In this study we measure apoptosis of CD4+CD25+high T cells to see if it could contribute to reduced suppressive activity of these cells.In experimental models, Type 1 diabetes T1D can be prevented by adoptive transfer of CD4+CD25+ FoxP3+ suppressor or regulatory T cells. Recent studies have found a suppression defect of CD4+CD25+high and CD4+CD25− T cells were co-cultured in a suppression assay to assess the function of the former cells. We found that recent-onset T1D subjects show increased apoptosis of CD4+CD25+high T cells when compared to both control and long-standing T1D subjects p<0.0001 for both groups. Subjects at high risk for developing T1D 2–3Ab+ve show a similar trend p<0.02 and p<0.01, respectively. On the contrary, in long-standing T1D and T2D subjects, CD4+CD25+high T cell apoptosis is at the same level as in control subjects p = NS. Simultaneous intracellular staining of the active form of caspase 3 and FoxP3 confirmed recent-onset FoxP3+ve CD4+CD25+high T cells committed to apoptosis at a higher percentage 15.3±2.2 compared to FoxP3+ve CD4+CD25+high T cells in control subjects 6.1±1.7 p<0.002. Compared to control subjects, both recent-onset T1D and high at-risk subjects had significantly decreased function of CD4+CD25+high T cells p = 0.0007 and p = 0.007, respectively.T-cell apoptosis was evaluated in children and adolescent 35 females/40 males subjects comprising recent-onset and long-standing T1D subjects and their first-degree relatives, who are at variable risk to develop T1D. YOPRO1/7AAD and intracellular staining of the active form of caspase 3 were used to evaluate apoptosis. Isolated CD4+CD25+high T cells in recent-onset T1D subjects and in subjects at high risk for the disease. This high level of CD4+CD25+high T-cell apoptosis could be a contributing factor to markedly decreased suppressive potential of these cells in recent-onset T1D subjects.There is a higher level of ongoing apoptosis in CD4+CD25+ Twenty years ago, T1D was hypothesized to be a chronic autoimmune disorder by Eisenbarthin vivo and in vitro experiments, exact identification and isolation has been and continues to be a problem. The FACS isolation method that gives the high number of most potent Tregs is based on the highest surface expression of CD25 IL2-Ralphahigh T cells are enriched in cells with regulatory properties. It has been shown in animal models that CD4+CD25+high T cells can prevent murine T1Dhigh also include activated CD4+CD25+ T cells that do not generally show regulatory properties. Nevertheless, high expression of CD25 on CD4+ cells has been shown to mark a small number of highly suppressive T cells. Although a specific molecule for regulatory T cells is lacking, the transcription factor FoxP3 - forkhead box P3, has been identified as necessary for their developmentAlthough the existence and function of regulatory T cells is confirmed in both high T cells in T1D patientshigh T cells in T1D patients, there is no compelling evidence identifying an underlying cause. Spontaneous apoptosis of Tregs in human T1D patients as a mechanism has not been fully investigated.Several studies with healthy control subjects and T1D patients have reported decreased regulatory function of CD4+CD25+high T cells in relation to their function. Healthy control and several T1D-related clinical groups were studied. These included recent-onset T1D subjects and unrelated, unaffected subjects with one, two and three positive T1D specific auto-antibodies aAbs to GAD, IA-2 and insulin. Those with two or more aAbs are at higher risk for developing T1D in the future. We also studied longstanding T1D and T2D patients. Our results implicate a role of increased apoptosis of CD4+CD25+high T cells in the pathogenesis of human T1D. Furthermore, intriguing data presented here shows that Tregs FoxP3+ve T cells within this population contribute most to the increased apoptosis. However, a strong inverse relationship between apoptosis of CD4+CD25+high T cells and their function needs to be investigated on a larger sample size in different clinical groups.In this study, we measured apoptosis of CD4+CD25+Seventy-five Wisconsin study subjects 67 Caucasian, 5 Asian, 2 African-American and 1 Hispanic of both genders 35 females and 40 males were recruited through the diabetes clinics at Children's Hospital of Wisconsin CHW, Froedtert Hospital FMLH and other local hospitals and clinics that had over 1600 T1D families, through ascertainment of a proband with the T1D onset before age of 17. In families with a child proband, siblings and parents were also asked to join the study. However, just one person from each family was included in our study. Control subjects were recruited by posting flyers in areas of the hospitals that might be visited by potential “healthy” subjects, i.e. orthopedic, dental, and eye clinics. Inclusion criteria for control subjects were random blood glucose less then 110 mg/dl, no history of any autoimmune disorder including T1D in the family and an absence of T1D- specific aAbs. Although healthy subjects of all ages were included n = 28, age-matched control subjects formed a separate clinical group n = 10 because of the possible age effect on number and function of isolated CD4+CD25+high T cellsAll participants had aAbs and glucose levels measured at the recruitment visit. An accurate method for the detection of multiple T1D auto-antibodies developed by Hagopian and collaborators was employedhigh using the Fluorochrome Minus One FMO method, that allows a more precise definition of cells having fluorescence above the background levelhigh T cells, known to be enriched for TregsRecent-onset and long-standing T1D and T2D subjects had HbA1c and insulin dosage recorded at each visit. Peripheral blood mononuclear cells PBMC were collected using vacutainers with ACD solution B of trisodium citrate and isolation was done using Ficoll-Hypaque density gradient centrifugation following the manufacturer's protocol Amersham Pharmacia, Uppsala, Sweden. Cells were counted and stained with a cocktail of fluorochrome-conjugated monoclonal antibodies in PBS APC-αCD4 clone RPA-T4, APC-Cy7-αCD25 clone M-A251, FITC-αCD14 clone M5E2, FITC-αCD32 clone FLI8.26, FITC-αCD116 clone M5D12, PE-Cy7-CD8 clone RPA-T8 all from BD Pharmingen, San Diego, CA and sorted on FACSAria BD Biosciences, San Diego, CA. Cells were gated first for live lymphocyte, eliminating dead cells and debris. Next, two gates were set up to eliminate non-CD4 T cells FITC- and PECy7-conjugated antibodies. CD4+ T cells were further gated as CD4+CD25- and CD4+CD25+high T cellsApoptosis in all samples was measured using our recently published method high T cells is their capacity to suppress proliferation of responder T cells. This capacity is measured via a proliferation assay, where responder CD4+CD25− and suppressor within the CD4+CD25+high population T cells are stimulated in vitro separately and together in co-culture under defined conditions. The proliferation assay was performed using 10,000 responder T cells CD4+CD25− and 10,000 irradiated PBMCs 5000rad along with 1,000 CD4+CD25+high T cells seeded in a 96-well plate. Stimulation was with aCD3-coated beads 1 ug/ml, 3 beads/cell for 5 days under 5% CO2 and saturated humidity. Cells were left in culture after pulsing 1 µCi of [H3]-thymidine for an additional 16 hours, then harvested and after adding scintillation liquid, read for counts per minute cpm. Suppression % of Tregs was calculated as [cpm of responders in single culture − cpm of cells in co-culture/cpm of responders in single culture]×100.The only reliable feature of CD4+CD25+The Mann-U-Whitney and Tukey-Kramer tests were used to compare results between clinical groups with p value ≤0.05 considered significant. GraphPad software was used for data presentation. We also performed Kruskal-Wallis in addition to a one-way ANOVA.high T cells.We used a FACS machine for cell separation in this study. The sorter allows specific isolation of a cell population from a heterogeneous T-cell mixture, especially if the surface marker is used as a tag. To minimize the number of activated T cells that express high levels of CD25, we only collected 1% of cells with the highest CD25 expression. CD4+CD25+high were collected and analyzed ex vivo for 1. proliferation/suppression; 2. apoptosis determination using nucleic acid stains YOPRO1/7AAD; 3. intracellular staining for FoxP3 and 4. intracellular staining for aaCas3. The CD4+CD25− T cells are used as responders in a suppression assay and they also served as an internal control for the measurement of apoptosis.In all clinical groups studied, two T-cell subsets CD4+CD25− and CD4+CD25+high T cells were analyzed for apoptosis using the YOPRO1/7AAD stain combinationhigh T-cell apoptosis was significantly different across the clinical groups Kruskal-Wallis p<0.0001; ANOVA F = 16.07 df 6, 78, p<0.0001, high T-cell apoptosis in recent-onset T1D subjects differed significantly from all other clinical groups except from the high at-risk group high is the highest in recent-onset T1D subjects 11.4±1.1 followed by high at-risk subjects 8.6±1.5. The lowest measured CD4+CD25+high T-cell apoptosis level is among the longstanding T2D 3.5±0.4 and is similar to CD4+CD25+high T-cell apoptosis among both the control and longstanding T1D subjects 3.9±0.5 and 4.0±0.5 respectively. When compared to control subjects, CD4+CD25+high T-cell apoptosis of recent-onset T1D showed the strongest statistical difference p<0.0001. We were also able to recruit ten age-matched control subjects to rule out the possibility that increased age was associated with reduced apoptosis of CD4+CD25+high T cells. Compared to the age-matched control group, recent-onset T1D subjects showed a significant increase in CD4+CD25+high T cell apoptosis p<0.0001. While not as noticeable, the high at-risk clinical group both 2 Ab+ and 3 Ab+ also showed a significant increase in CD4+CD25+high T-cell apoptosis when compared to random control subjects p = 0.008.Within a uniformly short time after FACS isolation, in all studied subjects, fresh CD4+CD25− and CD4+CD25+high T-cell apoptosis isolated from the same subjects measured by two different apoptosis assays. The apoptosis results were similar. Furthermore, correlation of apoptosis results measured by these two methods was significant Spearman's rank correlation coefficient r = 0.59 95% CI = 0.28–0.79, p = 0.0006. A higher percentage of CD4+CD25+high T cells in recent-onset T1D subjects produced more active caspase 3 than the same T-cell subtype in control subjects, confirming the ongoing apoptosis detected by YOPRO1/7AAD. Control subjects had significantly lower apoptosis level from both recent-onset T1D and high at-risk subjects as measured by intracellular staining of aaCas3 p = 0.0006 and p = 0.009 respectively.In order to confirm our results using YOPRO1/7AAD, we used a second apoptosis detection method. Thus, an aliquot of isolated T cells was stained for the presence of active caspase 3. high T cells comprise FoxP3+ Tregshigh T cells committed to apoptosis at a higher percentage 15.3±2.2 compared to FoxP3+ve CD4+CD25+high T cells in control subjects 6.1±1.7; high T cells in both control and longstanding T1D subjects did not show differences in the apoptosis rate, FoxP3+ve CD4+CD25+high in recent-onset T1D subjects were significantly more apoptotic than FoxP3-ve CD4+CD25+high T cells from the same individual Since CD4+CD25+high T cells were also tested for their suppressive function using standardized in vitro suppression assays. Analysis of CD4+CD25−/CD4+CD25+high T cell co-culture at a ratio of 1∶10 indicated that CD4+CD25+high T cells in control subjects showed average suppression of 46.0.±5.9% while high at-risk and recent-onset T1D subjects showed similar suppression of 10.±9.9% and 9.9±5.5% respectively, high T cells p = 0.0007 and p = 0.007, respectively. Although suppressive potential of CD4+CD25+high T cells isolated from longstanding T1D subjects was higher than in recent-onset T1D subjects, it was significantly lower when compared to healthy controls p = 0.04.FACS-isolated CD4+CD25+high T cells p<0.0001. Although exogenous insulin levels are significantly different because of the honeymoon phase p = 0.004, our data for CD4+CD25− T cells do not show apoptosis differences, which confirm a non-significant role of insulin or any other factor in the protection from or genesis of apoptosis. Age-dependent differences in the frequency of have been shown beforehigh T cells dependent on donor age were observed Mann-Whitney U-test p = 0.19.One must be aware of other factors associated with increased apoptosis of T cells in the clinical setting. These include ambient glucosehigh T cells in T1D families and control subjects. Although a recent study shows that 2–3% of the highest CD25-expressing T cells can be safely sorted as Tregsin vitro, is monitored by suppression of the proliferation of responder T cells. In fact, recent studies in vivo have confirmed that Tregs probably act in a similar manner by inhibiting the interaction between antigen-presenting cells and responder T cells, leading to reduced proliferation of the latter cellshigh dysfunction in T1D In this study, we aimed to measure apoptosis and function of CD4+CD25+high T cells from T1D families and control subjects. The highest levels of apoptosis were found in recent-onset T1D subjects and in individuals at high risk 2+3Ab+ve for disease We present evidence that increased apoptosis of Tregs could potentially play a role in the pathogenesis of human T1D, a major autoimmune disease. Apoptosis was measured in freshly isolated CD4+CD25+high T cells that is similar to control subjects. The percentage of apoptosis increases with the number of Abs detected in the serum, reaching the highest level in subjects at the onset of the disease staying high during the period immediately following diagnosis. It is during this period that requirements for exogenous insulin drop dramatically and beta cell function improves “honeymoon” phase. Later on, when the autoimmune destruction is complete and insulin requirements increase, CD4+CD25+high T-cell apoptosis goes back to the level detected in healthy control subjects.Our data suggest that individuals who are positive for one Ab low risk to develop T1D have a low apoptosis level of CD4+CD25+high T-cell fraction using YOPRO1/7-AAD high T cells high T cells that contribute most to the apoptosis seen in highest CD25-expressing T cells.Our results with the new stain combination are confirmed with a second, independent, apoptosis detection method on the same samples. The second method was the intracellular staining for the active form of caspase 3. Generated results parallel the apoptosis data in the CD4+CD25+high T cells have reduced in vitro suppressive capacity in T1D. Some of the possible causes include insufficient amount of ambient IL2high T cells development or non-functional TGF-βR on the surface of responder T cells high T cells after FACS isolation but under specific conditions Fritzsching et al., 2005 high T-cell fraction with respect to autoimmune disease.Even though under intense investigation during the last several years, it has not been completely determined why CD4+CD25+in vitro suppression is the only functional test, we have developed a robust assay Jana et al, in preparation. High suppression in control subjects using our high co-culture ratio ensures that functionally these are true suppressor cells. Significant suppression differences between control and recent-onset T1D subjects following the same experimental conditions, point to a functional impairment of regulatory cells seen in the recent-onset T1D clinical group high T cellsHow does increased apoptosis of Tregs affect their function? Since high T cells leads to reduced suppression and the beginning of T1D. Although other tests for the prediction of T1D exist family history and HLA genotyping, we believe that this easy-to-use method that captures early apoptosis of CD4+CD25+high T cells may be of value as a marker for individuals at T1D risk and that it opens up a new area in T1D investigation.The novel stain combination for apoptosis measurement used here for the first time in a study involving diabetic patients correlates well with the number of diabetes-specific auto-antibodies. A future longitudinal study on more subjects will give direct proof that ongoing apoptosis of CD4+CD25+


To compare the effect of a sustained inflation followed by an incremental mean airway pressure trial during conventional and high-frequency oscillatory ventilation on oxygenation and hemodynamics in a large porcine model of early acute respiratory distress syndrome.2 decreased to less than 60 mmHg. After a stabilisation period of 60 minutes, the animals were randomly assigned to two groups: Group 1 : FIO2 = 1.0, PEEP = 5 cmH2O, VT = 6 ml/kg, respiratory rate = 30/min, I:E = 1:1; group 2 : FIO2 = 1.0, Bias flow = 30 l/min, Amplitude = 60 cmH2O, Frequency = 6 Hz, I:E = 1:1. A sustained inflation  followed by an incremental mean airway pressure (mPaw) trial (steps of 3 cmH2O every 15 minutes) were performed in both groups until PaO2 no longer increased. This was regarded as full lung inflation. The mPaw was decreased by 3 cmH2O and the animals reached the end of the study protocol. Gas exchange and hemodynamic data were collected at each step.Severe lung injury (Ali) was induced in 18 healthy pigs  by repeated saline lung lavage until PaO2/FiO2-Index  and PaCO2-reduction  during HFOV compared to lung injury and PCV. Augmentation of mPaw improved gas exchange and pulmonary shunt fraction in both groups, but at a significant lower mPaw in the HFOV treated animals. Cardiac output was continuously deteriorating during the recruitment manoeuvre in both study groups .The SI led to a significant improvement of the PaO2-elimination in an "open lung" approach.A sustained inflation followed by an incremental mean airway pressure trial in HFOV improved oxygenation at a lower mPaw than during conventional lung protective ventilation. HFOV but not PCV resulted in normocapnia, suggesting that during HFOV there are alternatives to tidal ventilation to achieve CO Current treatment goals in patients with acute respiratory distress syndrome (ARDS) are to maintain adequate oxygenation while minimizing ventilator-associated lung injury (VILI) ,2. Sever2/FiO2-Ratio in the HFOV group, while applying a significantly higher mPaw [High-frequency oscillatory ventilation (HFOV) is an alternative to conventional lung protective ventilation. The application of tidal volumes less than 6 ml/kg body weight combined with a high mean airway pressure (mPaw) to avoid lung overdistension and derecruitment, provides a basis for an alternative lung protective ventilatory strategy ,14. In aher mPaw . In smalher mPaw -18. HFOV2O) followed by an incremental and matched mean positive airway pressure augmentation in a large porcine model of severe ARDS with two different lung protective ventilation strategies  on gas exchange and hemodynamics. We hypothesized, that HFOV will reverse hypoxia at a lower mPaw than conventional lung protective ventilation, thereby aiming for a more lung protective recruitment strategy.The aim of this study was to evaluate the immediate effect of a sustained inflation . Anaesthesia and muscle relaxation were maintained with continuous infusion of 5–10 mg/kg/h thiopental, 0.01 mg/kg/h fentanyl and 0.1 mg/kg/h pancuronium throughout the experiment. Pressure controlled ventilation (PCV) was adjusted with a PEEP of 5 cmH2O, a tidal volume of 6 ml/kg, a respiratory rate (RR) of 30/min and an inspiratory to exspiratory ratio (I:E) of 1:1. The inspiratory oxygen fraction (FIO2) was kept at 1.0. Continuous electrocardiography , pulsoxymetry, capnography and distal tracheal pressure monitoring were performed. A 20-gauge arterial catheter  and a 9.0-French introducer sheath  were inserted in the left carotid artery and in the right internal jugular vein respectively by using real-time ultrasound guidance . A 7.5-French flow-directed pulmonary artery catheter  was inserted under transduced pressure guidance. The core temperature, as determined by the pulmonary artery catheter, was maintained at 38.0° ± 0.5°C during the experiment by using a heating pad. After a bolus of 500 ml colloid solution  prior the recruitment manoeuvre a continuous infusion of 4–5 ml/kg/h balanced electrolyte solution was administered for adequate hydration.Eighteen healthy female pigs , Pietrain breed, were fasted for 24 hours with free water access. After intramuscular premedication with ketamin (10 mg/kg), xylazine hydrochloride (1 mg/kg) and atropine (25 μg/kg) an intravenous line was obtained, and anaesthesia was induced with 10 mg/kg thiopental. All animals were positioned supine and orally intubated with a cuffed 8.5-mm ID endotracheal tube with an additional side lumen . Heart rate (HR) was traced by the electrocardiogram. Cardiac output (CO) was measured by using standard thermodilution techniques and expressed as the mean of three consecutive measurements , each consisting of a 10 ml bolus of ice-cold saline into the right atrium randomly during the respiratory cycles. Blood samples were drawn from systemic arterial and pulmonary arterial lines and were immediately analyzed for oxygen pressure (PO2), carbon dioxide pressure (PCO2) and pH using standard blood gas electrodes . In each sample, hemoglobin and oxygen saturation (SO2) were measured using specific spectroscopy . Arterial (CaO2), mixed venous (CvO2) and pulmonary capillary (CCO2) oxygen contents (ml/dl) were calculated using the standard formula: Oxygen content = hemoglobin concentration × 1.34 × (% O2 saturation/100) + PO2 × 0.0031. Oxygen delivery (DO2) and consumption (VO2) were determined using the conventional formulas DO2 = CO × CaO2 × 10 and VO2 = CO × (CaO2 - CvO2) × 10. The standard shunt equation was used to calculate the amount of venous admixture (Qs/Qt): (CCO2 - CaO2)/(CCO2 - CvO2). Mean airway pressure was measured at the side lumen ending at the tubes tip using an air filled pressure transducer  referenced to atmospheric pressure. The oxygenation index was calculated using the following formula: OI = (FiO2 × mPaw × 100)/PaO2[All hemodynamic data were taken in the supine position referenced to atmospheric pressure at the mid-chest level. Mean arterial pressure (MAP), mean pulmonary artery pressure (MPAP), central venous pressure (CVP) and pulmonary artery occlusion pressure (PCWP) were recorded (Servomed100)/PaO2.baseline) were performed. Severe lung injury was induced by bilateral pulmonary lavages with 30 ml/kg isotonic saline (38°C), repeated every 10 minutes until PaO2 decreased to less than 60 mmHg and remained stable for 60 minutes with unchanged ventilatory parameters.After instrumentation the animals were stabilized for 30 min in the supine position and baseline measurements  were obtained and the animals were randomly assigned to the following treatment groups (n = 9/group):After stabilisation post injury measurements 2. High-frequency oscillatory ventilation (HFOV): FIO2O) in the PCV group and increasing the mean airway pressure (mPaw) to 50 cmH2O without oscillation for 60 s in the HFOV group. In both groups, the mPaw was then adjusted 3 cmH2O higher than after acute lung injury (TAli) and increased in steps of 3 cmH2O until PaO2 no longer increased or began to decline. This was regarded as full lung inflation or the beginning of lung overdistension. Afterwards mPaw was reduced by 3 cmH2O and the animals reached the end of the study protocol [2O. Pulmonary artery blood gases were measured at following time points: TBaseline, TAli, T60 and T120. At the end of the study the animals were killed using an intravenous overdose of sodium thiopental and T61® . The study protocol and time course of the whole experiment is outlined in Fig. A sustained inflation (SI) was applied by an exspiratory hold for 60 s . Two-way analysis of variance (ANOVA) for repeated measurements was used for data analysis. Student-Newman-Keuls post hoc test was used for comparison of significant ANOVA results within and between the groups. P values of less than 0.05 were considered significant. Because of different numbers of animals during the time points T2-, OI-, PaCO2 and CO-changes during the experimental period are displayed in figures Detailed data regarding hemodynamics, gas exchange and respiratory parameters are presented in tables 2, OI, PaO2/FiO2-Index, VO2, SvO2, mPaw, PIP, MAP, CVP, MPAP, Qs/Qt – ratio and CO: pHa decreased from 7.46 ± 0.08 to 7.35 ± 0.06, PaCO2 increased from 39.8 ± 5.9 to 54.9 ± 11.8 mmHg, OI increased from 1.7 ± 0.2 to 27.6 ± 8.5, PaO2/FiO2-Index declined from 564 ± 41.9 to 57 ± 12.2 mmHg, VO2 increased from 174 ± 40.0 to 212 ± 61.5 ml/min./m2, SvO2 decreased from 83 ± 5.0 to 61 ± 11.0 %, mPaw increased from 9.5 ± 0.5 to 14.9 ± 1.3 cmH2O, PIP increased from 14.1 ± 1.2 to 24.8 ± 2.6 cmH2O, MAP increased from 73 ± 11 to 93 ± 10 mmHg, CVP increased from 5 ± 2.3 to 7 ± 2.4 mmHg, MPAP increased from 18 ± 3.8 to 36 ± 5.5 mmHg, Qs/Qt increased from 8 ± 4 to 53 ± 8 %, CO increased from 4.6 ± 1.4 to 6.4 ± 1.8 l/min. No significant differences could be detected between the 2 groups for the parameters tested at time points TBaseline and TAli  with significant changes p ≤ 0.05) in pHa, PaCO8 ± 3 wit in pHa, 2/FiO2-ratio in the HFOV group compared to TAli and the PCV group from T15 to T75  Combination of HFOV and SI with rising mPaw improved oxygenation and resulted in a significant reduction of pulmonary shunt fraction at a lower mPaw than during conventional lung protective ventilation. 2) The SI and rising mPaw resulted in normocapnia in the HFOV but not in the PCV group 3) Both groups exhibited a continuously deterioration in cardiac output with rising mPaw.2O for 60 seconds and afterwards set the mPaw 3 cmH2O higher than after lung injury. The PaO2/FiO2-Ratio increased and the OI decreased significantly in the HFOV, but not in the PCV group  declare that they have no competing interests.RMM conceived the study, collected and analysed the data and drafted the manuscript. MK and BZ collected data and performed the statistical analysis. CW and NR participated in the design of the study. JB designed the study protocol and helped writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66α and p66β. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems.We made loss of function mutants in the mouse p66α gene . We found that mp66α is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66α in gene silencing.mp66α is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing. Epigenetic changes in gene expression, such as occurring during imprinting and X chromosome inactivation, can result from DNA methylation and from chromatin remodeling. The latter includes histone modifications such as deacetylation Drosophila as a genetic modifier of Wingless signaling Another protein in the NuRD/Mi2 chromatin complex is termed p66, cloned in To characterize the activity of p66 and assess its importance in mouse development, we have made loss of function mutants in the mouse p66α gene . We report here that the gene is required for normal embryogenesis.By a combination of EST and cDNA mapping, we assembled a 6.4 kb cDNA contig for mp66α. The transcript theoretically encodes a protein of 630 amino acids with 87% identity and 93% identity or similarity with human p66α . The proWe confirmed homologous recombination in 11% of the neomycin-resistant R1 embryonic stem cells . NortherWe examined intercross embryos at days e8.5 (embryogenesis 8.5) through e12.5. Overall, we found a wide variety of defects. Virtually every organ of candidate mutant embryos could be affected, resulting in dramatically smaller embryos and advanced stages of necrosis. We could not identify particular defects or abnormalities that would suggest a specific step in embryogenesis being dependent on mp66α. We present this analysis in We found that by day e12.5, 61% of embryos were either wild-type or heterozygous, while 39% were in such an advanced stage of necrosis that they could not be examined or genotyped. For embryonic days 8.5–10.5, we initially genotyped 82 whole animals using a Southern blot protocol to account for all animals in a litter. We obtained ratios close to normal Mendelian ratios: +/+, 0.96; +/−, 1.92; −/−,0.84 . HoweverAn examination of 83 homozygous null e7.5–e10.5 embryos from additional litters revealed a range of defects, delayed development, and much smaller size than litter mates , but witA normal day 8.5 embryo has 8–12 somites, is in the process of turning, has a paired heart primordial fusing anteriorly, and has closed neural folds at somites 4–5 Abnormalities in mp66α null embryos around day 9.5 included malformed neural folds that were ruffled and not fused, failure of closure of anterior neuropore, missing or excessively large blood vessels in the yolk sac, turned but small underdeveloped embryos, and embryos that failed to turn despite having 20 or more somites .Given the evidence that p66 is part of a repressor complex, we were interested in asking whether specific genes are affected by the absence of mp66α in embryos. We carried out an Affymetrix microarray analysis of 4 pairs of null vs. wild type day 8.5 siblings that had achieved an equal level of development as judged during dissection.Over 45 genes were identified as being increased in the null embryos of at least three but usually all four matched sibling pairs . Four ofBecause of the presumed role of p66 proteins in epigenetic gene repression, we compared the mp66α null embryonic phenotype with published mutants in other genes implicated in this process: the DNA methyltransferases, Dnmt1, the maintenance methyltransferase, and Dnmt3a and Dnmt 3b, the de novo methyltransferases, and the histone methyltransferases G9a and SUV39H The mp66α null embryos were very similar to the Dnmt1 null, Dnmt3a/3b double null, and the G9a null embryos. The Dnmt1 null embryo Xenopus and mammalian cells. By making mice mutant for mp66α, we show that the gene is essential for normal development.The NuRD/Mi2 complex, which includes p66 is found in all fetal and adult tissues and its components are expressed throughout early embryonic development from at least the 8–16 cell stage onward The mp66α−/− mutation results in an embryonic lethal phenotype at around day e9.5. Because methylated DNA binding proteins or activities are capable of repressing reporter genes by recruiting histone deacetylase activity, the loss of HDAC due to loss of mp66α should result in a DNA methyltransferase knockout phenotype. The maintenance methyltransferase (Dnmt1)−/− embryo dies by the eight somite stage, around day e8.5), while the de novo Dnmt3a/3b methyltransferase double null embryo dies before e11.5, with a variety of developmental abnormalities The mp66α null phenotype appears to be less severe than the knock-out phenotypes of two individual NuRD/Mi2 components. The MBD3 null embryo is less developed than Dnmt−/− embryo and the HDAC1null embryo dies prior to e9.5 with a cell proliferation defect On the other hand, we found that the mp66α phenotype is also not as severe as that due to loss of all methylation. mp66α−/− embryonic stem cells were capable of differentiating into embryoid bodies at normal rates (data not shown), while Dnmt1 homozygous mutant ES cells are reported to undergo more limited differentiation at slower rates If DNA methylation were dependent upon H3-K9 methylation, and if mp66α were required for H3-K9 deacetylation, one would expect to see loss of DNA methylation in mp66α−/− ES cells, even in a hypomethylated state. We used probe for minor satellite methylation and an IAP probe to assess methylation of dispersed repetitive elements. DNA methylation at all these loci was normal in the ES cell cultures (data not shown).Given the fact that methylated DNA binding activities and proteins have been shown to repress reporter genes through histone deacetylase activity, it has been puzzling that the MeCP2 and MBD2 knockouts have given viable, mild phenotypes Xenopus p66α start site We used the 3′ UTR sequence of an existing mp66α clone to identify additional mp66α clones in the NCBI mouse EST database, of which only two extended beyond the UTR . We sequenced these and assembled a sequence  that extTM Long Template PCR System (Boehringer Mannheim) but scaled down to a 25 µL reaction volume.We probed a BAC library  derived from mouse strain 129/Sv and obtained 4 positive clones for mp66α . Except for the 37 kb intron, we mapped the genomic structure using long-range PCR with primers made to the cDNA at regular intervals, and sequenced the amplimers into exon/intron boundaries when introns were detected. We identified critical sites for cloning and verified their presence in the 129/Sv genomic DNA we was using for the construct. For the long-range PCR we used the level 2 protocol of the ExpandWe made the targeting construct by cutting a 1.4 kb short homologous arm and a 6.3 kb long homologous arm from the 129/Sv BAC clone 95E12, then used a shuttle vector to place these arms into the targeting vector, ploxPNT. The targeting vector was designed to replace exons 2–4 of mp66α with a floxed pgkneo cassette. The homologous arms for the targeting construct were cut from BAC 95E12, placed into Bluescript SK-, and finally into the targeting vector ploxPNT  [33]. A http://www.mshri.on.ca/nagy/) in 1991 from 129/Svx129/SV-CP stock and has agouti coat color in the F1 generation. ES cells were grown in gelatinized dishes on neomycin resistant γ irradiated mouse embryonic fibroblasts in ES complete medium consisting of DME-H21 (UCSF#AA400) supplemented with 20% fetal bovine serum , 0.1 mM non-essential amino acids (Gibco#11140-0500), 1× penicillin-streptomycin (Gibco #15140-122), 1 mM sodium pyruvate (Gibco#11360-070), 100 µM β-Mercaptoethanol (Sigma # M-7522), and 2000 U/mL murine LIF  (Stanford Transgenic Facility protocol) Gene targeting was performed on passage #15 R1 ES cells purchased from the Stanford Transgenic Research Facility. This line was developed by the Andras Nagy lab , 2-32 (5′-gccgggtattcgagtggaggaggt-3′), 2-Q (5′-aatcacatcaggcgagggaggtct-3′), and N-5 (5′-ttggctacccgtgatattgctgaa-3′). N-5 is found only in the pgk-neo cassette while 2-Q is only in the targeted region; primers 2-30 and 2-32 flank the targeted region. A 25 µL PCR reaction consisting of 0.4 µM primer 2-30, 0.2 µM each remaining primer, 0.8 mM dNTPs, 1.5 U Taq DNA polymerase , 1× Mg-free supplied PCR buffer, 4.5 mM MgClCorrectly targeted ES cells were injected into C57BL/6 blastyocysts We wanted to eliminate possible confounding effects of the pgk promoter on neighboring genes. To do this, we crossed the heterozygous mp66α females to a CMVCre recombinase male to produce offspring mosaic for the pgkneo deletion. These were then crossed to wild type animals to obtain pure Δpgk mp66α lines for both lines 26 and 110 . These rTo remove the floxed pgk-neo cassette, we crossed heterozygous mp66α females of both lines, 110 and 26, to a CMV-Cre male on a C57BL/129/SV background. Offspring receiving the CMV-Cre transgene were mosaic for the deletion so these were crossed again to wild type to produce pure Δmp66α lines on a mixed background. The Δmp66α110 and Δmp66α26 lines were then maintained in the heterozygous condition on a mixed background as stock.Because aberrant RNA is produced in the null animals, we could genotype the embryonic RNA using RT-PCR. Individual RNA from three litters was examined and genotyped for quality and mp66α genotype. Extraembryonic membranes were included with each embryo. Four sibling pairs were chosen from the three litters for microarray analysis. An Affymetrix protocol was used so that each embryo produced an individual set of expression data that could be compared as desired, and all sample pairs were prepared simultaneously. Sex was determined by examining the expression data. Two pairs were entirely female while two other pairs were mixed. The data for each pair was examined in parallel for each gene and genes were not listed unless they were above a for at least three of the four pairs. There was one male each of null and wild type, so these were compared to each other also. The wild type male and wild type females were compared to confirm known male/female differences and to include them in interpretation. In all cases examined for known genes, the results were sex appropriate.PolyA-RNA was then isolated using the spin column protocol of the Oligotex mRNA midi kit (Qiagen). For microarray analysis, the RNA was quantitated in a Beckman DU530 spectrophotometer and labeled cDNA was made using the Cyscribe labeling kit. The lab reference RNA, (1/4 four-week whole brain : 3/4 e 12.5 complete embryo), was labeled with Cy3 and the ES cell RNA was labeled with Cy5. 0.25 M NaOH was added to degrade RNA during a 15 minute 37°C incubation, then 0.7 M HEPES free acid was added to neutralize. Final purification was carried out with the CyScribe GFX Purification Kit. Absorbance at 550 nm(Cy3) and 650 nm (Cy5) was determined to quantitate incorporation of label, so that equimolar amounts of Cy3 and Cy5 could be used for hybridization. Mouse microarrays were purchased from the SFGF facility, and were stabilized, immobilized, and pre-hybridized as follows. Slides were etched in one corner to identify side of slide printed with the array, and then rehydrated, array side down, over 45°C 1× SSC, then snap-dried on a 100°C surface until the vapor disappeared, then UV cross-linked at 65 mJoules. To prehybridize, arrays were incubated with 50% formamide, 3× SSC, 0.1%SDS, 0.1 mg/mL BSA for 45 minutes at 42°C, immersed in milliQ water to remove SDS, and dried by centrifugation at 600 rpm for 2 minutes.TM humidified with 3× SSC and submersed in a 65°C water bath for 14–18 hours. The slides were then washed in foil covered slide dishes as follows: 2× SSC, 0.2% SDS at 55°C, 10 minutes, removing cover slips when immersed; 1× SSC. 0.2% SDS at 55°C, 10 minutes; 0.1× SSC, 0.2% SDS at RT, 10 minutes; 0.1× SSC at RT, 10 minutes; distilled water for 10 seconds followed immediately by centrifugation drying as above. The slides were then immediately scanned with a GenePix scanner with photomultiplier tube voltages in each channel adjusted to a ratio of between 0.8–1.2. Images were then gridded and flagged using the GenePix Pro 4.0 Software, then sent to the Stanford Microarray Database.For hybridization, 40 pmol of reference and ES cell DNA were mixed, 20 ug each of mouse Cot1 DNA, polyA RNA, and tRNA were added, and the sample was concentrated to 28 µL in a foil covered speed-vac. 6 µL 20× SSC and 0.5 µL 20% SDS were added, samples were denatured at 100°C for 2 minutes, and then spun at 14,000 rpm in a microfuge for 15 minutes. The mixture was then pipetted onto the microarray slide and covered. Hybridization was carried out in a Gene Machines HybChamber


The acidic stores may comprise the contractile vacuole network (CV), the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca2+-store was investigated.cAMP-induced Ca2+. Release of Ca2+ was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca2+-transport and Ca2+-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca2+-compartment we wanted to know whether or not it takes part in cAMP-induced Ca2+-influx. We made use of the LvsA--mutant expected to display reduced Ca2+-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca2+-influx into whole cells.Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release CaDictyostelium represent a highly efficient acidic Ca2+-store that is required for cAMP-induced Ca2+-influx.The contractile vacuoles in  Dictyostelium consists of tubes and bladders. It transiently fuses with the plasma membrane to expel water and ions and thereby serves as an efficient osmoregulatory organelle 2+ev) of 0.53 ± 0.02 μM uptake ceased to 0.10 ± 0.05 μM [Ca]2+ev in the presence of 32 ± 12 μg protein. In agreement with previous results obtained for acidic Ca2+-stores the calmodulin antagonist W7 as well as arachidonic acid (AA) caused Ca2+-release in P0E. The response to W7 (30–100 μM) amounted to 21.4 ± 10.2 nmol Ca2+/mg protein (n = 4) and to AA (10–20 μM) 23.9 ± 7.2 nmol Ca2+/mg protein (n = 4).Since both purifications had given a strong enrichment of the GFP-label as well as in Ca2+-uptake by P1E, the eluate of calnexin-GFP-labeled cells containing ER, is shown. In contrast to P0E P1E was less responsive to AA. In three out of four experiments no Ca2+-release was measured. The one that responded to AA had a lower purification factor with respect to GFP fluorescence compared with the other three, 3.8 versus 7.8 ± 0.96 (n = 3). IP3, most of the times, was inefficient to elicit Ca2+-release. In general, Ca2+-uptake was slower in P1E as compared to P0E starting at 0.56 ± 0.15 μM [Ca]2+ev and ceased to extravesicular Ca2+-concentrations of about 0.36 ± 0.08 μM in the presence of 27 ± 12 μg protein (n = 4), indicating that the CV is more relevant in maintaining a basal cytosolic Ca2+-concentration of 50 to 60 nM i and stimulus-induced [Ca2+]i-transients ev at the beginning of the experiment is not known and Ca2+-ATPase activity is increasing with increasing Ca2+-concentrations the results shown in Table 2+]ev in order to relate the outcome of different experiments to each other. This was done by using a calibration curve at different [Ca2+]ev obtained with either P0 or P1, respectively, and adjusting the measured value to a [Ca2+]ev of 1 μM. The Ca2+-ATPase activities of both, P0 and P1, increased by a factor of about 3.3 from 0.5 μM to 1.5 μM [Ca2+]ev. Statistical data are expressed throughout as the mean ± S.D.Caescribed . In brie+-ATPase antibody (kindly provided by Margaret Clarke) preferentially reacting with the 37/42 kD subunits of the proton pump.Seven microgram of protein per lane were resolved by SDS-PAGE in 12.5% gels, blotted and immunoblotted with a V-type H2+-influx was measured as described [- and DH1) during every washing step was shortened without any loss of cells. Protein concentrations were determined with the Coomassie protein assay reagent (Pierce) using bovine serum albumin as standard. Endosomal content was determined by fluid phase uptake of RITC-dextran as described, except that labeling was performed in phosphate buffer pH 6.0 [cAMP-induced Caescribed . The cenr pH 6.0 .AA: arachidonic acid; BHQ: 2,5-di(tert-butyl)-1,4-hydroquinone; CV: contractile vacuole; CMA: concanamycin A; ER: endoplasmic reticulum; Tg: thapsigargin2+-influx and responses of living cells to AA; CS cultured the strains and prepared the figures. ADL contributed the strains LvsA- and DH1 and participated in the preparation of the manuscript as did AMT, who also contributed the GFP-strains and Figure DM performed the organelle experiments and in collaboration with CS and DFL designed the experiments and wrote the manuscript. DFL measured receptor-mediated Ca


The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysisIncubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA), relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 × PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0× PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 × PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 × PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucineIn a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway. Starvation induces muscle wasting by decreasing the rate of muscle protein synthesis and increasing protein degradation. A decrease in plasma concentrations of amino acids, and in particular branched-chain amino acids, is thought to synergise with lower levels of insulin to signal the starvation-induced decrease in protein synthesis  but the in vitro -tyrosine at all of the concentrations employed with the half-maximal effect on proteolysis of approximately 10 -8 M (P < 0.01)  to the media decreased protein degradation (18 to 21%) at both concentrations of amino acids  and sub-physiological concentration of amino acids (0.2 × PC AA) were used to determine the interaction between amino acids and insulin in the regulation of skeletal muscle protein degradation. Protein degradation was increased by 9–12% when differentiated myotubes were incubated with the amino acid limiting 0.2 × PC AA media compared with 1.0 × PC AA, in both the absence and presence of insulin  to determine the role of the 26S proteasome pathway in responses to varying amino acid concentration. MG132 inhibited proteolysis by about 75% in incubations with both physiological and sub-physiological concentrations of amino acids, but importantly, the inhibitor abolished the difference in proteolysis between the two amino acid levels . Insulin addition to media containing either the low or physiological amino acid concentrations further decreased C2 mRNA abundance in the myotubes, compared to that observed in the absence of insulin (P < 0.05). Incubation with the 1.0 × PC AA compared with 0.2 × PC AA elicited an inhibitory effect on the expression of C8 in the differentiated C2 C12 incubations but this was not statistically significant.We next assessed the specific effect of leucine on myotube protein degradation by supplementing the amino acid limiting 0.2 × PC AA media with three levels of leucine ranging from sub-physiological to supra-physiological  and 750 μM). In the absence of insulin, increasing the concentration of leucine in the media tended to inhibit myotube protein degradation but this effect was not significant (P > 0.05). In the presence of insulin, proteolysis was progressively inhibited by the addition of increasing leucine concentration with both 150 and 750 μM leucine, being significantly lower compared to 30 μM leucine .The anabolic effect of insulin on muscle protein synthesis is acutely sensitive to amino acids . StudiesThis study is the first to report that the amino acids alone, or additively with insulin, decrease muscle protein degradation by acting on the ubiquitin dependent proteasome pathway. We also report that varying the concentration of leucine whilst maintaining all other amino acids at a low level, does not mimic the effect of varying all amino acid concentrations suggesting that the response to amino acids is more than a response to alterations in leucine as has been previously suggested .Increasing the media amino acid concentration from 0.2 × physiological levels to 1.0 × PC AA decreased myotube proteolysis as assayed by the release of protein-bound labelled tyrosine into the medium, but these effects were abolished by addition of the proteasome inhibitor MG132. Furthermore, the expression of both the 14-kDa E2 and C2 sub unit of 20 S proteasome was significantly increased during incubations with the sub-physiological amino acid incubation medium. The effect of amino acids was additive with insulin: maximal stimulation of protein degradation and expression of 14-kDa E2 and C2 were observed in the absence of insulin and at 0.2 × amino acids. Addition of insulin in the presence of amino acids strongly inhibited proteolysis and expression of 14-kDa E2 and C2. Depletion or supplementation of media leucine concentration influenced proteolysis but only in the presence of insulin. These results therefore strongly argue that insulin and amino acids interact to suppress myotube proteolysis by acting on the ubiquitin-proteasome pathway. The tyrosine release assay measures total myotube protein degradation and is therefore more representative of muscle protein degradation and is also able to determine small differences in turnover that are not possible with the measurement of specific proteins or their rate of ubiquitlylation.in vitro in the presence of 10 mM leucine -tyrosine/ml fusion medium for 48 hours in an atmosphere of 5% CO2 and 95% O2 at 37°C to label cellular proteins. Monolayers were rinsed with 1 × HBSS and incubated with serum free defined medium for two hours at 37°C to allow the degradation of short live proteins. Myotubes were then rinsed twice with 1 × HBSS and chased once again for 8 hours in defined medium containing 0.05% (w/v) bovine serum albumin (BSA) with or without insulin or other agents. At the end of incubation period media was removed into tubes containing 1 mg BSA and precipitated overnight (for convenience) with an equal volume of 20% (w/v) TCA at 4°C. Tubes were centrifuged at 3000 g at 4°C for 5 min and supernatant containing soluble proteins (A) was separated. TCA insoluble proteins (B) in the pellets and myotubes (C) in the plates were solubilised in 0.5 and 1.0 ml of tissue solubiliser (0.5 M NaOH and 0.1% Triton X-100), respectively. One hundred microlitres of each fraction of protein  was counted using Packard Scintillation counter. Percent protein degradation was calculated by dividing radioactivity in TCA soluble protein (A) by sum of radioactivity in TCA soluble (A), TCA insoluble (B) and in myotubes (C).Protein degradation was determined by monitoring the release of TCA soluble radioactive tyrosine into the culture medium . Fully dL-tyrosine. At the end of 8 h incubation period myotubes were rinsed with ice cold 1 × HBS and total RNA was extracted using Tri Reagent (SIGMA) according to the manufacturer's guidelines. The quantity and purity of RNA were assessed by observing absorption at 260 and 280 nm.Differentiated myotubes were used from day 5. Experimental conditions used for tyrosine release assay were reproduced except that cold tyrosine was used instead of 32P] dATP to a specific activity of 109 cpm/μg cDNA using Ambion Strip EZ DNA kit . Unincorporated radioactivity was removed by passing the synthesized probe through chromaspin – 30 column . Membranes were prehybridised with Quickhyb hybridisation solution  for 20 min at 65°C. Purified probe containing salmon sperm DNA (Ambion) was denatured at 90°C for 10 min and transferred to the hybridisation solution. Probe was then hybridised to the RNA on the blot for two hours at 65°C. To remove non-specific binding the blot was washed twice with 2 × SSC/0.1% SDS at 60°C for 15 min each and then once with 1× SSC/0.1% SDS at 65°C for 15 min. Autoradiographic signals were quantified by laser densitometry and expressed relative to the corresponding GAPDH mRNA value to normalise for equal loading and transfer of RNA. GAPDH was used because previous studies with nutrients and hormones have documented that mRNA level for it remains constant [Ten micrograms of RNA was run on 1% agarose formaldehyde denaturing gel, transferred to Genescreen™ membrane  and covalently fixed to the membrane by UV irradiation. Strippable probes, either of cDNA for 14-kDa E2 ubiquitin conjugating protein, C2 and C8 20 S proteasome subunits and glyceraldehyde 3 phosphate dehydrogenase  were synthesized with [α-2 and 95% O2 at 37°C. Next morning medium was replaced with 5 ml of fresh 1 × PC AA medium containing 0.05% (w/v) BSA and dishes were transferred back to the incubator at 37°C. After 60 min half of the dishes were treated with 2.2 × 10-8 M rapamycin for 20 min at 37°C in 5% CO2 and 95% O2. Following this half of the rapamycin treated and non-treated dishes were stimulated with 10-8 M insulin for another 20 min. Medium was aspirated and monolayers were rinsed with ice cold 1 × PBS. Cells were lysed by adding pre heated (95°C) buffer composed of 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 1.55% (w/v) dithiothretol (DTT), 10% (v/v) glycerol and 0.01% (w/v) bromophenol blue, on them. Cells were scraped of and immediately transferred in to 1.5 ml micro centrifuge tubes and passed through a 25 guage needle 5 times to break cellular nucleoprotein complexes. Cell extracts thus obtained were heated at 95°C for 5 min and stored at -80°C until further analyses.Myotubes from day 6 were incubated over night in 1 × PC AA medium containing 0.05% (w/v) BSA in a humidified atmosphere of 5% COEqual concentration of protein from cell extracts was resolved on 10% SDS polyacrylamide gel. Gel was run at a constant voltage of 80 for 3 hours and then transferred overnight on to polyvinylidene diflouride (PVDF) membrane (NEN) at a constant current of 300 mA at 4°C. Protein was heat fixed to the membrane at 65°C for 30 min to expose antibody binding epitopes on the antigen. Prior to immunodetection, heat fixed membrane was briefly soaked in 100% methanol, followed by a 5 min wash in 1 × TBS  at room temperature. Membrane was blocked in 1 × TBST buffer (1 × TBS containing 0.1% (v/v) Tween-20 and 5% (w/v) non fat dry milk) for 1.5 hour at room temperature. Membrane was incubated overnight with primary antibody, phospho-p70 S6 kinase (Thr 389) (New England Biolabs (UK) ltd., Hertfordshire, England), dilution 1: 1000 in 1 × TBST containing 5% (w/v) BSA at 4°C. The next morning, membrane was washed four times with 1 × TBST for 5 min each at room temperature and incubated with HRP-linked anti-rabbit secondary antibody, dilution 1: 2000 in 1 × TBST containing 5% (w/v) BSA at 25°C for an hour. Finally membrane was washed again as before and HRP-protein substrate complex was detected using lumiglo detection system (New England Biolabs) according to the manufacturer's instructions. Membrane was exposed to Amersham Hyperfilm and the intensity of bands observed was quantified by laser densitometry. The membrane was stripped at 55°C for 20 min in a buffer composed of 62 mM Tris-HCl, pH 8.0, 2% (w/v) SDS, 0.7% (v/v) β mercaptoethanol. The blot was then reprobed for anti-p 70 S6 kinase that can recognise both phospho and dephospho antigenic sites.Values are presented as means ± SEM. Differences of means were determined by Analysis of variance in a General Linear Model, running Minitab version 13.1. Post hoc comparisons were performed by Tukey's pair wise comparison for multiple treatments. p < 0.05 was accepted as a significant difference between treatments.FS designed, conducted the experiments & analysed the data and drafted the manuscript. MAL & DGH participated in the design of the studies, data interpretation and preparing the final manuscript. All authors read and approved the final manuscript.


Human nucleic acid-binding protein 1 and 2  form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS–PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein–protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3–hNABP1/2–C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown. Although DNA primarily exists as a duplex, there are many cellular processes like replication and transcription, where single-stranded DNA (ssDNA) is exposed, making it more vulnerable to damage and enzymatic degradation. Single-stranded DNA-binding proteins (SSBs) protect these structures from nuclease attack, and also recruit appropriate proteins like DNA repair enzymes and cell cycle regulatory proteins ,2. SSB pMany studies have shown that both hNABP1 and hNABP2 form an independent complex with two proteins, INTS3 (integrator complex subunit 3) and C9ORF80 –9. INTS3The structure of INTS3–hNABP2–C9ORF80 reveals that INTS3 acts as a scaffold to bridge hNABP2 and C9ORF80; however, only the N-terminus of INTS3 (1–500 amino acids) was used in this study . It has Despite hNABP1/2, especially hNABP2, has been studied extensively, the biochemical function of INTS3 and C9ORF80 remains to be elucidated. In the present study, we purified the recombinant proteins INTS3 and C9ORF80 to near homogeneity. Although both proteins exist as monomers, C9ORF80, but not INTS3, exhibits anomalous behavior in gel filtration and SDS–PAGE because of random coil conformation. INTS3 has higher affinity toward ssRNA than ssDNA, and C9ORF80 exhibited ssDNA binding but not with ssRNA. The C-terminus of INTS3 is required for its nucleic acid binding, while the N-terminus is involved in protein–protein interactions. In addition, we have also successfully purified the INTS3–hNABP1/2–C9ORF80 complex and found it also exhibited similar substrate affinity like hNABP1/2 alone; however, the complex has reduced affinity, which is because of INTS3 but not C9ORF80.INTS3  were amplified and cloned into the BamHI and XhoI sites of pGEX-6P-1 vector . The C-terminus of INTS3 was amplified and cloned into the Nde1 and Xho1 sites of pET28a vector (Novagen). The C9ORF80 was amplified and cloned into the NcoI and XhoI sites of pET28a vector. All plasmids were verified by DNA sequencing. The primers used for the amplification of genes are listed in Supplementary Table S1. The vector construction of hNABP1/2 were described recently  ATP using T4 polynucleotide kinase (NEB) at 37°C for 1 h. Unincorporated radionucleotides were removed by a G25 chromatography column . The labeled ssDNA or ssRNA substrates were kept at 4°C and ready to use. For the dsDNA substrate, a [γ-32P] ATP-labeled oligonucleotide was annealed to a 2.5-fold excess of the unlabeled complementary strands in annealing buffer  by heating at 95°C for 6 min and then cooling slowly to room temperature. For dsRNA and RNA : DNA hybrid, [γ-32P] ATP-labeled oligonucleotide was annealed to a 2.5-fold excess of the unlabeled complementary strands in annealing buffer  by heating at 95°C for 6 min and then cooling slowly to room temperature. All double-stranded substrates were purified by PAGE isolation, and their concentrations were determined by liquid scintillation counting before use.PAGE-purified oligonucleotides were purchased from Integrated DNA Technologies and are listed in Supplementary Table S1. Oligonucleotide was 5′-end-labeled with [B]/[AB], where [A], [B], and [AB] are the concentrations of proteins, nucleic acid, and protein–nucleic acid complex, respectively. However, as the concentration of nucleic acid (B) is very low when compared with that of proteins, the concentration of protein [A] used to shift 50% of nucleic acid  was used to determine the Kd [Protein/nucleic acid-binding mixtures (20 μl) contained the indicated concentrations of protein  and 0.5 nM of the specified e the Kd . The shig for 30 min at 4°C, and supernatant was collected. The supernatant was divided into equal two parts, and hNABP2 antibody or rabbit IgG was added and incubated for 1 h at 4°C. Following this, protein A beads were added, incubated for overnight at 4°C, and beads were collected by centrifugation at 500×g for 5 min. The beads were washed five times with IP buffer and proteins were eluted by incubation in 2× SDS loading dye at 80°C for 5 min. The eluted samples were separated on 10% SDS–PAGE gel using the Tricine buffer system. After visualized with silver staining , the bands that were present in hNABP2 pulldown, but absent from control IgG, were excised from the gel and destained as per the manufacturer's instruction, followed by in-gel digestion with trypsin. The samples were injected onto an Agilent Zorbax 300SB C8  custom-made chip for desalting and online mass spectrometric analysis. The extracted samples were loaded onto the enrichment column using 95% mobile phase A  and 5% mobile phase B  at a flow rate of 4 μl/min. Positive ESI mass spectra were acquired in 3200 m/z, 2 GHz extended dynamic range mode. The data extraction and protein confirmation were done using the Agilent MassHunter BioConfirm software and searched against the Swiss-Prot Human database. The search parameters included trypsin digestion, carbamidomethyl of cysteine residues as fixed modification, and methionine oxidation as variable modification.HeLa cells collected from 2 × 150 cm dish were resuspended in 5 ml of immunoprecipitation (IP) buffer  supplemented with protease inhibitor cocktail and 2 µM PMSF. The lysate was centrifuged at 100 000×E. coli [FLINTS3 into a pGEX-6P1 vector, and the expressed protein was purified by affinity and size-exclusion chromatography. The fractions obtained from glutathione sepharose beads . The identity of the INTS3 protein was further confirmed by Western blot using an anti-INTS3 antibody  and alcohol dehydrogenase (150 kDa)  [FL protein in peak 2 exists as a monomer in solution, and we used this fraction for our further experiments.The N-terminal fragment of INTS3 (1–500 aa) has been purified from E. coli , and itsE. coli . We clonse beads  were poose beads . Electrose beads , with twantibody  and massd 208 nm . AnalysiDiction)  has alsoFL have never been reported. Thus, to understand the nucleic acid-binding ability of INTS3FL, we incubated the protein with ssDNA (dT30) and ssRNA (rU30). We found that INTS3FL could bind both dT30 , while it exhibited 81% binding with rU30 at 12.8 µM . We observed two and three shifted bands with 60- and 90-mer oligo (dT), suggesting that INTS3 can bind 30 nucleotides in an orderly manner. We also examined INTS3FL binding with a random ssDNA sequence and found that the protein bound these nucleotides. Similarly, the affinity increased with the length of ssDNA  along with an unknown protein of molecular mass of 70 kDa  and SDS–PAGE (∼15 kDa) is much higher than predicted. Thus, we evaluated the secondary structure of C9ORF80 with PSIPRED  and POND30 and rU30, we found that C9ORF80 could bind with dT30 but barely with rU30 substrate . However, C9ORF80 did not bind dsRNA , dsDNA , or DNA : RNA hybrid .Similar to INTS3, the nucleic acid-binding activity of C9ORF80 protein has not been studied. Using dTubstrate . C9ORF80of 85 µM . AlthougFL bound ssRNA and ssDNA. Hence, we predicted the DNA-binding residues of INTS3FL using BindN [N, 1–513 amino acids) and C-terminal region   . We clonltration . When evand rU30 , while Iand rU30 , with Kdfor rU30 .Figure FL, INTS3N, and INTS3C proteins were purified with glutathione sepharose beads and their interactions with hNABP1/2 and C9ORF80 were evaluated. The results suggested that the INTS3N is primarily involved in the interaction with hNABP1/2 and C9ORF80  or ssRNA (rU30), and found that the INTS3FL–hNABP1–C9ORF80 complex exhibited higher binding affinity with dT30 than rU30 , and rU30 with increasing concentration of INTS3FL–hNABP2–C9ORF80 complex . The protein/nucleic acid complex was transferred to the nitrocellulose membrane, and proteins were detected with INTS3, hNABP1/2, and C9ORF80 antibodies. All three proteins, INTS3, hNABP1/2, and C9ORF80, were present in the complex (∼250 kDa), but INTS3 and C9ORF80 were not observed in the hNABP1/2 control, indicating that the complex itself is binding to the substrate. However, the complex exhibits reduced affinity compared with their individual hNABP1/2 proteins. For example, INTS3–hNABP1–C9ORF80 complex has a Kd value of 0.6 ± 0.09 µM with ssDNA . Similarly, the INTS3FL–hNABP2–C9ORF80 complex has a Kd value of 0.5 ± 0.07 µM with ssRNA .To evaluate the binding ability of the INTS3han rU30 . For inshan dT30 , it bounin alone , where hth ssDNA , while hth ssRNA , while t30) at a concentration of 0.2 and 0.4 µM, respectively. C9ORF80 did not affect hNABPs' binding ability with ssDNA, even at the ratio of 2 : 1 with hNABPs. This suggests that C9ORF80 is not critical for hNABP binding with ssDNA. On the contrary, INTS3 inhibited the binding ability of hNABPs with ssDNA at the ratio of 1 : 1 with hNABP. Further increasing the concentration of INTS3, it completely inhibited the DNA-binding ability of hNABPs.Since INTS3–hNABP1/2–C9ORF80 complex protein could bind ssDNA and ssRNA but with reduced affinity, we asked which one, INTS3 or C9ORF80, affected the binding ability of hNABP1 and hNABP2. Thus, we reconstituted this heterotrimeric complex by mixing them at various concentrations, and it was subjected to EMSA. hNABP1  and hNABSimilar to the DNA-binding results, C9ORF80 did not affect the binding ability of hNABP proteins with ssRNA . HoweverThe hNABP1/2 associates independently with INTS3 and C9ORF80 proteins ,8,16; hoThe RPA heterotrimer consists of RPA70, RPA32, and RPA14 subunits, with three OB-fold in RPA70 subunit and one in each of the RPA34 and RPA14 subunits. Previously, we have shown that hNABPs have a significant lower affinity to DNA than RPA , which mN fragment with C-terminal 6×His-tag was not successful, even though the protein was expressed (data not shown). However, we succeeded in purifying INTS3FL and INTS3N using N-terminal GST-tag, suggesting that the N-terminus of INTS3 might affect the conformation of the full-length INTS3 protein, resulting in inaccessibility of C-terminal tag for efficient binding with the beads in affinity chromatography. This might be the reason why, we found that, INTS3C has 5-fold higher affinity than INTS3FL  complex  has revealed that NABP2 protein interacts with nucleic acid and INTS3 via the N-terminus OB-fold, while the C9ORF80 interacts with INTS3, but not with NABP2 . In our  INTS3FL , becausent study , suggestIn our current study, the complex reduced the binding affinity of hNABPs by 5-fold toward ssDNA and ssRNA. INTS3 inhibits the hNABPs' binding with ssDNA and ssRNA. Since the OB-fold of hNABP2 is responsible for the interaction with INTS3 ,16 and bRecently, the Richard laboratory found that hNABP2 is associated with a large number of proteins with roles in mRNA metabolism and various chromatin-remodeling complexes . SimilarWe have purified INTS3 and C9ORF80 proteins and found both exist as a monomer in solution. INTS3 binds both ssDNA and ssRNA, but C9ORF80 binds ssDNA only. Moreover, we purified the INTS3–hNABP1/2–C9ORF80 heterotrimeric complex, and found that it binds ssDNA and ssRNA at reduced affinity, compared with hNABP1/2 alone. Collectively, our results indicate that INTS3 might function as a scaffold protein to recruit hNABP1/2 to DNA damage sites to maintain genome integrity.


D. rerio ZEM-2S embryonic cells. Immunopositive labeling for Opn4m1 was detected in the cell membrane whereas Opn4m2 labeling shows nuclear localization, which did not change in response to light. opn4m1, opn4m2, gr, per1b, and cry1b presented an oscillatory profile of expression in LD condition. In both DD and LD condition, dexamethasone (DEX) treatment shifted the peak expression of per1b and cry1b transcripts to ZT16, which corresponds to the highest opn4m1 expression. Interestingly, DEX promoted an increase of per1b expression when applied in LD condition but a decrease when the cells were kept under DD condition. Although DEX effects are divergent with different light conditions, the response resulted in clock synchronization in all cases. Taken together, these data demonstrate that D. rerio ZEM-2S cells possess a photosensitive system due to melanopsin expression which results in an oscillatory profile of clock genes in response to LD cycle. Moreover, we provide evidence that glucocorticoid acts as a circadian regulator of D. rerio peripheral clocks.Here we report, for the first time, the differential cellular distribution of two melanopsins (Opn4m1 and Opn4m2) and the effects of GR agonist, dexamethasone, on the expression of these opsins and clock genes, in the photosensitive Time is a fundamental variable for the manifestation of biological phenomena, and as such it has been systematically investigated since the pioneer chronobiology studies by Halberg . The genDanio rerio, the popular zebrafish, has been employed as an alternative model to study clock molecular machinery, due to the similarities with the mammalian clock regarding the genes and their feedback loops [ck loops , 19. On ck loops , 20 allo Xenopus laevis melanophores [ Opn4m, orthologue of mammalian melanopsin, and Opn4x, orthologue of the melanopsin initially cloned from X. laevis [ Opn4x locus during chromosomal reorganization [ Danio rerio retina, five melanopsin genes, opn4m1, opn4m2, opn4m3, opn4x1, and opn4x2 [ opn4m1 and opn4m2 are the mostly abundant melanopsins expressed in zebrafish embryonic cells (ZEM-2S) [The photopigments responsible for converting light into temporal information are assembled in a family of nonimage forming opsins named melanopsins, which were firstly discovered innophores , with sunophores –25, and nophores –28. Data. laevis , 25. Botd opn4x2 , are fou(ZEM-2S) . The hig(ZEM-2S) .Mammalian melanopsin is expressed by a small subset of retinal ganglion cells and translates light stimulus into neural information, which reaches the suprachiasmatic nucleus (SCN) through the retinohypothalamic tract , 32. By  clock, bmal, per, and cry. Clock:Bmal dimer induces rhythmic expression of per and cry, while Per and Cry proteins repress their own expression by interacting with Clock:Bmal via the processes of blocking and displacement repressions [ bmal expression through the clock-controlled genes, Ror and Rev-erb, respectively. The completion of this feedback system takes about 24 h, thus generating a pattern of circadian rhythm [The molecular mechanisms of zebrafish circadian rhythm are similar to the well-known mammalian system and rely on core set of genes composed ofressions . In addin rhythm . period , 6 cryptochrome , 3 clock , and 3 bmal  genes have been cloned from D. rerio [ per2 and cry1a genes via D box-binding factor TEF (Thyrotroph Embryonic Factor) [However, some differences as the multiple clock genes and the light sensitivity make zebrafish a peculiar vertebrate model. FourD. rerio , 37, 38. Factor) –41, a si Factor) , 19. Per1 in rat fibroblasts [ Per1 gene possesses the glucocorticoid responsive element (GRE), indicating that GC-receptor complex may directly modulate Per1 expression [ Per1, Per2, Per3, Cry1, Cry2, Npas2 , and Bmal1 and of the clock-controlled genes, Rev-erb α/β and Dbp to GCs [ Rev-erbαtranscription [The synchronization between central and peripheral clocks raised the hypothesis that humoral factors are responsible for their communication. Hormones such as glucocorticoids (GCs) became strong candidates as regulatory agents of clock genes, as they are rhythmically produced and released in all vertebrates –44. One-roblasts . In factpression . Mouse pp to GCs . In rat cription .Although the literature strongly indicates regulation of mammalian biological clocks by GCs, the mechanisms and the relevance of this modulation in nonmammalian vertebrates are yet to be established. In zebrafish corticotrope-deficient larvae, it has been demonstrated that dexamethasone was able to rescue high-amplitude circadian cell cycle rhythms . Danio rerio embryonic cells ZEM-2S and the evoked regulation of melanopsins, GC receptor, per1b, and cry1b expression in this photosensitive cell line.Here, we investigated the putative role of GCs as synchronizing agents ofDanio rerio embryonic cells (ZEM-2S)  were kept in 50% Leibowitz L-15, 35% Dulbecco's Modified Eagle Medium (DMEM), 15% Ham's F12 medium , and 15 mM 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) , supplemented with 10% fetal calf serum , 1% antibiotic/antimycotic solution , and all-trans-retinal at final concentration of 10−7 M , pH 7.2, at 28°C.For the experimental protocols the serum concentration was reduced to 2%. Cells were handled in the dark under a red safelight . D. rerio Opn4m1 (MSGAAHSVRKGISC) and Opn4m2 (MSHHSSWRGHHCAPGC) were conjugated to keyhole limpet hemocyanin and inoculated into rabbits to obtain the primary antisera . The primary antisera were previously tested in a panel of dilutions ranging from 1 : 100 to 1 : 1000, and good staining was observed only at 1 : 100 and 1 : 250 for both antibodies. The antisera and the Cy3-labeled secondary anti-rabbit antibody  were diluted in incubation buffer .The 15 N-terminal sequences with an appended C-terminal cysteine ofThe expressions of Opn4m1 and Opn4m2 were evaluated in ZEM-2S cells kept in DD condition for 3 days and subject to 1 hour of white light pulse (650 lux) at the beginning of the 4th day. Control cells were kept in DD for the duration of the experiment. Twenty-four hours after the light pulse, cells were fixed with 4% paraformaldehyde  in phosphate buffered saline (PBS) at 4°C for 30 min. The blockade of nonspecific sites was made with 6% normal goat serum plus 22.52 mg/mL glycine  in PBS at 4°C for 1 h, followed by incubation with the primary antibodies  overnight at 4°C. The cells were then incubated with the secondary antibody for 1 h at room temperature, mounted with DAPI-Vectashield hard aqueous medium  and coverslipped. Photographs were taken in an inverted fluorescence microscope Axiovert 40CFL .6) were kept in LD cycle for 3 days and on the 4th day the cells were fixed with 4% paraformaldehyde at ZT4 and ZT16. The material was then subject to similar procedure as described in the immunocytochemistry section, incubated in anti-Opn4m1 or anti-Opn4m2 polyclonal antibodies at 1 : 250 and 1 : 100 dilutions, respectively, followed by incubation with a sheep FITC-conjugated anti-rabbit antibody at 1 : 200 dilution . Negative controls were incubated with FITC-conjugated anti-rabbit antibody, in the absence of the primary antibody. The analysis of 10,000 cell counts per sample was performed by IDEAS software  based on scatter plot of bright field area versus aspect ratio and expressed as normalized frequency based on FITC fluorescence intensity.To determine the expression of Opn4m1 and Opn4m2 proteins at light and dark phases, we quantified the fluorescence of cells using imaging flow cytometry . ZEM-2S cells (2.106 cells/25 cm2 flask) were seeded in 2% serum-supplemented medium and subject to DD or 12 : 12 LD cycle  for at least 5 days, at 28°C. On the following day, total RNA was extracted every 4 h over a day. Each protocol was repeated at least twice, with two or three flasks per time point to obtain n = 4 to 6.ZEM-2S cells  peaks in the dark phase.The medium of the control cells was changed simultaneously with DEX-treated cells, totaling up to 2, 6, or 10 medium changes, respectively. DEX concentration and application time were based on Gilchrest and colleagues , who rep pellet was resuspended in DEPC H2O  and treated with DNAse according to the manufacturer's protocol . RNA concentration (OD260) was determined in a spectrophotometer , and 2 µg was submitted to RT-PCR  using random primers. The reaction protocol was as follows: 5 min at 65°C and 1 min at 4°C; after the enzyme addition, 5 min at 25°C and 50 min at 50°C; and 15 min at 70°C in a thermocycler .Total RNA was extracted with TRIzol Reagent  according to the manufacturer's instructions. The RNA per1b, cry1b, and ribosomal 18S RNA or opn4m1, gr, and 18S RNA we prepared a mix of primers . Each experimental cDNA was run in triplicate in 96-well plates.Using TaqMan approach to simultaneously analyze the expression of opn4m2 and 18S RNA, the qPCR reactions contained iQ™ SYBR® Green Supermix 2X  or SYBR® GreenER™ qPCR SuperMix for iCycler® 2X  and specific primers, in final concentrations of 300 ηM for opn4m2 and 50 ηM for 18S RNA, respectively, in independent solutions. Primers and probes for Danio rerio were designed based on sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/Genbank) and synthesized by IDT . 18S RNA was used to normalize the values of specific genes, since it did not vary among time points under the various experimental conditions and has a stable expression in many tissues of zebrafish [Fory-axis and the log of cDNA dilutions in the x-axis. The efficiency for each primer pair was calculated according the equation: 10∧(−1/x) − 1∗100, in which x corresponds to the inclination angle of the linear regression curve. Values between 90% and 110% were considered as indicators of appropriate efficiency.Primers' efficiency was determined using serial dilutions  of a single cDNA sample. The triplicate mean CT of each gene was plotted in the All assays were performed in iCycler or iQ5  thermocycler, with the following protocols: multiplex, 1 cycle of 7 min at 95°C followed by 45 cycles of 30 sec at 95°C and 30 sec at 60°C; SYBR Green, 2 min at 50°C, 8:30 min at 95°C, 45 cycles of 15 sec at 95°C, 1 min at 60°C, 1 min at 95°C, 1 min at 55°C, and 80 cycles of 10 sec at 55°C with a gradual increase of 0.5°C. Negative controls without templates were routinely included.p ≤ 0.05.The ΔΔCT method was used to analyze relative changes in gene expression by qPCR. The CT was obtained comparing the number of cycles in control and experimental wells and among the various time points, by passing a threshold line through the geometric portions of the amplification curves. The ΔCT was calculated as the difference between the CT for 18S RNA and that for the gene of interest; the smallest value obtained in the control group or in a time point was then subtracted from each ΔCT, originating the ΔΔCT. This value was then used as the negative exponential of base 2. To assess temporal variation among different time points (ZTs), means were compared by one way analysis of variance (ANOVA) followed by Tukey test; to compare control and experimental groups along time, two way ANOVA followed by Bonferroni test was used. All data were analyzed and graphed by GraphPad Prism 5 software, and the differences were considered statistically significant when  D. rerio explained by genome duplication event in the teleost lineage may reflect either redundant or specialized functions performed by the multiple copies [The striking diversity of extra genes expressed ine copies . Howevere copies , 50. opn4 mRNAs were detected in earlier stages of development, as demonstrated in D. rerio ZEM-2S cells, where opn4m1 and opn4m2 genes are more expressed as compared to opn4m3, opn4x1, and opn4x2 genes, whose expression was negligible [ opn4m1 and opn4m2 genes were analyzed besides the glucocorticoid receptor (gr) gene which was also found to be expressed in this cell line.By contrast, not allgligible . Thereby D. rerio ZEM-2S cells using immunocytochemistry and imaging flow cytometry. Positive labeling for Opn4m1 . The differential response between nonmammalian and mammalian melanopsins may be due to the fact that mammals express only one melanopsin gene/protein, while D. rerio and X. laevis express multiple melanopsins. In D. rerio Opn4m1 found in the cell membrane likely works as a photosensor, while the nuclear melanopsin (Opn4m2) could function as a transcription factor. In addition, analyses of flow cytometry imaging revealed that the majority of the cells were positive for Opn4m1 (ZT4 91.9%  ± 7.5% versus ZT16 99.07%  ± 0.12%) and Opn4m2 (ZT4 99.75%  ± 0.1% versus ZT16 99.43%  ± 0.20%). However no difference in fluorescence intensity between ZTs was found , and 3 pulses of DEX remarkably increased mRNA levels keeping the peak in the beginning of the dark phase  has yet to be demonstrated in the melanopsin genes, its presence in opn4m1 but not in opn4m2 promoter might explain the differential response of these melanopsins to glucocorticoids.The oscillatory profiles of both melanopsins in LD cycle Figures  were altrk phase , but not and in d bmal2, ), as wel, [clock ) maintaiD cycles . Phylogepatterns . In partnditions , 18, 19. per1b expression in cells subject to medium changes in DD seems to oscillate with higher values at ZTs 0, 4, and 8, no statistical differences were found ; among them, opn4m1 and opn4m2 were reported as the most expressed melanopsins in ZEM-2S cells . The cellular compartments where Opn4m1 and Opn4m2 are expressed were identified here by immunocytochemistry. Surprisingly Opn4m2 was found in the nucleus when ZEM-2S cells were kept in DD conditions. So, we investigated the effects of white light pulse in promoting melanopsin translocation. Danio rerio ZEM-2S cells were kept for 3 days in DD condition and subject to 1 hour of white light pulse (650 lux) in the beginning of the 4th day. The incubation of ZEM-2S cells with anti-Opn4m2 at 1 : 100 dilution demonstrated that white light pulse did not altered Opn4m2 nuclear location. On the other hand, in mammalian melanoma cells, a short white light pulse (15 min) promotes melanopsin translocation from the nucleus to the cell membrane . Davies, W. I. L. et al. (2011). Functional diversity of melanopsins and their global expression in the teleost retina. Cell. Mol. Life Sci. 68(24), 4115–4132. Ramos, B. C. R., Moraes, M. N. C. M., Poletini, M. O., Lima, L. H. R. G., and Castrucci, A. M. L. (2014) From blue light to clock genes in zebrafish ZEM-2S cells. PLoS ONE 9 (9), Article ID e106252. de Assis, L. V. M., Moraes, M. N., da Silveira Cruz-Machado, S., and Castrucci, A. M. L. (2016) The effect of white light on normal and malignant murine melanocytes: a link between opsins, clock genes, and melanogenesis. Biochim. Biophys. Acta 1863(6), 1119–1133.


The ELISAs showed a broader working range than that of the LC3 Western blots (We analyzed autophagy/mitophagy flux rn blots . Table 2rn blots . Specifications TableValue of the data•The presented data indicated that the ELISA had smaller data distribution and was more repeatable to measure autophagy/mitophagy flux, compared with Western blot data.•These data could be helpful for many autophagy researchers to obtain more accurate and reproducible data using this ELISA technique.•These data could also be beneficial for researchers in other areas to adapt the ELISA-based assay strategy from the Western blot.1versus 1.4, the ratio of the highest O.D. value to the lowest)   . The val P<0.05, , which is ≤ 0.4) .Table 1T2in vitro and in vivo LC3-II turnover measured by Western blot in the presence and absence of an autophagy inhibitor as previously described We performed the autophagic flux assays using 2.1C2C12 myotubes grown on 10-cm dishes were incubated in amino acid and serum-free starvation buffer or treated with 10 μg/mL rapamycin or 25 μg/mL CCCP with and without 200 nM BafilomycinA1 for 8 h.2.2i.p. injected to mice daily for two days. Mice were also treated with and without 0.4 mg/kg/day colchicine administration for 48 h. Control mice received an equal volume of i.p. saline.Ten week old male wild-type C57BL/6 mice were divided into four groups: fed and starvation, or vehicle, rapamycin, and CCCP. Starvation was performed by removing food for 48 h. 10 mg/kg/day rapamycin, 4 mg/kg/day CCCP or vehicle was 2.3Subcellular fractionations from cultured cells or tissue were performed as previously described 2.4Mitochondrial fractionations from cultured cells or tissue were performed as previously described 2.5Following subcellular fractionation of mouse skeletal muscle cells and tissue, cytosolic, membrane, and mitochondrial fractions were analyzed through a sandwich ELISA using two LC3 antibodies, LC3 capture and HRP-conjugated LC3 detection antibodies as previously described 2.6LC3 protein turnover and other protein levels were measured using immunoblot techniques as previously described 2.7p<0.05.Data are presented as means±SD and were evaluated by a paired t-test or reliability analysis at Korean Government (NRF-2013-332-2013S1A5A8021905).This work was supported by the National Research Foundation of Korea Grant funded by the