ΔCT(vehicle-sample)/(E18S)ΔCT(vehicle-sample) [Vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 2.5. Six h p.i., total RNA was prepared using a RNeasy Mini Prep kit  according to the manufacturer's instructions. Samples were treated with DNase I (Ambion, Inc., Austin, TX), RNA concentration and purity were determined spectrophotometrically  . DiffereVehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 2.5. Twelve h p.i., the cells were harvested in 500 μl of 1X RIPA buffer containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and 1 mM PMSF. Lysates were sheared 3X with a 27G 1/2 needle and cell debris was pelleted by centrifugation at 14,000 r.p.m. at 4°C. Total protein concentrations from cleared supernatants were estimated with a Micro BCA™ Protein Assay Kit (Pierce, Rockford, IL), 50 μg of total protein were resolved on 10% SDS-polyacrylamide gels and transferred by blotting to PVDF membranes (Amersham Biosciences, Piscataway, NJ). Non-specific reactivity was blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and blots were incubated for 1 h at room temperature with a polyclonal antibody that recognizes the HCMV IE proteins (IE72/86), kindly provided by Daniel N. Streblow . The blo2 (Molecular Probes, Eugene, OR) for 30 minutes at 37°C, followed by a 2 minute incubation with 1 μM 4',6-diamidino-2-phenylindole, dihydrochloride  at room temperature. Cells were coverslipped and mounted in Prolong Antifade mounting medium (Molecular Probes), visualized on a Zeiss Axio Plan II microscope (Thornwood, NY) and images were analyzed with deconvolution SlideBook™ 4.0 Intelligent Imaging software (Intelligent Imaging Innovations, Denver, CO). To determine the number of HCMV-infected cells, three fields of view (100X) for each treatment group were considered and the percent of IE-positive cells was calculated as: (average number of IE-stained cells/average number of DAPI-stained cells)×100.Vehicle- and IFN-treated HFFs were infected with Towne-GFP at a MOI of 1.0. Five d p.i., cells were washed 3X with 1X PBS, fixed with 1:1 methanol/acetone for 10 minutes at room temperature, washed again with 1X PBS, and blocked with 4% BSA/PBS for 15 minutes at room temperature. Cells were incubated for 1 h at 37°C with a HCMV IE antibody  diluted 1:200 in 0.5% BSA/PBS. Cells were then stained with 1:50 Alexa Fluor 568-conjugated goat anti-mouse IgG F(ab')t test (GraphPad Prism© Home, San Diego, CA).Data are presented as the means ± standard error of the means (sem). Data from IFN-treated groups were compared to vehicle-treated groups and significant differences were determined by one-way analysis of variance (ANOVA) followed by Tukey's post hoc The author(s) declare that they have no competing interests.BS and HL conceived of the study, participated in the experimental design, performed all experiments and drafted the manuscript. RG and CM participated in the coordination and design of the study. All authors read and approved the final manuscript.


Workers of social insects prevent other workers laying eggs to increase colony efficiency and not -- as traditionally thought - purely because workers are more related to the queen of the colony The order and harmony that appears to bless the lives of many social insects, from ants to bees, has long fascinated naturalists. That individual workers seem to routinely sacrifice their own (reproductive) interests for the good of the colony has also piqued the interest of philosophers and kings, for obvious reasons. But scratch the surface and that blissful harmony reveals a complex feat of social engineering that is both exquisitely organized and potentially ruthless.One of the altruistic behaviors that social insects are famous for is that one or a few queens perform most or all of the reproduction in a colony, while workers are, for the most part, non-reproductive. The evolution of this social structure partly stems from the unusual sex determination system of social insects, in which unfertilized eggs (of either workers or queens) develop into males and fertilized eggs (produced only by queens) develop into female queens and workers. This creates unusual relationships between family members that affect how W. D. Hamilton's theory of “kin selection” operates in these species. Kin selection, as elegantly summarized by “Hamilton's Rule,” predicts that the altruistic behavior of workers—that is, investing in the reproduction of others in the colony rather than in their own reproduction—can evolve if the indirect reproductive payoff to workers (i.e., via reproduction by relatives) is higher than the cost of the missed opportunity for direct reproduction. Kin selection revolves around relatedness because relatedness determines the magnitude of indirect reproductive payoffs. However, based on a survey of 50 species of ants, wasps, and bees, Rob Hammond and Laurent Keller now demonstrate that the behavior in the colony cannot be accounted for simply based on relatedness patterns, but that it is necessary to consider how colony efficiency influences behavior.In some social insect colonies, workers do lay eggs, in a sense “cheating” on the other workers who are investing in the queen's reproduction rather than in their own. Such action can be severely penalized by other workers, who aggressively police the colony for the illicit offspring—or behavior—of their guilty colleagues. In honeybees, where this behavior was first shown, workers remove worker-laid eggs within hours by eating them, and, in some ants, more draconian methods lead to the mutilation of the culprit caught in the act of laying.Why workers police worker reproduction in some colonies and not in others can also be influenced by relatedness. If a queen is monogamous and mates only once, then each worker will actually be more related to her nephew (produced by a sister worker) than to her brother (produced by the queen); in this case, workers should tolerate other workers' male offspring. But if the queen mates more than twice (as in honeybees) or if there are multiple queens heading a colony, then the relationship between workers becomes diluted , and workers are more closely related to brothers than to nephews. In this case, workers should clamp down hard on any worker breeding and raise only the queen's sons (in addition to her daughters).But workers policing the reproduction of their fellow workers could also be advantageous if the energy invested by workers into laying eggs—which would otherwise be used in foraging and legitimate brood rearing—detracts from the overall efficiency and growth of the colony. Although there is some evidence for this “efficiency hypothesis,” it is widely accepted that the driving force behind policing is primarily explained by patterns of relatedness. By doing a detailed comparative phylogenetic analysis of different species, Hammond and Keller put the “relatedness hypothesis” to the test and—contrary to expectations—found evidence that this genetic incentive for workers to police the reproduction of other workers cannot account for its widespread prevalence among social insects.One prediction from the relatedness hypothesis is that the extent to which workers produce male offspring is determined by the relatedness of the workers. By contrast, the efficiency hypothesis predicts no such relationship. In line with this, Hammond and Keller's survey reveals that no matter how related workers are to each other, most males across this broad range of species are produced by queens. In other words, worker-policing does not depend on relatedness, so other factors—such as colony efficiency—must act as an important constraint on worker reproduction. This, Hammond and Keller emphasize, does not amount to showing that kin selection is unimportant—but it does mean that the harmony and regulation of reproduction in social insects is much more complex than expected from simple theoretical expectations based solely on relatedness.


A fundamental question in developmental biology is, how does a multicellular organism develop from a single cell? It's clear that one cell begets two, two beget four, and so on, but how do the newly created cells know which developmental fate to pick? Major insights into this question have come from identifying genes, molecules, and intercellular signaling pathways involved in a wide range of developmental processes. Operating in labyrinthine, often overlapping pathways, intercellular signals determine whether a cell divides, differentiates, migrates, and even lives or dies.Caenorhabditis elegans a popular developmental model. C. elegans can exist as either a male or a hermaphrodite, and for some biologists, the hermaphrodite vulva—which consists of just 22 cells—is the perfect system for working out key aspects of intercellular signaling and cell fate.Scientists prefer to work out such problems in organisms with a manageable number of cells for obvious reasons, making the 959-cell soil nematode In a new study, Alex Hajnal and colleagues challenge conventional thinking about vulval cell specification by identifying an enzyme that can amplify a signal's range and help turn three non-vulval precursors into vulval cells. Surprisingly, the enzyme, called ROM-1, accomplishes this feat by acting in the signal-receiving vulval precursor cells, rather than in the signal-sending cell that instructs the vulval cell fates.The worm vulva forms a bridge between its gonad and the opening to the outer epidermal layer, called the cuticle. In the current model of vulval formation, a group of twelve epidermal cells, called Pn.p cells, lines the ventral surface of the worm. Six of these cells, P3.p–P8.p, the vulval precursor cells (VPCs), have the potential to become vulval cells. During postembryonic development, the anchor cell in the larval gonad secretes an epidermal growth factor  that activates the EGFR/RAS/MAPK signaling pathway and induces just three of the precursors to differentiate into vulval cells. The VPC closest to the anchor cell, P6.p receives most of the signal, and differentiates into eight vulval cells that form the tube linking the uterus to the gonad. Positioned on either side of P6.p, P5.p and P7.p receive a slightly attenuated signal, which, combined with a lateral signal from P6.p, gives rise to seven vulval cells that form vulval structures. The other three vulval precursors, P3.p, P4.p, and P8.p, it was thought, are too far away to receive the vulval induction signal and fuse into the surrounding epidermis.rom genes were not required for normal vulval development, the authors had a closer look at their role in vulval cell fate specification. Since loss of ROM-1 reduces the severity of a defect (in this case, multiple vulvas) caused by hyperactivation of the EGFR/RAS/MAPK pathway but has no effect on the precursors closest to the anchor cell, the authors conclude that ROM-1 enhances the EGFR/RAS/MAPK pathway, allowing it to reach the distant P3.p, P4.p, and P8.p precursors.The LIN-3 epidermal growth factors sit nestled within the cell membrane and must be “processed” to become active, prompting Hajnal and colleagues to look for candidate enzymes that could be doing the processing. They investigated the Rhomboid family of proteases, which are known activators of epidermal growth factor transmembrane proteins, and found one, ROM-1, with the amino acid profile required for catalytic protease activity. After showing that LIN-3 exists in two variant forms of different lengths, the longer one carrying a stretch of 15 extra amino acids in the region that is cleaved off to yield an active growth factor. Hajnal and colleagues show that ROM-1 only acts on the longer form to regulate the EGFR/RAS/MAPK pathway—and that the ROM-1/LIN-3 interaction occurs in the VPCs, independently of the anchor cell. They go on to propose a two-step model of vulval cell specification in which ROM-1 “extends the range” of the anchor signal, relaying it from the proximal to the more distant precursor cells by promoting the secretion of the long version of LIN-3. In normal development, LIN-3 secretion by the VPCs may serve initially to maintain the differentiation potential of all the precursors, while the anchor cell signal may seal their fates at a later phase.


It has previously been shown that an anterior cruciate ligament (ACL) injury may lead to impaired postural control, and that the ability to maintain postural control is decreased by fatigue in healthy subjects. To our knowledge, no studies have reported the effect of fatigue on postural control in subjects with ACL injury. This study was aimed at examining the effect of fatigue on balance in single-limb stance in subjects with ACL injury, and to compare the effects, and the ability to maintain balance, with that of a control group of uninjured subjects.Thirty-six patients with unilateral, non-operated, non-acute ACL injury, and 24 uninjured subjects were examined with stabilometry before (pre-exercise) and immediately after (post-exercise) short-duration, sub-maximal cycling. In addition, the post-exercise measurements were compared, to evaluate the instantaneous ability to maintain balance and any possible recovery. The amplitude and average speed of center of pressure movements were registered in the frontal and sagittal planes. The paired t-test was used for the intra-group comparisons, and the independent t-test for the inter-group comparisons, with Bonferroni correction for multiple comparisons.No differences were found in the effects of exercise between the patients and the controls. Analysis of the post-exercise measurements revealed greater effects or a tendency towards greater effects on the injured leg than in the control group. The average speed was lower among the patients than in the control group.The results of the present study showed no differences in the effects of exercise between the patients and the controls. However, the patients seemed to react differently regarding ability to maintain balance in single-limb stance directly after exercise than the control group. The lower average speed among the patients may be an expression of different neuromuscular adaptive strategies than in uninjured subjects. The anterior cruciate ligament (ACL) is the most commonly injured ligament in the knee. The risk of future joint problems, in the form of functional limitations, secondary lesions, and arthrosis, is increased following such an injury. Secondary effects commonly seen after an ACL injury include defective neuromuscular function with reduced strength and functional performance, a different movement and activation pattern, defective proprioception and impaired postural control . ImpaireFatigue is caused by a combination of different physiological mechanisms occurring at both the central and peripheral levels , affectiTo our knowledge, no studies evaluating the effect of fatigue on postural control in subjects with ACL injury have been reported. The main purposes of this study were: 1) to examine the effect of short-duration, sub-maximal exercise performed on a cycle ergometer, on postural control, measured by stabilometry in single-limb stance on a force platform, in individuals with ACL injury in comparison with that of a control group of uninjured subjects, and 2) to explore the patients' instantaneous ability to maintain balance in single-limb stance after exercise, in comparison with that of the control group. Furthermore, the patients were compared to the control group in order to verify previous findings that postural control is affected in both legs by a unilateral ACL injury . No compThirty-six patients (18 men and 18 women) were included in the study. Inclusion criteria were: 1) age between 15 and 35 years, 2) unilateral, non-operated, non-acute ACL deficiency with or without associated lesions of other structures of the knee, 3) an uninjured contralateral extremity, back and neck, and 4) no history of neurological disease, vestibular or visual disturbance. Their mean age was 26 years (SD 5 years), mean height 174 cm (SD 9 cm), and mean body mass 72 kg (SD 13 kg). Their median activity level before injury was 6.5 (range 3 to 9) and on the test occasion 4 (range 1 to 9) according to the Tegner activity level scale . The meaThe measurements of twenty-four uninjured volunteers (11 men and 13 women) from a previous study , with no-1; and 2) number of movements exceeding 10 mm from the mean value of CP (DEV 10)1) average speed of CP movements in mm·s, giving a total of four variables (two variables in each plane). The mean value of CP is the average distance (mm) of the CP from the reference lines, and DEV 10 is the number of movements exceeding 10 mm from the mean value of CP. DEV 10 (n) reflects the deviation of CP (i.e., displacement of CP), and the average speed (mm·s-1) reflects the amplitude and frequency of CP movements. Figure Balance in single-limb stance was tested by means of a strain gauge force plate (33 × 38 cm) with the subject barefoot in a standardized position ,26,27 F. This meNo exertion at all, 13 = Somewhat hard, 15 = Hard, 17 = Very hard, 19 = Extremely hard, and 20 = Maximal exertion). The RPE scale was designed to increase linearly with exercise intensity and heart rate for work on a bicycle ergometer, and correlates closely with several physiological variables, including heart rate and blood lactate concentration [2max) results in a higher percentage of the maximum heart rate (HRmax); e.g., 75% of VO2max represents an intensity of 86% of HRmax [Short-duration, sub-maximal exercise was performed on a cycle ergometer. The subjects' heart rate was continuously recorded during the entire test. Borg's scale for Rating of Perceived Exertion  was used to assess the subjective effort level during exercise . On thisntration . A lineaof HRmax . The maxof HRmax . Effectsmax [max, perceived the exercise as hard or very hard , and had reached steady-state heart rate, i.e., after approximately 5 min.The rate of pedaling was kept constant at 60 revs/min. The level of exercise was calculated so as to be similar to that perceived during a general exercise session. The workload (W) was set individually, depending on the sex and physical condition of each subject, with the aim of reaching a heart rate above 60% of the predicted HRmax  in all sThe average of the right and left legs; i.e., (right+left)/2, was used for statistical analysis in the control group, since there were no clinically or statistically significant differences between the legs. The use of the mean value of both legs when performing parametric statistics can be questioned, since this may affect the data variability. It cannot, however, be excluded that a dominance of one or the other side exists, which is difficult to define , and themax; the mean level being 82% (SD 6%, range 66 to 92%) among the patients and 81% (SD 7%, range 68 to 99%) among the controls. The median power output produced by the patients and the control group at the end of fatiguing exercise was 125 W (range 75 to 200 W) and 150 W (range 100 to 200 W), respectively, and the mean value of perceived exertion, rated according to the RPE scale, was 15.8 (SD 1.1) and 15.4 (SD 0.9), respectively. The final heart rate attained among the patients and the control group was 159 beats/min (SD 11 beats/min) and 159 beats/min (14 beats/min), respectively, and the heart rate after the stabilometric assessment, approximately 3 1/2 minutes after exercise, was 112 beats/min (SD 14 beats/min) and 117 beats/min (SD 16 beats/min), respectively. No significant differences were found between the patients and controls with regard to the above-mentioned variables.All subjects exceeded the 60% value of the predicted HRHigher values were noted post- than pre-exercise in the FP and SP in the injured and uninjured legs, but only in the FP in the control group Table . No diffA lower value was observed in the third than in the first of the post-exercise measurements on the injured leg in the FP, but no differences were noted on the uninjured leg or in the control group Table . The injLower values were observed pre-exercise in the SP in the injured and uninjured legs of the patients than in the control group Table .A higher value was found post- than pre-exercise in the uninjured leg in the FP, and the post-exercise value tended to be higher in the injured leg and in the control group Table . No diffThe third of the post-exercise measurements was lower than the first in the injured leg in both planes, but no differences were found for the uninjured leg or in the control group Table . No diffNo differences were found between the injured leg and the control group, or between the uninjured leg and the control group Table .Short-duration, sub-maximal exercise on a cycle ergometer resulted in increased average speed in both planes, and in the amplitude of CP movements DEV 10) in the FP during balance in single-limb stance among the patients with ACL injury. In the intra-group comparisons, three of four variables showed higher values post- than pre-exercise in the uninjured leg, and two of four variables were higher post-exercise in the injured leg. In the control group, one of four variables was higher post- than pre-exercise , and therefore to the stabilization of the knee joint . DecreasThe lower average speed and lack of difference in DEV 10 in the patients compared to the control group, indicate sway movements at a lower speed with retained amplitudes to be neuromuscular adaptive strategies, rather than more rapid, smaller adjustments Table . These sThe results of the present study showed no differences in the effects of exercise between the patients and the controls. However, the injured leg was more affected or tended to be more affected directly after exercise than the control group, which indicates that patients with ACL injury react differently regarding their ability to maintain balance in single-limb stance after short-duration, sub-maximal cycling, than a control group of uninjured subjects. The patients used sway movements at a lower speed with retained amplitudes, which may be an expression of neuromuscular adaptive strategies.The author(s) declare that they have no competing interests.EA participated in the design of the study, participated in collecting the data, performed the statistical analysis, and drafted the manuscript. DR participated in collecting the data. EH participated in the progress and revision of the manuscript. TF participated in the design of the study, and in the progress and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases.In eukaryotes, PPP D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs.We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined. Reversible phosphorylation of proteins is a ubiquitous mechanism, indispensable for regulation of virtually any cellular function. Therefore, protein kinases and phosphatases are of paramount importance for normal functioning of all metabolic and signalling pathways. In eukaryotes, PPP family is one of the two known protein phosphatase families specific for Ser and Thr -4. UnlikThree motifs (GDXHG, GDXXDRG and GNH(E/D)), highly conserved in the N-terminal subdomains of the catalytic domains of all PPP phosphatases ,11, contPhyscomitrella patens, showing no similarities to the known PPP phosphatases beyond the presence of the GDXHG and GDXXDRG motifs. Detection of homologous cDNA sequences from Arabidopsis and rice suggested the presence of an unknown PPP group in plants, distinct from "conventional" eukaryotic PPP phosphatases [In a previous work  we identphatases .We have now taken advantage of a much greater representation (as compared to 1999) of sequence databases for various species to further explore this initial observation. We present the evidence for the existence in eukaryotes of three "non-conventional" branches of the PPP family. We also identify a previously unrecognised conserved motif in the PPP catalytic domain, which can be used as a signature of "bacterial"-type PPP phosphatases.Arabidopsis sequences, At3g09960 and At3g09970, were retrieved using the P. patens fragment [Two fragment  as a quefragment . They ar-Proteobacteria, the closest matches being with Rhizobiales, Rhodobacterales and Rhodospirillaceae. Therefore, we designate this group as Rhizobiales / Rhodobacterales / Rhodospirillaceae-like phosphatases, or Rhilphs  and 2 (RLP2), respectively.Among prokaryotes, related sequences were detected in some αs Figure , and theAll residues that are expected to bind metal ions in the catalytic centre are conserved in Rhilphs Figure . RhilphsArabidopsis gene At1g07010 was reported in an independent study [While this work was in progress, a phosphatase encoded by an nt study .Arabidopsis RLP2 as a query to see whether Arabidopsis genome encodes additional "bacterial-like" PPPs. These searches identified two more genes for putative PPP phosphatases, only distantly related to Rhilphs and to any other members of the family , in Apicomplexa, Trypanosomatidae, and in a red alga Porphyra yezoensis  and psychrophilic bacteria Alteromonadales (γ-Proteobacteria): uncharacterised phosphatases from Shewanella oneidensis and Colwellia psychrerythraea and a Tyr-specific phosphatase PPI from Shewanella sp. [Shewanella-like" phosphatases, or Shelphs, and the products of the two prototype Arabidopsis genes At1g07010 and At1g18480 as "Shewanella-like" phosphatases 1 (SLP1) and 2 (SLP2), respectively.The most closely related prokaryotic phosphatases were detected in ella sp. . TherefoA.thaliana SLP1 and corresponding Shelph isoform from rice have chloroplast targeting sequence, which could not be detected in A.thaliana SLP2 and corresponding isoform from Medicago truncatula.Like in Rhilphs, all residues that are expected to bind metal ions in the catalytic centre are conserved in Shelphs  than to other PPP groups. Therefore, we tentatively designate them as ApaH-like phosphatases, or Alphs , in Try Figures .Alphs share a distinctive common structural feature. In the GDXXDRG motif, absolutely conserved in other PPPs, the second Asp  is replIn order to better understand the relationship of "bacterial-like" PPP phosphatases in eukaryotes to each other and to bacterial PPPs, we attempted to extend our previous phylogenetic analysis of eukaryotic PPP phosphatases  by incluTo circumvent the ambiguity of the results, we used Neighbor-Net , a neighC-terminal regions of the catalytic domain of all "conventional" eukaryotic PPP phosphatases share a highly conserved (with minor variations) SAPNY motif, located in the β12-β13 loop. This loop and the Tyr residue of the SAPNY motif in particular are implicated in interaction with regulators and inhibitors ,30-32. βIn addition, we note the presence of another characteristic feature of bacterial and "bacterial-like" PPPs: the His residue  coordinating one of the metal ions in the catalytic centre is preceded by an absolutely conserved Gly; this residue is conserved in some archaeal but not in "conventional" eukaryotic PPPs Figure .In this report, we have documented the presence in different eukaryotic lineages of the genes that encode PPP phosphatases resembling those of bacterial origin, rather than "conventional" eukaryotic members of the family. Catalytic domains of these "bacterial-like" phosphatases are characterised by relatively conserved structure of the N-terminal subdomains, but very diverse organisation of the C-terminal subdomains, where conserved motifs and residues forming the active centre are separated by sequences of various length that share little or no similarity between different clades Figure . In mostRhizobiales. The absence of related sequences in eukaryotes other than land plants suggests that Rhilphs may have been acquired after plants started colonising land. Although bacterial lineage that could be the source for plant Rhilphs could not be unambiguously identified by phylogenetic reconstruction, Rhizobiales  appet genome  or even t genome , a situat genome . PossiblApicomplexa, Trypanosomatidae, as well as in some fungi. The similarity between proteins from Apicomplexa and Trypanosomatidae and those from plants is well documented. Trypanosomatidae are related to photosynthetic euglenoids and are thought to have lost plastids secondarily [Apicomplexa and Trypanosomatidae is not surprising and probably reflects the presence of Shelphs in a common ancestor of photosynthetic eukaryotes. The presence of chloroplast targeting sequence in SLP1 suggests a possible origin of Shelphs from a bacterial precursor of the chloroplast (however it should be noted that Shelphs are absent from cyanobacteria); alternatively, this sequence may have appeared secondarily. Protein Ser/Thr phosphorylation / dephosphorylation is essential for regulation of photosynthesis, and unidentified okadaic acid-insensitive protein phosphatases in chloroplasts have been reported [Phosphatases of another group, designated as Shelphs, are found in green plants, in a red alga, in ondarily . Apicompondarily . Thus, treported ,41. SLP1S. pombe, but not in a number of other ascomycetes, whose genomes have been completed. Current data do not permit to discriminate between (i) the presence of Shelphs in a common ancestor of eukaryotes and their loss in such lineages as animals and many fungi, and (ii) independent acquisition of Shelphs from bacteria by an ancestor of photosynthetic eukaryotes and by fungi. Further sequencing of eukaryotic genomes may shed light on the evolutionary history of this PPP group.The origin of fungal Shelphs is unclear. Curiously, they are found in basidiomycetes and in an ascomycete The third group of "bacterial-like" phosphatases detected in eukaryotes, designated here as Alphs, appears to be distantly related to bacterial diadenosine tetraphosphatases ApaH. Patchy distribution in several eukaryotic kingdoms suggests that Alphs were probably present in the common ancestor of eukaryotes, but were independently lost in many lineages, including insects, vertebrates and flowering plants. A characteristic modification of the conserved GDXXDRG motif shared only with ApaH further supports a suggestion that Alphs may represent a divergent branch of diadenosine tetraphosphatases, rather than protein phosphatases. However, relatedness of eukaryotic Alphs to bacterial diadenosine tetraphosphatases remains hypothetical, since Alph sequences are too divergent from ApaH, as well as from each other, to permit a reliable phylogenetic reconstruction.Diadenosine oligophosphates are considered as emerging signalling molecules in both intra- and intercellular signalling in eukaryotes ,43. In pin vivo, although they may be able to dephosphorylate phosphoTyr-containing substrates in vitro . Thi. Thiin vro (e.g. . Howeverro (e.g. ,13,47-49specific , it woulspecific ).per se, taking into account extreme structural diversity of bacterial PPP phosphatases. It indicates that (I/L/V)D(S/T)G was probably present as the fourth "universal" signature motif in the common ancestor of PPP phosphatases, and was lost in the common lineage of archaeal and "conventional" eukaryotic PPPs. An alternative possibility could be that the (I/L/V)D(S/T)G motif was acquired by a bacterium and propagated by lateral gene transfer, replacing the ancestral SAPNY-related motif. However this scenario seems less likely, since the (I/L/V)D(S/T)G motif is present, with minor variations, in virtually all bacterial phosphatases, despite their great diversity, and is replaced by SAPNY-related sequences only in archaeal and "conventional" eukaryotic PPPs.Three motifs, GDXHG, GDXXDRG and GNH(E/D) form the diagnostic signature of all PPP phosphatases ,11. We dnd position of (I/L/V)D(S/T)G is highly conserved and can only be replaced by Glu, indicating that the negative charge is essential. The presence of a highly conserved Gly in the 4th position indicates that flexibility of the polypeptide chain is likely to be important. The crystal structure of bacteriophage λ phosphatase  shows tvs. Ser/Thr specificity.It would be tempting to speculate that this difference in the region just downstream of the β9 (β12) may be responsible for a feature that is common to all "bacterial"-type but not to eukaryotic / archaeal PPPs. One such feature is the Ser/Thr specificity of the latter group. The Tyr residue of the SAPNY motif has been suggested to provide a bulky phenol ring in the β12-β13 loop, sufficient to sterically block access of phosphoTyr-containing substrates to the active site . HoweverSo far, eukaryotic PPP phosphatases were considered as a well-defined monophyletic group of enzymes, specifically dephosphorylating phosphoSer and phosphoThr, while a much more structurally and enzymatically diverse PPP phosphatases were known to be present in prokaryotes. Our findings demonstrate that, in addition to "conventional" eukaryotic PPP Ser/Thr-specific protein phosphatases, many eukaryotes possess very diverse "bacterial-like" PPP phosphatases. Enzymatic characteristics, physiological roles and evolutionary history of these phosphatases have yet to be revealed.Chlamydomonas reinhardtii draft genome [Sequence similarity searches were conducted using BlastP or TBlastN  at NCBI t genome . In all t genome .Multiple alignments were generated using CLUSTAL W  at KyotoThe presence of signal peptides and targeting sequences was analyzed using TargetP  at the tRhizobiales / Rhodobacterales / Rhodospirillaceae – like phosphatase; Shelph (SLP), Shewanella – like phosphatase.EST, expressed sequence tag, Alph, ApaH – like phosphatase; PPP, protein phosphatases of the P family; Rhilph (RLP), Both authors contributed equally to this work.Neighbor-Net analysis of the conserved N-terminal subdomains (starting 5 amino acid residues before conserved GDXHG and ending 25 residues after GNH(E/D) of 104 bacterial, archaeal and eukaryotic PPP phosphatases. This version of Figure Click here for fileDistinct conserved motifs in the C-termini of bacterial and "bacterial-like" PPP phosphatases from eukaryotes as opposed to archaeal and eukaryotic PPP phosphatases. This is an expanded version of Figure Click here for file