The aims of this study were to determine if pregnant women consumed the recommended vitamin D through diet alone or through diet and supplements, and if they achieved the current reference range vitamin D status when their reported dietary intake met the current recommendations.2, 25(OH)D3, 3-epi-25(OH)D3 concentrations. Dietary data were obtained from questionnaires including a Supplement Intake Questionnaire and a 24-hour recall of the previous day’s diet.Data and banked blood samples collected in second trimester from a subset of 537 women in the APrON (Alberta Pregnant Outcomes and Nutrition) study cohort were examined. Frozen collected plasma were assayed using LC-MS/MS (liquid chromatography-tandem mass spectrometry) to determine 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3) were identified in all of the 537 plasma samples;3-epi-25(OH)D3 contributed 5% of the total vitamin D. The median (IQR) total 25(OH)D (D2+D3) was 92.7 (30.4) nmol/L and 20% of women had 25(OH)D concentration < 75 nmol/L. The median (IQR) reported vitamin D intake from diet and supplements was 600 (472) IU/day. There was a significant relationship between maternal reported dietary vitamin D intake (diet and supplement) and 25(OH)D and 3-epi-25(OH)D3 concentrations in an adjusted linear regression model.Participants were 87% Caucasian; mean (SD) age of 31.3 (4.3); BMI 25.8 (4.7); 58% were primiparous; 90% had education beyond high school; 80% had a family income higher than CAN $70,000/year. 25(OH)DWe demonstrated the current RDA (600 IU/ day) may not be adequate to achieve vitamin D status >75 nmol/L in some pregnant women who are residing in higher latitudes  in Alberta, Canada and the current vitamin D recommendations for Canadian pregnant women need to be re-evaluated. Vitamin D plays an important role in promoting healthy pregnancy and fetal development, and poor vitamin D status has been associated with adverse maternal and infant health . There iDespite these recommendations, a recent study showed one-quarter of Canadians ages 6–79 years) did not meet the Recommended Daily Allowance  through food sources alone. However, the daily use of vitamin D supplements contributed to a better 25-hydroxycholecalciferol (25(OH)D) status  years di.2) and 25(OH)D3, as both metabolites are metabolically active and both are available as dietary supplements and in fortified foods. In addition, recent studies have shown the epimeric form of 25(OH)D3 contributes significantly to measured 25(OH)D3 when methods are unable to separate the less active epimer [3) from vitamin D status calculation underestimates the classification of pregnant women and their newborns at risk for vitamin D insufficiency (>75 nmol/L) [The best clinical laboratory indicator of vitamin D status is the blood concentration of 25(OH)D . It is ie epimer , 12. We  nmol/L) .Although the literature regarding vitamin D in pregnancy is growing rapidly, several gaps in our knowledge remain, including the prevalence of vitamin D deficiency/ insufficiency during pregnancy and the intake of vitamin D during pregnancy needed to ensure optimal status. Our aims in the current study were to determine: a) if pregnant women were consuming the recommended vitamin D through diet alone or through diet and supplements, and b) if pregnant women can achieve the current reference range vitamin D status when their reported dietary intake of vitamin D met the current recommendations.Alberta Pregnancy Outcomes and Nutrition; www.ApronStudy.ca) is a longitudinal cohort of pregnant women and their children residing in Calgary and Edmonton . We used the APrON cohort data and analyzed the banked blood samples collected from the first 537 women recruited to APrON in their second trimester of pregnancy . APrON recruited a total of 2191 women between March 2009 and July 2012, primarily through in-person contact at maternity clinics. Full details of the APrON study are described elsewhere [This study is a secondary analysis of an established maternal and infant prospective cohort study. The APrON study  was administered by a trained nutrition professional to each participant once during each trimester of pregnancy and once at 3 months postpartum to determine current Natural Health Product (NHP) use including vitamin D. The SIQ was designed specifically for the APrON study and was initially used in a pilot study that included of fifty women in the APrON study who completed the questionnaire during their first and second visits to assess efficacy and the detail of information obtained . Women w2, 25(OH)D3, and 3-epi-25(OH)D3 and demonstrates an intra-assay coefficient of variability of 4.6% at 28.3 nmol/L, 3.3% at 83.9 nmol/L, and 3.9% at 5.8 nmol/L for 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3, respectively. Information on processing blood samples and integrity of plasma samples has been previously reported [2, 25(OH)D3, and 3-epi-25(OH)D3 in plasma were determined using LC-MS/MS  standards and Vitamin D External Quality Assessment Scheme (DEQAS) procedures were followed), as previously described [Maternal blood was collected at each clinic visit by a certified phlebotomist. The majority of APrON participants were recruited in their second trimester; therefore, we used the blood samples taken from their second trimester for this study . All frozen collected plasma was assayed alongside plasma quality control samples and standard reference materials using a clinically validated LC-MS/MS assay at the laboratory of Doctor’s Data Inc. , which mreported . The conescribed .2, 25(OH)D3, and 3-epi-25(OH)D3 in terms of means and standard deviations (SD) and medians and interquartile ranges (IQR). The prevalence rates of vitamin D deficiency and insufficiency in women were estimated as percentages with 95% confidence interval (CI). Plasma concentrations of 25(OH)D at <25 nmol/L [3 and 3-epi-25(OH)D3 was determined using the Pearson correlation coefficient test. Repeated measures ANOVAs were used to examine the difference between the reported dietary intakes across the three trimesters. As reported dietary and supplemental vitamin D intakes were not normally distributed, the natural log transformation of the skewed variables was used for the test.Results are presented for plasma 25(OH)D5 nmol/L , <50 nmo5 nmol/L , and <755 nmol/L  were use2 + D3) concentration and their reported dietary vitamin D intake in supplements and diet adjusted for the potential covariates of maternal age, maternal BMI at second trimester, season (summer from May 01-Oct 31 and winter from Nov 01-Apr 30), and race (Caucasian vs. non-Caucasian) [3 and 3-epi-25(OH)D3) were not normally distributed, the natural log transformation of the skewed variables was used for the test.A multiple linear regression analysis was used to examine the association between women’s plasma 25(OH)D (Ducasian) . Again, vs. ≥ 50 and <75 vs. ≥ 75 nmol/L) as a function of reported vitamin D intake, adjusted for the same covariates (odds ratios and their corresponding 95% CIs). All analyses were conducted using Stata statistical software (version 14) and SPSS (version 22). P-values < 0.05 were considered statistically significant.In addition, plasma levels were dichotomized as deficient/insufficient versus adequate, and multiple logistic regression analysis was used to estimate the odds of insufficiency . vs. second vs. third) (P<0.001), resulting in a significant increase in total reported vitamin D intake from trimester one to trimester three (P = 0.004). Fifty-four percent of pregnant women reported taking 600 and higher IU/day of vitamin D through their diet and supplements in the second trimester of pregnancy; maternal characteristics were not different between those who reported taking <600 IU/day vitamin D vs. ≥600 IU/day, except university degree  (data are not shown).A repeated measures ANOVA showed no significant difference in log transformed mean reported vitamin D intake from diet  (P = 0.13) between time-points. However, there was a significant difference in log transformed mean reported intake from supplements between time points D2, 25(OH)D3and 3-epi-25(OH)D3concentrations. All samples were found to have detectable concentrations of 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. Median (25th-75th) maternal plasma concentration for 25(OH)D2 was 2.9 (1.7–4.4) nmol/L. Vitamin D supplements in Canada are almost all vitamin D3; however, vitamin D2 is still available by prescription and may be obtained from some food sources. Median (25th-75th) maternal plasma 25(OH)D3 was 89.2 (74.4–106) nmol/L, and for plasma 3-epi-25(OH)D3 was 5.2 (4.1–6.7) nmol/L (3) plasma concentrations <25 nmol/L, 12  women had plasma concentrations <50 nmol/L, and 100  women had concentrations in the <75 nmol/L range. The relationship between 25(OH)D3 and 3-epi-25(OH)D3 is shown in ) nmol/L . No partThe effect of 3-epi-25(OH)D3on the classification of 25(OH)D sufficiency. Three-epi-25(OH)D3 was detected (>1.0 nmol/L) in all maternal samples. Three-epi-25(OH)D3 comprised 5.0% of total 25(OH)D. When 3-epi-25(OH)D3 was excluded from the vitamin D estimations, 19% of women were considered to have levels <75 nmol/L; however, with 3-epi-25(OH)D3 included, the percentage of women with vitamin D concentration of <75 nmol/L decreased to 12%. The observed difference in the risk of insufficiency was significant (P<0.001). As we had very small numbers of participants with 25(OH)D concentrations <50 nmol/L, the estimate of insufficiency at this level with 3-epi-25(OH)D3 excluded was 2%, compared to 1.5% when it was included (P = 0.125).Association between women’s plasma 25(OH)D and 3-epi-25(OH)D3concentration and maternal reported vitamin D intake. The relationship between 25(OH)D and 3-epi-25(OH)D3 and vitamin D intake are shown in 3 and total reported vitamin D intake during the second trimester was significant , as well as maternal BMI at second trimester and race in an adjusted linear regression model (3 was significantly associated with reported vitamin D intake from supplements but not diet (data not shown). Women with plasma concentration of <75 nmol/L reported consuming less vitamin D (diet + supplements) compared to those with plasma concentration ≥ 75 nmol/L (median (IQR): 492 (320) IU/day vs. 622 (540) IU/day). In addition, when plasma levels were dichotomized at <50 vs. ≥ 50 and <75 vs. ≥ 75 nmol/L in a multiple logistic regression model, higher reported intake of vitamin D was associated with greater odds of vitamin D sufficiency during the second trimester within both ranges . Maternal age, BMI, season, and ethnicity were not significant effect modifiers or confounders in the model (data not shown).In addition, the association between maternal plasma 3-epi-25(OH)Don model . Plasma To our knowledge, this is the largest study to date to show 44% of pregnant women with a higher socioeconomic status reported vitamin D intake (diet and supplements) not meeting the dietary reference intakes (DRI) recomendations. Using LC-MS/MS, we separated epimer from estimation of vitamin D status and were able to accurately estimate the prevalence of vitamin D insufficiency in our pregnant women. We also found that 20% of pregnant women had vitamin D concentraion <75 nmol/L, despite more than half of these women reporting taking more than 600 IU/day of vitamin D (the current RDA) based on their reported intake from diet and supplements.In Northern countries such as Canada, the latitude of the country further reduces the potential contribution of dermal synthesis to the maintenance of appropriate vitamin D concentrations, placing women at greater risk for vitamin D deficiency/ insufficiency , 22. The3 in all our pregnant women’s plasma samples across pregnancy (second trimester), and that including this epimer in the measurement of vitamin D status significantly influenced the classification of prevalence of vitamin D sufficiency in pregnancy. This finding is consistent with our previous study showing epimer can alter vitamin D status estimation of pregnant women at the time of delivery and in their cord blood [3 [3 was vitamin D intake. The fact that season significantly affected 25(OH)D concentration but not 3-epi-25(OH)D3 suggests that vitamin D supplements could be a source for 3-epi-25(OH)D3 as shown in a previous study [3-epimerization pathway has not yet been elucidated, there are indications that it has some anti-proliferative activity and differentiation activity only at levels of approximately 10%-30%, respectively, compared to non-epimeric vitamin D metabolites [3-epimer should not be included in calculating 25(OH)D concentrations in serum. Clinical data has indicated that 9% of infants and 3% of adults would be misclassified as sufficient if the epimer had not been identified in the quantification [3 in the assessment of vitamin D status in pregnant women, given the fact that the cut-off for vitamin D sufficiency in pregnancy is unclear.In addition, we demonstrated the presence of 3-epi-25(OH)Drd blood . Althougblood [3 , 30, in abolites , 12. Thification . These fOne of our study’s limitations is that the cohort was skewed toward women with annual income >$70,000 (2014 Canadian dollars), which could influence the generalizability of study findings to the entire population. In our study, only 20% of women had 25(OH)D <75 nmol/L, which is much lower than what is reported in other Canadian studies. The Canadian Health Measures Survey (CHMS) found that 63% of non-pregnant women aged 20–39 years had 25(OH)D <75 nmol/L , and a r3 concentration. A Canadian study [3 concentration but fall sun exposure was not, which may be related to lower vitamin D-weighted climatological UV in Toronto during the fall. Although Calgary averages the greatest number of days/year with sunlight in Canada, study of adult participants in Calgary found a modest edffect of season on 25(OH)D concentration and serum PTH [Another limitation of our study is the lack of information on sun exposure, skin color, and clothing coverage of participants. We used data on season as a proxy for sun exposure in our study. Studies , 35 havean study  of youngerum PTH . We showerum PTH  are to aIn conclusion, we found a 20% rate of vitamin D insufficiency (<75 nmol/L) in a large, well-nourished cohort of pregnant women. Fifty percent of women who were classified as vitamin D insufficient (<75 nmol/L) consumed the RDA from both diet and supplements. This finding extends the literature suggesting that 600 IU/day may not be enough to achieve vitamin D status >75 nmol/L in pregnant women residing in higher latitudes with limited sun exposure. This cohort provided a vehicle for exploring this association, and problems noted in this best-case scenario group of educated and health-conscious women suggest the importance of interventions aimed at all Canadian pregnant women. These findings, together with existing data, highlight the need for re-evaluation of our current vitamin D recommendations for pregnant women residing in countries with higher latitude.


Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×1012 virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3+ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4+ or CD8+ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4+ and CD8+ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4+ and CD8+ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4+ or CD8+ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype.The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×10 Infection with papillomaviruses can cause benign warts (papillomas) on skin and mucosae of humans and animals but also malignancies, especially anogenital carcinomas and, in genetically predisposed or immunocompromised individuals, cutaneous squamous cell cancers. Control and clearance of these viruses are thought to be mediated by the cellular immune system, however experimental determination for the necessary cellular effector(s) is lacking. The recently identified mouse papillomavirus  is known to induce papilloma formation on skin of immunodeficient mice. However, its pathogenesis in immunocompetent mice is unclear. Our study shows that in an immunocompetent setting, MusPV1 generally causes asymptomatic skin infections, but no lesion outgrowth. Visible papillomas were consistently observed after profound immunosuppression in some, but not other, strains of mice. By selective removal of distinct cellular immune populations and employing genetically modified mice, we could show that T cells are pivotal for controlling MusPV1 infection and disease. We further show that surprising differences in the T cell subsets are required for protection in different strains of immunocompetent mice. This implies that unanticipated effector mechanisms can control virus infection and pathogenesis in specific genetic backgrounds. The findings may help to explain the wide of range of pathologic outcomes of infection by a specific human papillomavirus type in immunocompetent people. Papillomaviruses (PV) are DNA tumor viruses that infect stratified squamous epithelia of the skin and mucous membranes of humans and many other vertebrate species Although neutralizing antibodies against the viral capsid proteins are sufficient to prevent PV infections, cell-mediated immunity is generally thought to control the infections once they become established. For instance, individuals with underlying T cell deficiencies, but not B cell deficiencies, often have difficulties in controlling and clearing HPV-induced neoplasia nu/Foxn1nuFoxn1 nude laboratory mouse colony nu/Foxn1nuFoxn1, nu/Foxn1nuFoxn1 and SCID SHO mice in vitro synthesized full-length genomic DNA of MusPV1, giving rise to non-regressing cutaneous papillomas from which high titers of authentic, infectious MusPV1 virions were isolated and serially passaged The recent identification of MusPV1 , which is the first domestic mouse PV, provides an excellent opportunity to investigate the immune mechanisms that control PV infection in a mammalian species whose immunology has been well characterized. MusPV1 was found initially in an inbred NMRI-ba/ba mice, which have an unknown mutation that results in thin short hair, they induced small papules at most injection sites. These lesions, which were not characterized further, took at least three weeks to develop, and regressed spontaneously by 8 weeks post-inoculation In contrast to these reports of MusPV1 in immunodeficient mice, the study of MusPV1 in immunocompetent mice has been limited. The initial report of MusPV1 noted that when cell-free extracts from papillomas in the immunodeficient mice were used to inoculate the dorsal skin of S/RV/Cri-In this study we aimed at characterizing MusPV1 infection in different immunocompetent murine strains and sought to determine the key immunologic players primarily responsible for control of cutaneous PV infection and papilloma induction. Our results revealed asymptomatic MusPV1 infection in these immunocompetents and demonstrated that profound immunosuppression can render these strains that had various H-2 haplotypes susceptible to MusPV1-induced papilloma formation of the skin. This is reminiscent of infection with HPV of genus beta that also predominantly induces asymptomatic skin infections in immunocompetent individuals but can induce visible lesions after immunosuppression. The observed differences in the efficiency of papilloma outgrowth between the individual murine strains corresponded partially to their previously reported susceptibility to chemical-induced skin papillomatosis d, but vary in their sensitivity to chemical carcinogenesis, as is also true for the two H2b strains, C57BL/6 and 129S6/SvEv. The SENCAR strain was included because it was selectively bred for high susceptibility to skin tumor induction by chemical carcinogens. All strains were infected with 6×1010 MusPV1 virions per animal on pre-scarified tail skin, using a previously optimized procedure for PV infection of mouse skin Several inbred immunocompetent strains of mice, namely FVB/NCr, BALB/cAnNCr, DBA/2NCr, A/JCr, C57BL/6NCr (C57BL/6), 129S6/SvEv, C3H/HeJCr, and as well the outbred Cr:ORL SENCAR (SENCAR) were evaluated for their susceptibility to papilloma induction by MusPV1 . The inb10 MusPV1 virions and continued for additional 4 weeks, during which the mice were evaluated for papilloma formation. The results uncovered a hierarchy of susceptibility to MusPV1-induced papillomas among the strains . After this period, papilloma outgrowth was not observed in any of the animals (data not shown). MusPV1 E1∧E4 spliced transcripts, a marker for PV infection In a rabbit model, persistent PV genome expression was reported at mucosal sites of infection following lesion-regression b, the former strain was resistant to MusPV1-induced papilloma formation, while the latter strain had intermediate susceptibility. In addition, DBA/2NCr was resistant, while BALB/cAnNCr had intermediate sensitivity, although both strains are H2d. By contrast, we noted some correlation between the observed susceptibility to MusPV1-induced papillomas and their previously reported susceptibility to chemical carcinogenesis  (11 MusPV1 virions. All lesions spontaneously regressed within 1–2 weeks after formation (corresponding to 3–5 weeks post-infection). Consistently, lesions did not arise with lower amounts of inocula ranging from 1×1010 to 1×108 virions. The transient papillomas in the immunocompetent SENCAR mice obtained after inoculation with 1×1012 MusPV1 virions were morphologically and histologically similar to papillomas that formed under CsA immunosuppression and to those previously reported 12 MusPV1 virions, further supporting the higher resistance of this particular strain to MusPV1 pathogenesis  , even inogenesis .10 MusPV1 virions (corresponding to 5 weeks of CsA administration) from the virally inoculated sites of all strains, except for DBA/2NCr and 129S6/SvEv  was analyzed for the presence of CD4+ and CD8+ T cells. By immunofluorescent staining (IFS), higher numbers of both T cell subsets were present in the MusPV1-infected tissue, independent of whether the mice had been treated with CsA, although L1 expression was only detected in the infected CsA-treated mice  recognizing murine CD3. Depletion was started at various time points, from one week prior to viral infection (day −7) to 7 weeks (day +49) after infection, and it was maintained for seven weeks for all groups. The mice were infected on day 0 with 7.3×1010 MusPV1 virions, and the number and size of lesions were determined for each group after 7 weeks of immunodepletion  from the CD3-depleted mice  and every 2 weeks by flow cytometric analysis , and the depletion monitored similarly (+ T cells (0/5) (+ T cells (0/5)   or of onls (0/5)  did not ls (0/5) .When crude skin extracts taken from the mice 6 weeks after infection were evaluated by western blotting for the presence of L1 protein, it was only detected in the two groups that developed papillomas, CD3 depletion and the combined CD4+8 depletion .Given that the positive results with C57BL/6 mice were seen with CD3 depletion or combined CD4+8 depletion induced by mAb, further analysis was focused on depletion induced by these mAb. At 6 weeks after virus inoculation, high levels of MusPV1 E1∧E4 spliced transcripts, as a measure of persistent infection, were found in infected skin tissues of CD3- and combined CD4+8-depleted animals , the two+ cells were seen in skin sites from CD3-depleted, CD4+8-depleted, or CD4-depleted mice, although they were detected in CD8-depleted mice   for CD4ce (0/5)  and the ce (0/5)  were resce (0/5) , and didce (0/5) .+ T cells are specifically involved in early, presumably innate, immune responses that initially control viral gene expression.However, analysis of MusPV1 E1∧E4 spliced transcripts, as a measure of infection, at earlier time points  revealed10 MusPV1 virions and a 3.5 month observation period -T cell population, also failed to form papillomas after inoculation with 6.8×10n period , indicat12 virions) seems to overcome early control of infection in the highly susceptible SENCAR strain, allowing for transient papilloma formation. In the more resistant C57BL/6 strain, papilloma outgrowth was not observed after this stringent challenge, supporting the conclusion that this strain is better able to control infection than SENCAR mice.In this study, we have determined that a variety of immunocompetent mouse strains are resistant to papilloma induction by MusPV1, although a limited degree of virus expression can be detected at 4 weeks after infection. However, immunosuppression induced by CsA uncovered a strain-dependent hierarchy in the degree of susceptibility to papilloma formation and virus production. Papilloma formation correlated more closely with expression of E1∧E4 spliced transcripts than with expression of the major L1 capsid protein, as clinically normal virally inoculated skin sites in the resistant C57BL/6 mouse had relatively high levels of L1, but low E1∧E4 levels. Inoculation with what we assume are super-physiological amounts of MusPV1 virions  that is the key player responsible for virus control and papilloma regression. Observational studies in humans have found that regression of anogenital warts is accompanied by a massive infiltration of CD4+ T cell infiltrate in the dermal and the epithelial compartment, as well as an intraepithelial one composed of CD8+ T cells, whether the animals developed papillomas as a result of the immunosuppressive CsA treatment or had clinically normal skin because they had not been immunosuppressed. Thus, the infiltrate was similar, independent of whether the mouse developed lesions, indicating that functional properties of the T cells, rather than epithelial trafficking, were primarily affected by CsA treatment.In our murine system, MusPV1 infection of the sensitive SENCAR mouse induced a dense CD4+ or CD8+ T cells allowed papillomatosis. By contrast, combined depletion of CD4+ and CD8+ T cells was necessary for papilloma development in C57BL/6. These discrepant results were confirmed using genetically modified C57BL/6 mice deficient in either CD4+ or CD8+ T cells.The availability of many immunological reagents for the domestic mouse made it possible to critically evaluate individual T cell subsets in the control of PV infection and associated neoplastic disease. In SENCAR, depletion of either CD4+ T cells, especially cytotoxic ones. However, in many well-characterized systems, CD4+ T help is necessary both for induction of primary CD8+ T cell responses and for their proliferation, activation, and differentiation into effector cytotoxic T lymphocytes (CTL) + T help, CD8+ T cells often fail to acquire antiviral effector functions, including the ability to produce antiviral cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and cytotoxic molecules, such as perforin and granzymes. In this scenario, CD4+ T cells would contribute to protection indirectly, rather than directly providing a critical T cell effector function. However, the T cell subset depletion and knock-out data in C57BL/6 data make it clear that protective antiviral CD8+ T cell responses to a PV infection can develop in the absence of CD4+ T help, and that CD4+ T cell-dependent effector functions can control PV infection in the absence of CD8+ T cell functions.Clearance of virally-infected epithelial cells and regression of epithelial neoplasia have most often been attributed to CD8+ T helper-independent CD8+ CTL responses can be elicited by some pathogens, such as ectromelia virus + T cells were required for efficient local recruitment of herpes simplex virus-specific CD8+ T cells to the vaginal epithelium in a murine model of herpes virus infection + T cells infiltrated the skin sites inoculated with MusPV1 whether or not the mice contained CD4+ T cells, and vice versa. The discrepant conclusions between our study and the murine herpes virus infection report + T cells in the herpes virus study and the de novo generation of the T cells by in situ virus infection in the current study.There is an increasing body of evidence that CD4+ T cell activation in the absence of CD4+ T cell help have been proposed, such as direct signaling through CD40 present on antigen-presenting cells + T cell responses via direct engagement of CD40 on activated CD8+ T cells Various mechanisms for CD8+ T cells could independently control PV infection by either a cytokine-mediated mechanism or by direct cytotoxicity + T lymphocytes, which may represent a new CD4+ lineage, can contribute to the control of certain viral infections + T cells are capable of in vivo cytolytic killing of peptide pulsed MHC II-positive lymphocytes + T cells.CD4+ T cells would likely require a direct interaction of the T cells with MHC class II molecules on the infected keratinocytes . We did detect CD4+ T cells within the epithelium at sites of infection, in addition to being present in the dermis, and keratinocytes can express class II molecules under certain inflammatory conditions, e.g. in response to (IFN)-γ + T cells are activated by cross-presentation of viral antigens by professional antigen presenting cells, and PV infection is controlled via soluble factors produced by the CD4+ T cells.Cytolysis by CD4Although the details of immune recognition of MusPV1 remain to be determined, it seems likely that T cells control MusPV1 infection by both innate and adaptive immune mechanisms. The timing of papilloma regression after suspending CsA treatment and of spontaneous regression after high dose challenge in the SENCAR mice is consistent with induction of an antigen-specific adaptive response. However, the early control viral gene transcription in C57BL/6 mice at week 1–2, that is maintained in the CD8 KO mice but lost in the CD4 KO mice , strongl+ and CD8+ T cells. One possibility is that both the CD4+ and the CD8+ T cells in C57BL/6 are more potent in their ability to confer resistance than in SENCAR, making the presence of one of the T cell subsets sufficient to keep C57BL/6 resistant. This situation could arise if there were a single factor common to both T cell subsets that is more potent in C57BL/6 than in SENCAR, or if there were separate CD4+-specific and CD8+-specific factors. Alternatively, there could be a factor that, although it is extrinsic to CD4+ and CD8+ T cells, can cooperate with either subset in making a mouse resistant to papillomatosis. The observed strain-dependent difference could then be explained if the activity of this putative non-CD4/non-CD8 factor(s) is sufficiently more potent in C57BL/6 than in SENCAR that it can cooperate with CD4+ or CD8+ T cells to confer resistance in C57BL/6 but not in SENCAR.Our findings raise several additional interesting issues. One is what may account for the different requirements for protection in C57BL/6 and SENCAR by CD4+ and CD8+ cells in DMBA/TPA tumorigenesis in C57BL/6.A second issue is the partial correlation between the reported strain-dependent susceptibility to chemical-induced papillomatosis and the robustness of persistent infection and papillomatosis after CsA treatment found in this study. The observed partial correlations may be due to yet undetermined strain-specific genetic factors allowing for control of skin tumor susceptibility/resistance. The effects of immunosuppression on 7,12-dimethylbenz[a]anthracene (DMBA)/phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) tumorigenesis have been studied to a limited degree. In C3H/HeN, compared with wt mice, CD8 KO mice had an increased number of tumors, but numbers were decreased in CD4 KO mice A third issue is whether the characteristics of MusPV1 infection documented herein make it an attractive model of any HPVs. At present MusPV1 infection appears to most closely resemble that of genus beta HPVs. These cutaneous HPVs have been found in immunocompetent individuals in clinically normal skin and plucked hairs where they predominantly induce asymptomatic skin infections q), BALB/cAnNCr (H2d), DBA/2NCr (H2d), A/JCr (H2a), C57BL/6NCr (C57BL/6) (H2b), 129S6/SvEv (H2b), C3H/HeJCr (H2k); immunodeficient athymic NCr nu/nu; CD4-, CD8-, CD1d-deficient C57BL/6.The following mice (H-2 haplotypes are given in parentheses) were obtained from the National Institutes of Health or The Jackson Laboratories : immunocompetent outbred Cr:ORL SENCAR; immunocompetent inbred strains FVB/NCr .http://home.ccr.cancer.gov/Lco) Crude extracts of papillomatous tissues were prepared as previously described In vivo infection with purified MusPV1 virions was performed on pre-scarified skin of the animals' tails as previously published For systemic immunosuppression, CsA  was diluted under sterile conditions with PBS and administered subcutaneously to the animals five times per week at a dose of 75 mg/kg body weight in a volume of 0.1 ml. Treatment was started one week prior to infection and maintained for additional 4 weeks post-infection for a total of 5 weeks.In vivo depletion of T cells was achieved by intraperitoneal administration of mAbs : anti-mouse CD4 (clone GK1.5), anti-mouse CD8a (clone 53-6.72), anti-mouse CD3 (clone 17A2) in a dose of 0.5 mg per mouse in a volume of 0.1 ml. Rat IgG2b (clone LTF-2) or rat IgG2a (clone 2A3) mAbs were used as appropriate isotype controls. Depletion was performed on three consecutive days starting at indicated time points and the depleted state maintained by administration of mAbs twice a week for a period of 7 weeks. Depletion was verified by flow cytometry analyses as described below.d and H2b) were already represented by BALB/cAnNCr and C57BL/6 mice, respectively. The depletion experiments in Cr:ORL SENCAR mice (n = 9 per group) and in C57BL/6 mice (n = 5 per group) were repeated twice. The experiments employing C57BL/6 KO mice (n = 5 per group) were repeated twice.The CsA experiments  were repeated twice for FVB/NCr, A/JCr and C3H/HeJCr mice, three times for BALB/cAnNCr and C57BL/6 mice, and 17 times for Cr:ORL SENCAR. The experiments using DBA/2NCr and 129S6/SvEv mice were tested only once as these haplotypes , treated with DNAse I (Qiagen), and reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers' instructions. Real-time PCR using primers and probe specific for MusPV1 E1∧E4 spliced transcripts was performed in an ABI PRISM 7900HT Sequence Detection System, as previously reported + or CD8+ T cells, respectively. To determine localization of MusPV1 L1 in relation to basal keratinocytes, sections were co-stained with a phycoerythrin-conjugated anti-CD49f antibody . Nuclei were visualized by mounting sections with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI) (LifeTechnologies). All microscopy analyses were performed on a Zeiss LSM 510 UV system and color levels of images were processed equally in Adobe Photoshop across experiments.Skin necropsies were snap frozen in Tissue-Tek OCT Compound freezing medium (Sakura Finetek USA Inc.) and HE- and IFS performed on ethanol-fixed tissue sections of 6 µm thickness 2 euthanized mice. To obtain single cell suspensions, spleens and lymph nodes were enzymatically digested with DNAse I  with and without Collagenase A , respectively, and processed as described previously Spleens, iliac and inguinal lymph nodes, and tail skin were harvested from COBlood was collected in heparinized tubes and erythrocytes removed by ACK lysis.After blocking of Fc receptors by incubation with anti-mouse CD16/CD32 antibody (BD Pharmingen), surface-stainings were performed on single cell suspensions from tissues and blood using anti-mouse CD4-PerCP/Cy5.5  and anti-mouse CD8a-FITC labeled antibodies . Cells were fixed with Cytofix/Cytoperm (BD Biosciences) and acquisitions of flow cytometric data performed on a FACSCanto with FACSDiva software (BD Biosciences) Statistical analyses (Mann Whitney tests) were performed using the GraphPad Prism Software 6.00 for Windows.Figure S1(Related to) Cyclosporin A administration promotes strain-dependent MusPV1-induced papilloma formation and lesion maintenance; Lesion size over time after cessation of cyclosporin A. (A) 129S6/SvEv mice were intermediately susceptible and (B) DBA/2NCr mice resistant to MusPV1-induced papillomatosis while under cyclosporin A treatment. (C) Lesion size over time after cessation of cyclosporin A treatment (corresponding to 4 weeks post-infection) in four representative MusPV1-infected Cr:ORL SENCAR mice. Two representative mice with smaller papillomas of 2 and 4 mm in size after cessation of cyclosporin A treatment  and two representative mice with larger papillomas of 9 mm (green and blue lines) are shown.(PPTX)Click here for additional data file.Figure S2Evaluation of latency 5 months post-infection in MusPV1-infected mouse strains. Mice  previously inoculated with 6×1010 MusPV1 virions per animal were subjected to cyclosporin A administration at 4 months post-infection for a period of 4 weeks. After this period (corresponding to 5 months post-infection) mice did not develop visible lesions. Both, MusPV1 E1∧E4 spliced transcripts and the viral genome were undetectable in skin tissues taken from the inoculation sites. Absolute copy numbers of the MusPV1 genome, when detectable, in these samples are shown as numbers above each bar. As controls, skin tissues harvested 4 weeks post-infection from cyclosporin A-treated/MusPV1-infected Cr:ORL SENCAR mice (n = 4) were included in the analysis (mean ± SEM shown).(PPTX)Click here for additional data file.Figure S312 MusPV1 virions in Cr:ORL SENCAR mice.Transient papilloma development after inoculation with 1×10 (A) Small transient papillomas developed 2–3 weeks after infection with 1×1012 MusPV1 in Cr:ORL SENCAR mice. One representative mouse at week 3 post-infection shown. (B) The lesions showed histological features consistent with papillomas. Hematoxylin-eosin stained tissue section (magnification 4×) of a representative mouse. (C) Determination of MusPV1-specific E1∧E4 spliced transcripts relative to beta-actin revealed low, but detectable amounts of E1∧E4 in the papillomas at 3 weeks after infection with 1×1012 MusPV1 virions (M), which were absent in mock-infected littermates (0). Data from one representative mouse per group are shown; real time PCR reactions were performed in triplicate (mean ± SEM shown). (D) Immunofluorescent staining of a papilloma taken 3 weeks post-infection revealed punctate, cytoplasmic MusPV1 L1 staining  in the basal and lower spinous layers, and nuclear L1 staining in the upper spinous and granular layers of the epithelium. A phycoerythrin-conjugated anti-CD49f antibody (red) was used for co-staining of basal keratinocytes to faciliate orientation. (E) Skin tissues taken from the tail skin of a mock-infected littermate showed anti-CD49f staining, but lacked MusPV1 L1 staining. (F) The transient papillomas of Cr:ORL SENCAR mice contained infectious MusPV1 virions that were able to induce papilloma formation on the tail of an athymic nude NCr mouse after experimental transmission. (G) C57BL/6 mice did not develop papillomas after inoculation with 1×1012 MusPV1 virions (representative mouse at 3 weeks post-infection shown).(PPTX)Click here for additional data file.Figure S4+ and CD8+ T cell depletion in Cr:ORL SENCAR mice.Monitoring of CD4 Flow cytometry analyses were performed at indicated time points in the peripheral blood of (A) CD4- and (B) CD8-depleted MusPV1-infected Cr:ORL SENCAR mice and verified the depleted state. (C) Isotype-depleted/MusPV1-infected, (D) non-depleted/MusPV1-infected and (E) mock-infected littermates served as controls.(PPTX)Click here for additional data file.Figure S5+ and CD8+ T cell depletion in C57BL/6NCr mice.Monitoring of CD4 At indicated time points during (A) CD3 depletion, (B) single CD4 depletion, (C) single CD8 depletion and (D) combined CD4+8 depletion flow cytometry analyses verified the depleted state in the blood of MusPV1-infected C57BL/6NCr mice. (E) Isotype-depleted/MusPV1-infected, (F) non-depleted/MusPV1-infected and (G) mock-infected littermates served as controls.(PPTX)Click here for additional data file.Table S1(Transient) papilloma development in immunocompetent Cr:ORL SENCAR mice. MusPV1 virions were serially diluted , and decreasing doses applied to individual immunocompetent Cr:ORL SENCAR mice. After an observation period of 2.5 weeks post-infection mice were evaluated for papilloma formation.(PPTX)Click here for additional data file.


The complexes formed by BCL10, MALT1 and specific members of the family of CARMA proteins (CBM complex), have recently focused much attention because they represent a central hub regulating activation of the transcription factor NF-κB following various cellular stimulations. In this manuscript, we report the functional characterization of a Danio rerio 241 amino acids polypeptide ortholog of the Caspase recruiting domain (CARD)-containing protein BCL10. Biochemical studies show that zebrafish Bcl10 (zBcl10) dimerizes and binds to components of the CBM complex. Fluorescence microscopy observations demonstrate that zBcl10 forms cytoplasmic filaments similar to that formed by human BCL10 (hBCL10). Functionally, in human cells zBcl10 is more effective in activating NF-κB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that zBcl10 can functionally replace the human protein. Finally, we show that the zebrafish cell line PAC2 is suitable to carry out reporter assays for monitoring the activation state of NF- kB transcription factor. In conclusion, this work shows that zebrafish may excellently serve as a model organism to study complex and intricate signal transduction pathways, such as those that control NF-κB activation. NF-κB is an inducible and ubiquitously expressed transcription factor for genes involved in immune and inflammatory responses, cell survival, cell adhesion, differentiation, and growth , 2. GiveGenetic disruption of the BCL10 locus in murine strains results in immunodeficiency, having these genetically modified mice profound defects in humoral and cellular immune responses . In factThe biological function of BCL10 is explicated through formation of the CBM complex, a molecular complex that includes one of three members of the family of CARMA proteins and the protein MALT1 . The thrDanio rerio) genome have reported the presence of several CARD domain containing proteins encoded by the genome of this organism, including NOD1 and NOD2, RIPK2, PYCARD, CARD9, CARMA1, CARMA2, CARMA3 and BCL10 [Recently, extensive analysis of the zebrafish  and in the appropriate European Directives , adhering to the Guide for the Care and Use of Laboratory Animals . All the in vivo experimental activities were approved by the Animal Ethics Committee (CESA) of Biogem .Total RNA samples were extracted from whole 6-day larvae using Trizol RNA isolation reagent (GIBCO-BRL) according to the manufacturer’s instructions. 1 μg of total RNA was primed with oligo(dT) and reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions to generate a first-strand cDNA. Primers used to amplify zBcl10 were the following: forward 5’-ATGGATGTTACTCACCTG-3’ and reverse 5’-GACGTTTACGGAGACAAA-3’. PCR conditions were as follows: 98°C for 30 s, 30 cycles , and then 72°C for 5 min. Thermo Scientific® Phusion High-Fidelity DNA Polymerase (New England BioLabs) was used as amplifying polymerase, according to the manufacturer’s instructions. The RT-PCR product of the expected size was gel purified and cloned into pcDNA3 expression vector (Addgene), provided with an amino-terminal HA or FLAG epitope using standard cloning methodologies and confirmed by sequencing. Three independent clones deriving from three different PCR amplifications gave the same nucleotides sequence, corresponding to that deposited in GenBank with access number XM_002660692.The expression vectors used in this study have been previously described , 23–27http://www.ncbi.nlm.nih.gov/blast), and the multiple sequence alignment was created with ClustalW program  with default settings. Phylogenetic analyses with bootstrapping (100 replicates) were obtained by the Neighbor-joining method using complete deletion and the p-distance amino acid model in MEGA [The zBcl10 protein sequence was analyzed by using the BLAST algorithm at the NCBI web site ( in MEGA .HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The PAC2 fibroblast line, isolated from 24-h post-fertilization zebrafish embryos, was kindly provided by Dr. Isidoro [Short hairpin RNAs targeting hBCL10 were the following:shBCL10 #2: 5’-CCACCAGATCTACAGTTAGAACTCGAGTTCTAACTGTAGATCTGGTGGC-3’shBCL10 #3: 5’-CCTTAAGATCACGTACTGTTTCTCGAGAAACAGTACGTGATCTTAAGG-3’shBCL10 #4: 5’-CTTGTCGAACATCAAGTAGAACTCGAGTTCTACTTGATGTTCGACAAG-3’shBCL10 #5: 5’-GTTGAATCTATTCGGCGAGAACTCGAGTTCTCGCCGAATAGATTCAAC-3’Retroviral infections were carried out as previously described , 31.To assess for NF-κB activation, HEK293 were co-transfected in 6-well plates with 0.2 μg of pNF-κB-luc (Clontech), 0.1 μg of pRSV-βGal (Addgene) plus each expression plasmid. When necessary, the total amount of transfected plasmidic DNA (2 μg) was kept constant by adding empty vector. pNF-κB-luc encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the NFκB transcriptional response element. The plasmid RSV-βGal, expressing β-galactosidase, was added to the transfection mixture in order to normalize for the efficiency of transfection. After transfection and treatments, luciferase activity was determined with Luciferase Assay System (Promega). For measurement of β-galactosidase activity, 20 μl of cell lysates diluted 100-fold with 0.1 M potassium phosphate buffer was mixed with 200 μl of Galactone  that was diluted 100-fold with 0.1 M potassium phosphate and 1 mM magnesium chloride, pH 7.8, for 1 hr at room temperature. Then, β-galactosidase activity was measured after addition of 300 μl of Emerald (Tropix). Luciferase activity was normalized on β-galactosidase activity and expressed in arbitrary units.SC5611, generated against an epitope corresponding to amino acids 1–197 of human BCL10), Santa Cruz Biotechnology. The calf-intestinal alkaline phosphatase was purchased from Roche.Cell lysates were made in lysis buffer  and a mixture of proteases inhibitors  according to the manufacturer’s instructions. Proteins were separated by SDS–PAGE, transferred onto nitrocellulose membrane, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Blots were developed using the ECL system (Amersham Biosciences). For co-immunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Amersham Biosciences) for 2 hrs at 4°C. immunocomplexes were extensively washed, resolved by SDS–PAGE, and analyzed by immunoblot assay. Sources of antisera and monoclonal antibodies were the following: anti-FLAG, anti-β-Actin, Sigma; anti-HA and anti-BCL10  Nonidet P-40, 150 mm NaCl, 50 mm Hepes, pH 7.4, 5 mm EDTA, 10% (v/v) glycerol, and complete protease inhibitor mixture (Roche). After homogenization and centrifugation , protein concentration of supernatant was determined by BCA protein assay (Pierce). A 15 μg sample of whole cell extract was separated on SDS–polyacrylamide gel and transferred to membranes. Filters were blocked for 2 hrs in 3% nonfat dry milk in phosphate-buffered saline (PBS) with 0.3% Tween 20. Western blot analysis was performed using a rabbit anti-BCL10 antisera, followed by horseradish-peroxidase-conjugated mouse anti-rabbit antibody (Amersham Biosciences). Signal was developed using an enhanced chemiluminescence method (Amersham Biosciences) according to the manufacturer’s instructions.4 HEK293 were grown to 50% confluence and transfected in six-well chamber slides . Sixteen hours after transfection, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then permeabilized in PBS/0.1% Triton X-100. Cells were incubated for 30 min in 5% FCS–PBS with anti-FLAG antibody (Sigma-Aldrich) followed by several washes with 5% FCS–PBS, and then incubating for 30 min with secondary antibody in 5% FCS–PBS. All steps were done at room temperature.1 x 10The determination and the analysis of the zebrafish genome revealed the existence, in zebrafish, of a sequence similar to that encoding for the human protein BCL10 –22. ThusWhen analyzed in immunoblot assay, HA-tagged zBcl10 expressed in mammalian cells migrates as a 35 kDa protein , also resh isoform CMV promoter and tandem repeats of the NFκB transcriptional response element see  section.To exclude the possibility that NF-κB activation mediated by zBcl10 was due to its interaction and subsequent oligomerization of hBCL10, we abolished expression of hBCL10 in the human cell line HEK293 through retrovirus-mediated trasduction of short hairpin RNAs (shRNA) targeting hBCL10. As shown in Fluorescence microscopy experiments and structural studies have shown that the NF-κB activity produced by hBCL10 is regulated through formation of cytosolic filamentous structures , 33. We sh, CARMA2shE138A, which in humans is responsible for genetic psoriasis [shE138A induces hyperactivation of NF-κB compared to wt CARMA2sh. Since NF-κB transcribes genes that mediate the inflammatory response, such hyperactivation is thought to contribute to the manifestation of the psoriatic phenotype. Interestingly, hyperactivation of NF-κB following expression of CARMA2shE138A was also observed in PAC2 cells, confirming the existence of common regulatory mechanisms for NF-κB activation.Finally, we examined whether zBcl10 is able to activate NF-κB in zebrafish cells. For this, we used the the same NF-κB-luciferase assay we have used for mammalian cells Figs.–5. IndeeThere are several reasons that make particularly interesting the work here presented. The first one is represented by the possibility of using zebrafish as a model system to study the signal transduction pathways that modulate the activation state of the transcription factor NF-κB. Given the importance of this transcription factor in both normal cell biology and autoimmune, immunoproliferative and tumoral disorders, the possibility of using a model organism so flexible and informative such as zebrafish, certainly represents a field to massively explore further. This possibility is further supported by the extensive genomic knowledge on zebrafish we are acquiring in these days. Recently, one of these studies has revealed the presence of genes encoding for several putative CARD-containing proteins in the zebrafish genome, including the three CARMA proteins . It woulSecondly, it is interesting to note that the serine residues with inhibitory function present in hBCL10   are not shE138A, behave exactly the same way when analyzed in zebrafish Click here for additional data file.


Cysteine S-glutathionylation in eNOS reductase domain also induces O2.- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O2.- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS), a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS), which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD). Both WT eNOS and S1179D eNOS generated significant amount of O2.- in the absence of BH4 and L-arginine. The capacity of O2.- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1). O2.- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4). This BH4 un-inhibited O2.- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI). Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O2.- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O2.- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM). In addition to the heme center of eNOS oxygenase domain, we confirmed another O2.- generation site in the eNOS reductase domain and characterized its regulatory properties.In addition to superoxide (O N-myristoylation and post-translational cysteine palmitoylation in the nuclear unit and Golgi, respectively arginine was from NEN . NADPH, tetrahydrobiopterin, NG-nitro-L-arginine methyl ester (L-NAME), diphenyleneiodonium (DPI) and other reagents were purchased from Sigma unless otherwise indicated. Both bovine WT eNOS pcDNA3 and S1179D eNOS pcDNA3 plasmids were purchased from www.addgene.com.Calcium chloride (CaATGGGCAACTTGAAGAGTGTGGGCCAG-3’5’- CGGAATTCAAC and the backward primer is TCATCAGGGGCCGGGGGTGTCTGG-3’5’-GCTCTAGA. The bovine eNOS reductase domain (521–1205) PCR forward primer was: AAAGCAACCATCCTGTACG-3’5’-CGGAATTCAAC and the backward primer is: TCA TCAGGGGCCGGGGGTGTCTGG-3’5’-GCTCTAGA (start codon and stop codon was underlined). The PCR products were digested with EcoRI and XbaI and the product was subcloned into pCW plasmid arginine to L-[14C]citrulline conversion was monitored in a total volume of 200 μl of buffer containing 50 mM Tris-HCl, pH 7.6, 2 μM L-[14C]arginine, 0.5 mM NADPH, 0.5 mM Ca2+, 10 μg/ml calmodulin, 10 μM BH4, and 10μg/ml purified eNOS. After five minutes incubation at 37°C, the reaction was terminated by adding 3 ml of ice-cold stop buffer . L-[14C]Citrulline was separated by passing reaction mixtures through Dowex AG 50W-X8  cation exchange columns and quantitated by liquid scintillation counting[ENOS-catalyzation of L-[ counting.2+, 10 μg/ml calmodulin, 400nM purified eNOS, and 20 mM spin trap DEPMPO. EPR spectra were recorded in a disposable micropipette  at room temperature (23°C) with a Bruker EMX spectrometer operating at X-band with a high sensitive (HS) cavity using a modulation frequency of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20mW, and microwave frequency of 9.863GHz as described [6.Spin-trapping measurements of oxygen free radicals were performed in 50 mM Tris-HCl buffer, pH 7.6, containing 0.5 mM NADPH, 0.5 mM Caescribed , 25. TheNADPH oxidation was measured spectrophotometrically at 340 nm . The reat-test. Differences were considered to be statistically significant at P < 0.05.Data are expressed as means ± SE. Comparisons were made using a two-tailed paired or unpaired Student's Both wild type and S1179D eNOS were expressed and purified from E. Coli. In a culture of 2 liters, approximately 3.0–4.0 mg of eNOS was typically recovered using 2'5'-ADP Sepharose 4B chromatography. Further purification was performed using FPLC. The final purified products were identified by SDS-PAGE gel . Previou2.-[2.- generation from eNOS, we first characterized our DEPMPO and xanthine oxidase/xanthine measurement system. DEPMPO which has a higher affinity for O2.- than DMPO and its O2.- adduct, DEPMPO-OOH is also more stable than the DMPO-O2.- adduct[2.- generation system[2.- scavenger, 100nM BH4 did not affect the DEPMPO-OOH adduct signal generated from XO/X system (2.- until the concentration reached 500nM (data not shown). Moreover, DEPMPO-OOH adduct signals from both the XO/X system and from eNOS were significantly abolished by 200 units/mL SOD, indicating that these DEPMPO-OOH adduct signals were products of O2.- .BH4 is a critical regulator for Ofrom eNOS. A promi2+, CaM and BH4 to generating NO. ENOS generates O2.- in the absence of L-arginine and BH4. In this case the electrons are delivered to heme center and oxygen is oxidized into O2.-. In this experiment we first examined whether Ca2+ and CaM affect O2.- generation from WT eNOS and S1179D eNOS. EPR spectra showed that there was a strong DEPMPO-OOH adducts signal for WT eNOS. ENOS still generates a significant amount of O2.- in the absence of Ca2+ and CaM . In Ca2+ and CaM depleted conditions, S1179D eNOS also generated a significant amount of O2.- , a substrate for NO generation of eNOS which also binds in heme center, the DEPMPO-OOH adduct signals generated were 38367±4841.58 and 47520±1533.9 respectively, with no significant difference when compared to controls  at a Ca2+/CaM depletion condition. As shown in 2+ and CaM.Early studies showed that BH4 and L-arginine were the determinant factors regulating NO or Ome center–33. Next2.- release from the eNOS reductase domain, we subcloned the bovine eNOS reductase domain from WT eNOS plasmid and expressed it in E.Coli. 1μg purified eNOS reductase domain protein was identified on the SDS-page gel . However, S1179D eNOS consumed a significantly higher amount of NADPH than WT eNOS and eNOS reductase domain (5.35± 0.58 μM/h, P<0.05 compare to WT eNOS and reductase domain, respectively. As eNOS needs to consume NADPH to generate free electron, NADPH consumption is the indirect index consisting with O2.- from the FMN and FAD/NADPH binding site of the reductase domain. Second, O2.-generation from eNOS reductase domain is not regulated by eNOS cofactors BH4, L-arginine, and Ca2+ /CaM. However, O2.- generation from eNOS reductase domain is regulated by eNOS phosphorylation. Third, purified eNOS reductase domain protein provided direct evidence that the O2.- generation center is located in the FMN and FAD/NADPH binding sites of the eNOS reductase domain. Finally, NADPH consumption rate also indicate that eNOS reductase domain has an electron leakage site and generation of O2.-.In this study, we obtained three important findings. First, spin trapping experiments presented definitive evidence that eNOS generates O2.- generation from nNOS[2.- generation from nNOS [2.- generation from eNOS. This O2.- generation was speculated from eNOS reductase domain as they locate in the FAD/NADPH binding subdomain[2.- generated from the FMN and FAD/NADPH binding sites of eNOS. Overall, previous studies and our current study indicated all three isoforms of NOSs generate superoxide from their reductase domain.Although eNOS, nNOS and iNOS have different oxygenase domains and enzyme activities, they have similar structure in their reductase domains those function like NADPH-cytochrome P-450 . Previoufrom nNOS–35. Pharrom nNOS . Moreovesubdomain. In the 2+ /CaM, we also investigated the impact of these factors on the function of the eNOS reductase domain. Distinct from O2.- generation from the heme center of the eNOS oxygenase domain, BH4, L-arginine and Ca2+ /CaM did not affect O2.- generation from eNOS reductase domain. As DPI, a flavoprotein inhibitor, inhibited O2.- generation, the generation site may be the flavin center of the FMN and FAD/NADPH binding subdomains.As eNOS function is controlled by many factors, including BH4, L-arginine and Ca2.- generation from eNOS reductase domain. Our results show that mutation of Ser1179D eNOS significantly increases O2.- generation from both eNOS oxygenase domain and eNOS reductase domain (2.-. We are on the way to determining the importance of electron redistribution between the eNOS oxygenase domain and eNOS reductase domain caused by eNOS Ser1179 phosphorylation.Unlike the effect of eNOS cofactors on eNOS reductase domain, eNOS Ser1179 phosphorylation enhances Oe domain . NADPH c2.-. At physiological conditions, O2.- generation from the oxygenase domain and reductase domain in WT eNOS are 65.7 ± 10.3% and 34.2±1.8%, respectively (Figs 2+/CaM and by purified eNOS reductase domain also indicated that the electron was consumed by eNOS reductase domain (2.-is generated in the eNOS reductase domain (1/3 of total O2.-generation from eNOS, 2.-generation center in the eNOS reductase domain, its potential binding cofactor or chaperone protein remain unclear.Previous observations have found that the eNOS reductase domain binds with NADPH and generates an electron, which is delivered into the heme center of the oxygenase domain. In thise domain . Althoug2.- generation site in the eNOS reductase domain. This site is not regulated by BH4, L-arginine and Ca2+/CaM, but it is enhanced by eNOS phosphorylation. The O2.- generation capacity of the eNOS reductase domain is around 1/3 of the total eNOS O2.- generation capacity. Studies with pharmacologic inhibitors and cloned eNOS constructs indicate that this O2.- generation site is located in the FMN and FAD/NADPH binding region of eNOS reductase domain. These novel findings will aid the understanding of eNOS function and may help in the development of new therapeutic strategy for the patients suffering eNOS dysfunction related diseases.In conclusion, we offer direct evidence to identify a new O


Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity.Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27 Endometrial cancer (EC) is the most common gynecological malignancy. In the United States alone, 54, 870 new EC diagnoses and 10, 170 fatalities are expected in 2015. Hysterectomy with or without adjuvant treatment for localized EC yields a 5-year survival rate of approximately 95%. However, treatment outcome is poor in the 28% of EC cases that are diagnosed in an advanced stage .Recent research on the effects of androgens on endometrial tissue might have important implications for EC treatment. Polycystic ovarian syndrome (PCOS), an androgen-related disease characterized by prolonged periods of anovulation, has long been associated with increased risk of EC. PCOS and EC share many of the same risk factors, and androgens might also promote the development of EC tumors. A recent Australian study found that women with PCOS were at a four times higher risk of developing EC than women without PCOS. This risk of EC was associated with symptoms of androgen excess, such as hirsutism  and irregular periods  [The androgen receptor (AR), a nuclear transcription factor, regulates EC progression. AR can inhibit the growth of endometrial epithelial cells in culture, and this effect is blocked by an AR antagonist . Howeverin vitro and in vivo [Enzymes that regulate reversible histone lysine methylation also might play an important role in EC . One suc in vivo –12. Fina in vivo . These oin vitro and in vivo studies to identify the effects of KDM4 enzyme activity on AR signaling and EC progression. Levels of the four KDM4 proteins were decreased using siRNA in different cellular models of EC, and resulting changes in AR signaling and EC progression were measured using qRT-PCR, immunoassays, and measurements of cellular migration and proliferation. Additionally, known target genes of AR were probed in these cell lines to determine specific downstream molecular effects of manipulating KDM4 levels. Because KDM4 enzymes are important regulators of histone methylation, epigenetic changes were also examined in transfected cells. The use of cell lines with both high and low baseline AR expression AR allowed us to identify distinct roles for KDM4 proteins in EC. Xenograft experiments in which mice were injected with EC cells with either normal or reduced levels of specific KDM4 proteins confirmed their effects in vivo. Finally, tissues and genetic information from human patients with EC were examined to confirm the potential clinical relevance of our findings regarding KDM4 and AR.Here, we conducted a variety of To help establish whether AR signaling affects EC progression, we used cBioPortal to examine cross-cancer alteration summaries of AR, which included AR amplification, mutation, and deletion Figure . EC patiWe used siRNAs to decrease expression of each KDM4 methylase in order to determine whether these proteins affect AR signaling and EC cells. RT-PCR revealed that depletion of KDM4B down-regulated expression of the AR-dependent gene c-myc Figure  and 2B iTo test the effects of KDM4B knockdown on EC progression, MFE-296 cells were transfected with siRNA against AR, KDM4B, both AR and KDM4B, or a negative control (NC). Proliferation of MFE-296 cells decreased after knockdown of either AR or KDM4B. When both AR and KDM4B were knocked down, proliferation was further inhibited in MTT and clonogenic growth assays Figure  and 3B. in vivo. The final mean weight and volume of tumors in the MFE-296/shKDM4B group were significantly lower than those in the MFE-296/NC group  in AN3CA cells, which have low levels of AR Figure . KDM4B dkip1/p16/PTEN/p53) [kip1 gene  –17 aftere Figure  in respokip1 promoter in response to androgens. DHT treatment also recruited AR to the p27kip1 promoter  or NC (AN3CA/NC)  Figure . The fin) Figure . Xenogra3 Figure . Western3 Figure  and 2C.P < 0.05, **P < 0.01, Figure P < 0.05, Table Given the roles of KDM4B and KDM4A in EC cell AR signaling, expression of these two enzymes was assessed in human EC clinical material Figure . In totan = 333) in an independent dataset from TCGA network. Although UCEC patients with KDM4B and KDM4A alteration seemed to have poorer DFS rates, this effect did not reach statistical difference  of uterine corpus endometrioid carcinoma (UCEC) patients (kip1) and accelerates EC progression  signaling axis that regulates AR target genes  2008–0064). All efforts were made to minimize animal suffering.Tissue samples from 76 cases of uterine EC were obtained from patients who underwent surgery between June 1, 2011 and April 30, 2013 at the International Peace Maternity & Child Health Hospital affiliated with the Shanghai Jiao Tong University School of Medicine. The stages and histological grades of these tumors were established according to the criteria of the Federation International of Gynecology and Obstetrics (FIGO) surgical staging system (2009) . Two ind2 at 37°C.Human EC cell lines MFE-296, KLE, HEC-1B, and AN3CA were purchased from the Chinese Academy of Sciences Committee Type Culture Collection cell bank. MFE-296 cells are derived from a moderately-differentiated primary endometrial adenocarcinoma (grade 2) with high level of AR , while A5 cells/well in 6-well plates. After the cells were grown overnight, they were transfected with JMJD1C(siJMJD1C) , siKDM5A, siKDM4A, siKDM4B, siKDM4C, siKDM4D, siSMAD4, siKDM1A, siAR, or siRNA negative control (siNC)  using Lipofectamine2000  according to the manufacturer's instructions. The sequences are listed in Table 2000 (Invitrogen) according to the manufacturer's instructions.Cell transient transfections were performed as we previously reported . In brie2 [Tissue specimens of normal endometrial cells were obtained from ten women with endometriosis who had given informed consent before surgery. These women had no endometrial hyperplasia or neoplasia, and had not received any hormonal medication or anti-inflammatory drugs for at least 3 months prior to surgery. The endometrial biopsies were minced into small pieces and digested with 0.2% collagenase (Gibco). Cells were pelleted by centrifugation, resuspended in Dulbecco's modified Eagle's medium (DMEM)/F12 containing 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% FBS, and plated in 100-mm diameter culture dishes, then grown at 37°C in 5% CO2 .−7 M for different periods of time (0–24 h). Vehicle contained 0.1% absolute ethanol/phenol red–free DMEM/F12.Cells were incubated in phenol red–free DMEM/F12 (Gibco) overnight, with the final concentration of ethanol at 0.1% 5α-dihydrotestosterone (DHT)  was added to the cell culture media at concentrations of 10−7 M (100 nM) DHT and 24 h of incubation elicited the strongest expression of AR and its target genes [To investigate the impact of KDM4A and KDM4B combined with DHT treatment, cells that had been transfected with siKDM4A, siKDM4B, or siNC for 36 h were incubated with phenol red–free DMEM/F12 for 12 h. DHT or vehicle was then added according to the methods mentioned above. Previous dose-response experiments revealed that 10® realplex according to the manufacturer's instructions. Each sample was assayed in triplicate in each of three independent experiments. The primer sequences are listed in Table Total RNA was extracted from cultured cells using Trizol Reagent (Invitrogen). RNA was converted to cDNA with a one-step Prime Script RT reagent kit , and quantitative real-time polymerase chain reaction (PCR) was performed by using SYBR Premix Ex Taq (TaKaRa) in an Eppendorf Mastercyclerkip1 . Protein bands were visualized by enhanced chemiluminescence , and protein expression was normalized against β-actin .For Western blot assays, conducted as previously described , antibodTotal protein was extracted from MFE-296 cells. After protein quantification, 500 μg of each cell lysate was added to 10 μl of AR or KDM4B antibody, shaken at 4°C overnight, and then added to 30 μl of Protein A + G Agarose , shaken at 4°C for 4 h, centrifuged at 2500 × g for 5 min, and washed with a RIPA kit (Beyotime) to collect the immunoprecipitate-bound agarose beads. Each immunoprecipitate was denatured with 20 μl of 1× SDS-PAGE loading buffer at 100°C for 5 min. Each supernatant was subjected to SDS-PAGE (8% acrylamide). Other steps were as described in the Western blotting section.2000 according to the manufacturer's protocol. Cells were harvested and subjected to immunoprecipitation using AR antibody (Santa Cruz), followed by Western blotting with KDM4B antibody (Bethyl Laboratories) and KDM4A antibody.To determine whether KDM4A or KDM4B can interact with AR in AN3CA cells, AN3CA cells grown in DMEM/F12 media containing 10% fetal calf serum were transfected with 1 mg of PWP1/GFP/Neo-AR or 1 mg of its negative control PWP1/GFP/Neo after 72 h using Lipofectaminekip1 promoter between − 1523~− 1294 includes the AR binding site. Enrichment was calculated by using the comparative Ct method. Primers used (including p27kip1 promoter targeting the range − 1523~− 1294) are shown in Table Chromatin immunoprecipitation (ChIP) assays were performed as previously described , 39 usin3 cells/well in 96-well plates. Then, 20 μl of 3--2,5-diphenyltetrazolium bromide  was added to each well. Wells were subsequently incubated at 37°C for 4 h. Absorbance was measured at 490 nm using a Spectra Max 190 microplate reader . In colony formation assays, cells were seeded at 120 cells/well (MFE-296 cells) or 200 cells/well (AN3CA cells) in 6-well plates. After 14 days, cell colonies were stained with 0.1% crystal violet for 1 h and counted.In MTT assays, cells were plated at 3 × 105/well (for the migration assay) or 2 × 105/well (for the invasion assay) in invasion chambers  with or without matrigel coating for invasion or migration assays. Complete medium was added to the lower chamber as a chemoattractant. After 5 h (MFE-296) or 24 h (AN3CA) of incubation for the migration assay, or after 24 h (MFE-296) or 48 h (AN3CA) of incubation for the invasion assay, cells were fixed with 4% paraformaldehyde for 1 h. Cells on the apical side of each insert were removed by mechanical scraping. Cells that migrated to basal side of the membrane were stained with 0.5% crystal violet and counted at 200× magnification. Cells migration and invasion assays were repeated at least three times.Cells were plated serum-free at a density of 1 × 10The lentivirus vector down-expressing KDM4B was prepared for MFE-296 cells using shRNA targeting KDM4B  or a negative control vector  according to the manufacturer's protocol. The lentivirus vector down-expressing KDM4A was prepared for AN3CA cells using a shRNA targeting KDM4A  or a negative control vector  according to the manufacturer's protocol. shRNA lentiviral particles were treated in six-well plates in the presence of polybrene (6 μg/ml). Cells were treated with puromycin (2 μg/ml) to generate stable KDM4B and KDM4A knockdown clones, and several puromycin-resistant KDM4B and KDM4A knockdown clones were harvested by ring selection. The shRNA oligo sequences are provided in Table 6 cells of one of the following cell types: MFE-296/shKDM4B, MFE-296/shNC, AN3CA/shKDM4A, or AN3CA/shNC. Tumor size was monitored every 4 days by measuring the length and width with calipers, and tumor volumes were calculated with the formula: tumor volume (cm3) = (the longest diameter) × (the shortest diameter)2 × 0.5. The tumors were removed and weighed after 42 days (for the two MFE-296 groups) or after 49 days (for the two AN3CA groups).For the xenograft experiments, 20 5-week-old female BALB/c nude mice  were randomly divided into four groups. The mice were injected subcutaneously in the nape with 5 × 10Paraffin-embedded normal endometrial, atypical hyperplasias, and EC tissue sections (4 μm) were processed for immunohistochemical analysis as previously described . Brieflykip1 antibody , and ki-67 antibody  were used.In addition, tissue specimens (4 μm) of mouse xenografts were routinely prepared and immunohistochemically analyzed by using the standard avidin-biotin techniques. KDM4B antibody, c-myc antibody , KDM4A antibody, p27http://cbioportal.org) provides a Web resource for exploring, visualizing, and analyzing multidimensional cancer genomics data. Specifically, because of interests in the gene set of AR, we entered AR gene symbols, and then a report of genomic data across all cancer types was presented in a concise graphical summary called a cancer genomics of cross-cancer alteration summary of AR. We then selected endometrial carcinoma, entered AR and KDM4B gene symbols, and a network view of the AR neighborhood in EC was shown. All genes were ranked by the frequency of genomic alteration within the EC study, and less frequently altered genes were automatically pruned from the network.The cBioPortal for Cancer Genomics /disease-free survival (DFS) of EC patients using published data from The Cancer Genome Atlas (TCGA) network [P values from a log-rank test.We performed an  network , 44. Clit test was used for comparison between two groups, and one-way ANOVA followed by the post-hoc Tukey test were used for comparison among multiple groups. Correlation was analyzed by the Spearman test, and OS was assessed by using the standard log-rank test and the Kaplan-Meier method. All P values were two-tailed, and P values < 0.05 indicated a statistically significant difference. Each experiment was performed at least three times independently.Data were presented as mean ± SD and analyzed by SPSS 16.0 software . The Student


Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1 When cells divide they need to ensure that a complete copy of their genetic material is transmitted to both daughter cells. Cells have evolved many controls to ensure that every division is carried out with very high fidelity. The spindle checkpoint is one such control, which acts as a surveillance system during mitosis. Defects in this checkpoint control lead to unequal segregation of DNA/chromosomes, termed aneuploidy, which is responsible for human birth defects and is very common in tumour cells.Mph1 kinase. We show that Mps1Mph1-dependent modification of Mad3 and Mad2 acts to delay cell division in situations where the genetic material would not be equally inherited by daughter cells. This delay enables the cell to correct any problems within the division machinery and thus avoid aneuploidy.The molecular components of the spindle checkpoint, identified initially through yeast genetics, include several protein kinases. Surprisingly few of their substrates have been identified. Here we identify the checkpoint protein Mad3 as an important substrate of the Mps1 Slp1 .Six litres of yeast cells were grown at 30°C in concentrated rich medium to high densities that could still support active proliferation and mitotic arrests. Mitotically arrested cells  were obtained by rapid cooling on iced-water and shifting cultures to 18°C for 7 hours. Cells were harvested by centrifugation at 3,500g for 8 minutes at 4°C. Pelleted cells were frozen into pea-sized drops using liquid nitrogen and stored at -80°C until further processing. Approximately 25 g of cell mass was disrupted using a mixing mill  with grinding balls under cryogenic conditions (5 cycles of 3 minutes at 30 Hz). Yeast lysates were reconstituted in lysis buffer A containing 50mM Bis-Tris Propane-HCl pH 7.6, 10% (v/v) glycerol, 100 mM KCl, 5 mM EGTA and 1% Triton X100 supplemented with a ‘complete EDTA-free protease inhibitor’ tablet (per 50 ml), 1mM Pefabloc SC, 10 μg/mL leupeptin/pepstatin/chymostatin , 20 mM β-glycerophosphate, 5mM NaNProteins were eluted from Dynabeads by two x 5 min incubations with 150 μl of 100mM glycine-HCl (pH 2.5) at room temperature and precipitated with 100 μl of cold 100% (w/v) trichloroacetic acid. Proteins were pelleted at 4°C by centrifugation for 30 min at 22,000g. Supernatant was removed and pellets were washed twice with 500 μl of ice-cold acetone. and air-dried.Proteins were reduced, alkylated and digested with trypsin. Peptides were pressure vessel loaded onto a biphasic (C18-SCX) column. Peptides were separated by MuDPIT  using an Aligent 1100 quaternery LC with a 1:1000 split flow system. Peptides were eluted into an LTQ mass spectrometer (Thermo Fisher Scientific) at spray voltage of 2.6 kV. Eight data-dependant ms/ms were collected per primary scan. MS3 scans were triggered with neutral loss values of 32.50, 49.00 and 98.00.Slp1 and radiolabelled [Met S35] APC/C substrate, Securincut2 were generated by in vitro translation using the TNT Quick Coupled Transcription/Translation kit (Promega) according to manufacturer’s instructions using 1μg of pHY22-Cdc20Slp1 and pHY22-Securincut2 plasmids also provided by Hiro Yamano. Lid1-TAP APC/C was affinity purified using IgG agarose beads  as previously described [pombe cells carrying the ts slp1-362 mutation [slp1.Fission yeast genes E1 (Uba1) and E2s  inserted in pMAL and pET16b plasmids respectively were kindly provided by Hiro Yamano . These were used to recombinantly express proteins in BL21 RIL cells as described above for the Mad3 constructs. MBP-Uba1 and His-tagged E2s were then affinity purified using either amylose (NEB) or talon resin (Clonetech). MBP-Uba1 was subjected to further purification by size exclusion chromatography using a Sephadex S200 column. Full-length APC/C activator Cdc20 escribed ,79 from mutation  to allowIn vitro APC/C ubiquitination assays were performed following a method based on assays previously described for budding, fission yeast and human proteins [2, and 0.2 mM DTT. The ubiquitination mix was incubated at 23°C for 20 min. 0.5 μM APC/C, 4 ul and 2 ul of Cdc20Slp1 and SecurinCut2 respectively were then added to the mix in a total reaction volume of 20 μl. Reactions were carried out at 23°C for 45 min and stopped by adding 4xSDS gel buffer. Samples were subjected to SDS-PAGE electrophoresis and visualised by radiography using the Typhoon phosphoimager. The ubiquitination of SecurinCut2 by APC/C was analysed using the ImageQuant software  by quantifying the decrease in intensity of the unmodified Securin band  Fission yeast Mad3p alignments. These fission yeast sequences are available at the Broad Institute . The two sets of mutations (generating N9A and C9A) are highlighted on the S. pombe sequence with red boxes. T82 (a putative Aurora site) is in green. (B) Phosphosites identified. The superscript indicates the number with number of unique spectra/peptides in which the phosphorylated residue was identified. The residues highlighted in red (identified in vitro and confirmed in vivo) were only observed in mitotic samples.(PDF)Click here for additional data file.S2 FigThese serve as examples of MS/MS spectra used to identify and assign phosphorylation of peptides. Fragment ions containing the peptide’s N- (b-ions) or C- (y-ions) termini are labelled. A number of the identified peptides show neutral loss of phosphoric acid from the full-length peptide, supporting the conclusion of peptide phosphorylation. Significant fragmentation has also occurred allowing peptide sequencing and assignment of the phosphorylated residue.(PDF)Click here for additional data file.S3 Figcdc25 strains containing wild-type mad3, or mad3-S289A, or mps1-kd mutations were synchronised in G2, released in mitosis and then challenged to arrest in response to 75μg/ml of the anti-microtubule drug carbendazim (CBZ). Failure to maintain mitotic checkpoint arrest leads to septation. This was scored at 15 minute time-intervals, by methanol-fixing cells and staining with calcofluor. The mad3-S289A allele behaved like wild-type.(A) Strains were plated on rich (YES) media containing the indicated concentrations of the microtubule disrupting drug benomyl, and grown at 30°C for 3 days. (B) (PDF)Click here for additional data file.S4 Figcdc25 strains containing mad3-C9A, mad2-S92A, mad3-C9Aandmad2-S92A, or mps1-kd mutations and GFP-tagged Mad3 were synchronised in G2, released in mitosis and then challenged to arrest in response to the anti-microtubule drug carbendazim (CBZ). Failure to maintain mitotic checkpoint arrest leads to septation. This was scored at 15 minute time-intervals, by methanol-fixing cells and staining with calcofluor. These images are from the 60 minute time-point and accompany the experiment presented in mad3-C9A cells the Mad3-GFP can still be detected on kinetochores after cells have septated. This observation is consistent with such cells failing to maintain their checkpoint arrest due to a “downstream” defect in APC/C binding, rather than a Mad3 defect in checkpoint signalling at kinetochores and/or the kinetochore attachments being satisfied in these mutant strains.(A) (PDF)Click here for additional data file.S5 Figcdc25 strains indicated were pre-synchronised in G2 by shifting to 36°C for 3.5 hours. They were then released at 25°C and time points taken every 15 minutes. Carbendazim (CBZ) was added after 20 minutes. Cells containing Mad3-GFP were fixed in methanol, stained with calcofluor and then scored for septation, which indicates a failure to maintain spindle checkpoint arrest. The images are from the 60 min time point, and represent the strains used in Figs (PDF)Click here for additional data file.S6 FigSlp1 is ~80% in the mps1-kd strain when compared to wild-type in this and similar experiments. This can partly account for the reduced levels of MCC in mps1-kd, but not in the mad3 phospho-mutants.The peak level of Cdc20(PDF)Click here for additional data file.S7 Fig(A) The recombinant Mad3-MBP fusions are all soluble and stable. The gel shown here was stained with coomassie. (B) Mad3 phospho-mimics are targeted to kinetochores. The indicated strains were synchronised in G2 using the cdc25 block, and then released into mitosis before imaging. Scale bar is 10 microns. (C) The mad3-3D/E and 4D/E phospho-mimic alleles are not significantly benomyl sensitive, but the mad3-6D/E and 7D/E alleles are sensitive. Strains were plated on rich (YES) media containing the indicated concentrations of the microtubule disrupting drug benomyl, and grown at 30°C for 3 days. (D) The mad3-3D/E and 4D/E phospho-mimic alleles are checkpoint competent, but the mad3-6D/E and 7D/E alleles are unable to maintain an nda3 arrest. nda3 strains containing the indicated mad3 alleles, or the mad2 deletion as a control, were shifted to 18°C for 4 and 6 hours. Plo1-GFP, which binds SPBs in mitosis, was used to score the mitotic index. The mad3-3D/E and -4D/E alleles arrest well. The mad3-6D/E and -7D/E alleles initially arrest (4 hours) but are unable to maintain the checkpoint arrest and by 6 hours have a significant defect. This indicates that having six or more constitutive phospho-mimic mutations near KEN2 perturbs Mad3p function.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.