OnabotulinumtoxinA (OnabotA) is considered effective in in patients with chronic migraine (CM) who failed on traditional therapies. This study was designed to evaluate the effect of OnabotA injection series on migraine outcome, negative emotional states and sleep quality in patients with CM.A total of 190 patients with CM (mean (SD) age: 39.3 (10.2) years; 87.9% were female) were included. Data on Pittsburgh sleep quality index (PSQI), headache frequency and severity, number of analgesics used, Migraine Disability Assessment Scale.(MIDAS) scores and Depression, Anxiety and Stress Scale (DASS-21) were evaluated at baseline (visit 1) and 4 consecutive follow up visits, each conducted after OnabotA injection series; at week 12 (visit 2), week 24 (visit 3), week 36 (visit 4) and week 48 (visit 5) to evaluate change from baseline to follow up.p = 0.002), headache severity , number of analgesics  and MIDAS scores . No significant change from baseline was noted in global PSOI and DASS-21 scores throughout the study.From baseline to visit 5, significant decrease was noted in least square (LS) mean headache frequency  contains supplementary material, which is available to authorized users. Chronic Migraine (CM) is a complex and severely disabling neurological disorder, characterized by occurrence of headache on ≥15 days per month for >3 months with at least five attacks fulfilling criteria of migraine without aura on ≥8 days per month , sleep quality [Pittsburgh sleep quality index (PSQI)] and negative emotional states  were evaluated at baseline and follow up visits. Medication overuse was baseline of simple analgesics on ≥ 15 days. Follow up period was composed of 4 consecutive visits each conducted after a new OnabotA injection series; at week 12 (visit 2), week 24 (visit 3), week 36 (visit 4) and week 48 (visit 5), respectively. Change from baseline to follow up was evaluated based on data from visit 1 to visit 3 (24 weeks) for PSQI, from visit 1 to visit 4 (36 weeks) for DASS-21 scores, while based on data from visit 1 to visit 5 (48 weeks) for headache frequency and severity, number of analgesics used and MIDAS score. Data at visit 2, visit 3, visit 4 and visit 5 refer to changes in parameters observed after 1CM was diagnosed based on International Classification of Headache Disorders (third revision) (ICHD-3 beta) diagnostic criteria that require headache occurring on ≥15 days per month for >3 months with at least five attacks fulfilling criteria of migraine without aura on ≥8 days per month [Administration of OnabotA was performed as 31 fixed-site, fixed-dose intramuscular injections applied at seven specified head and neck muscle points every 12 weeks for a minimum of 24 weeks (2 treatment cycles) according to injection scheme proposed in the PREEMPT studies , 29, 33.The MIDAS is a 5-question tool to quantitatively evaluate the headache-related disability in terms of the number of days in the past 3 months and activity limitations due to migraine. MDAS was developed by Stewart et al. and valiThe DASS-21 is a 21-item questionnaire which includes three self-report scales designed to measure the negative emotional states of depression, anxiety and stress. The Depression scale includes items that measure symptoms typically associated with dysphoric mood . The Anxiety scale includes items that are primarily related to symptoms of physical arousal, panic attacks, and fear . Finally, the Stress scale includes items that measure symptoms such as tension, irritability, and a tendency to overreact to stressful event. Each item is scored on a 4-point scale  to rate the extent to which they have experienced each state over the past week. Sum of the score of each item reveals the total score with higher scores indicating greater levels of distress. Each state is categorized into normal/mild/moderate/severe/extremely severe based on cut-off scores recommended for depression (0-4/5-6/7-10/11-13/14+), anxiety (0-3/4-5/6-7/8-9/10+) and stress (0-7/8-9/10-12/13-16/17+) –38. PsycThe PSQI, used to assess sleep quality, was developed by Buysse et al.  and valip < 0.05 was considered statistically significant.Statistical analysis was made using IBM SPSS Statistics . Change over time analysis was based on number of patients with available data on both baseline (visit 1) and follow-up visits (visit 2 to 5), and performed via repeated measures variance analysis and McNemar test for continuous and categorical variables, respectively. Since time to follow up visits showed variability among patients, change over time analysis of continuous variables were adjusted for time . No specific procedure was defined for missing data. Data were expressed as n(%), mean , median  and mean  where appropriate. Of 190 patients; 101 (53.2%) attended at least one follow-up visit. Overall 98 (51.6%) patients attended visit 2, 58 (30.5%) patient visit 3, 34 (17.9%) patients visit 4, and 20 (10.5%) patients attend visit 5, which were performed at week 12, week 24, week 36 and week 48, respectively.During the study period overall four injection series were applied per patient within a total treatment time of 48 weeks (median (IQR) 62.4 (53.8–85.2) weeks).Most of the participants were university graduates 41.1%), diagnosed with CM per se (48.9%) without triggers (76.8%) and for median (IQR) 3.0 (1.0–10.0) years. Family history for migraine was evident in 28.9% of patients; while most common previous medication was non steroid anti-inflammatory drugs (88.4%). Medication overuse was evident in 76.2% of patients  headache frequency was 17.9  at visit 1 and 6.8  at visit 5. Median (IQR) headache severity scores were 8.0 (7.0–9.0) and 7.0 (5.0–7.0), number of analgesics used were 20.0 (15.0–30.0) and 5.5 (2.0–10.0) and MIDAS scores were 57.0 (35.5–75.0) and 10.0 (2.0–15.0) at visit 1 and visit 5, respectively (Table p < 0.001), to 7.5 at visit 3 (p < 0.001), to 5.4 at visit 4 (p < 0.001) and to 8.4 at visit 5 (p = 0.002) (Table Mean headache frequency was significantly decreased from LS mean 19.5 at visit 1 to 6.8 at visit 2 (p < 0.001), to 5.8 at visit 3 and visit 4 (p < 0.001 for each) and to 6.1 at visit 5 (p = 0.017) (Table Mean headache severity was significantly decreased from 8.1 at baseline to 6.2 at visit 2 (p < 0.001), to 8.7 at visit 3 (p < 0.001), to 5.1 at visit 4 (p < 0.001) and to 10.4 at visit 5 (p = 0.023) , to 15.3 at visit 3 (p < 0.001), to 9.3 at visit 4 (p < 0.001) and to 18.5 at visit 5 (p < 0.001) , anxiety  and stress  in similar percentage of patients at visit 1, visit 2, visit 3 and visit 4, respectively , anxiety  and stress  was evident in remarkable percentage of patients throughout the study period, while severe-to-extremely severe depression , anxiety  and stress  were also noted in less than 15% of patients.Based on patients with valid data for both baseline and follow up visits, no significant change was noted in percentage of patients categorized to have normal anxiety, depression and stress scores at each follow up visit Table .Table 5COverall, median (IQR) global score at visit 1 and visit 5 were 9.0 (5.0–12.0) and 4.0 (1.0–7.0), respectively Table .Table 6Op = 0.002), sleep latency (from baseline 1.7 (1.4–2.0) to 1.1  and sleep disturbance (from baseline 1.7 (1.4–2.0) to 1.2 (0.9–1.6) at visit 2, p = 0.013) components improved significantly from baseline to visit 2  scores for subjective sleep quality (from baseline 1.7 (1.4–2.0) to 1.1 (0.8–1.5) at visit 2, Good sleep quality was noted at visit 1, visit 2 and visit 3 34.3, 40.0 and 57.1% of patients with normal depression scores at visit 1, in 38.7, 45.5 and 50.0% of patients with normal anxiety scores at visit 1, and in 33.3, 46.2 and 60.0% of patients with normal stress scores at visit 1, respectively  in some patients. In one patient who has a very thin cervical region, dysphagia and difficulty in swallowing appeared while regressed after the third week of therapy. Treatment was continued in this patient with omission of further injections to the cervical area.n = 2), addition of antiepileptic (n = 3) or SSRI (n = 9) medications and discontinuation of ongoing antiepileptic (n = 3) or SSRI (n = 1) medications.Treatment alterations included discontinuation of OnabotA therapy  . Given tIn terms of monthly headache frequency, headache severity, number of analgesics used and MIDAS scores our findings support the association of OnabotA therapy with rapid improvement in migraine parameters usually after the first session as reported in past studies , 51–53.The rapid improvement of migraine parameters after OnabotA therapy has been linked to the combined pharmacological and the placebo effect at the beginning of the treatment , 52, whiAlike to our findings, use of four OnabotA injections over 48 weeks in CM patients was reported to be associated with significant improvement in monthly headache days, migraine days and medication days that continued throughout the entire study period . NonetheLong-term efficacy of OnabotA in CM patients has recently been evaluated in some studies based on administration of OnabotA therapy for 2 years , 4 yearsBoth depression and anxiety have been proposed to be a risk factor for migraine chronification , 59, 60,Reduction in headache frequency via 24-week OnabotA therapy was reported to be associated with reduction in depression (via Beck Depression Inventory-II) and anxiety scores  in a past among CM patients . In factIn this regard, it seems notable that despite significant reductions obtained via OnabotA therapy in both monthly headache frequency and headache severity in our cohort, no significant change from baseline occurred in depression, anxiety and stress rates throughout the study.High rates of negative emotional states regardless of the ongoing OnabotA therapy in our patients seems notable given the impact of psychiatric comorbidities on disease prognosis, treatment, and clinical outcomes and higher prevalence of severe headache-related disability, headache impact and poor quality of life in migraineurs with than without psychiatric comorbidities , 13, 64.Identification of poor sleep quality at baseline in 72.9% of our patients seems consistent with high prevalence (30–79%) of co-morbid poor sleep quality among migraineurs , 66–68, In a past study on the effects of OnabotA on jaw motor events during sleep in sleep bruxism patients, no significant change from baseline was noted in usual sleep variables such as sleep efficiency, arousal index, sleep stages, or awakenings per hour during follow-up recordings .Similarly, our findings revealed no direct effect of OnabotA injection on overall sleep quality at week 12 and week 24, while significant improvement in subjective sleep quality, sleep latency and sleep disturbance at week 12. This seems notable given the reported association of higher migraine frequency with higher scores for certain domains of PSQI such as “cannot get to sleep within 30 minutes,” “wake up in the middle of the night or early morning,” and “bad dreams” among migraineurs .Sleep-related migraine is considered to be associated with a more severe and disabling clinical course given the increased mean attack severity and monthly use of symptomatic drugs in patients with than without sleep related migraine despite similar monthly headache frequency .Although data on sleep quality are available for only two OnabotA injections in our cohort, given that patients without negative emotional states at baseline showed improved sleep quality throughout the study, maintenance of co-morbid negative emotional states throughout the study seems to be associated with lack of improvement in overall sleep quality, despite significantly decreased migraine frequency.Nonetheless, it should also be noted that while the presence of negative emotional states such as depression was reported to be reciprocally associated with poor sleep quality , 71 and Certain limitations to this study should be considered. First, due to observational nature, non-randomized allocation and thereby the likelihood of main selection bias and confounding is possible. Second, although provide data on real-life clinical practice, potential lack of generalizability seems another important limitation due to single-center design of the study. Third, lack of intervention considering timing and number of follow up visits in accordance with the observational nature caused relatively limited follow-up data and the frequency of patient visits to be not uniform with considerable loss to follow up rate challenging analysis of sleep and mood variables. Fourth, analysis of data on negative emotional states and sleep quality were based on self-report rather than objective measures. However, our analysis was based on use of validated questionnaires along with evidence on high concordance between self-report instruments and clinical diagnosis of psychiatric disorders . Lack ofIn conclusion, our findings in a cohort of chronic migraineurs revealed that OnabotA injection was associated with significant improvement in migraine outcome leading to decrease in headache frequency and severity, number of analgesics used and MIDAS scores. While no significant change was noted in overall sleep quality and prevalence of negative emotional states with OnabotA injections, patients without negative emotional states at baseline showed improved sleep quality throughout the study. There is a need for future larger-scale long-term longitudinal studies addressing the durability as well as predictors of efficacy of OnabotA in CM patients.


Nature Communications6: Article number: 7808; DOI: 10.1038/ncomms8808 (2015); Published: 08062015; Updated: 120820162], and this should have read [(4/3) × pi × (radius)3]. As a result, the correct absolute adipocyte numbers are higher than those published. However, relative differences between all groups in this panel remain the same. None of these alterations change the original conclusions. The correct version of the figure appears below as In Figure 3 of this Article, marrow adipose tissue volumes (in panels b and c) and adipocyte numbers (in panel g) were calculated incorrectly. In Fig. 3b,c, regional MAT volumes in the ‘GP to T/F J' group were normalized incorrectly to both regional marrow volume and total marrow volume instead of normalizing to total marrow volume only. As a result, the correct MAT/marrow percentages in the ‘GP to T/F J' group are higher than those published. Furthermore, in Fig. 3g, the formula used for calculating adipocyte volumes was [(4/3) × pi × (radius)


Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3–6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC A receptors, mediate fast inhibitory neurotransmission in the mature central nervous system , together with GABA50 values of glycine, while inhibition of both kinases using H-89 (PKA) and Staurosporine aglycon (PKC) led to a decrease of EC50. Effects of kinase modulators were concentration-dependent and developed fully within 5–10 min after intracellular application. None of the kinase modulators had an immediate effect on maximum current responses. However, When the effects of PKA- and PKC activators was followed over 10 min, a significant reduction of Imax was observed compared to controls, while Imax was slightly increased relative to control in presence of kinase inhibitors. PKA modulators had no effect on GlyR trafficking, while stimulation of PKC by PMA accelerated receptor internalization and lowered cell surface expression of recombinant GlyRs. The opposite was observed with the PKC-inhibitor Staurosporine aglycon. Thus, phosphorylation was confirmed to be a specific and relevant modulatory element of GlyR activity.Here, we studied the effects of PKA and PKC modulators on inhibitory GlyRs expressed in HEK293 cells. Stimulation of PKA and PKC by Forskolin and phorbol-12-myristate-13-acetate (PMA), respectively, gave higher EC2 and 37°C in a water saturated atmosphere. For electrophysiological experiments, cells were plated on poly-L lysine treated glass coverslips in 6 cm dishes. Transfection was performed 1 day after cell passage using Gen-Carrier : 1.3 μg of receptor DNA, 1.3 μg of green fluorescent protein DNA, and 2.6 μl GenCarrier were used, following the manufacturer’s instructions. Measurements were performed 2–4 days after transfection. HEK293 cells used for protein expression and internalization analysis were transfected using a modified calcium phosphate precipitation method. In brief, a mixture of plasmid DNA, CaCl2 (2.5M), 0.1× TE buffer and 2× HBS  was applied onto the cells for 6 h. A GlyR α1 to GlyR β ratio of 1:2 was used for co-expression. Transfection medium was replaced by fresh medium after 6 h. Immunocytochemical stainings were always done 24 h after transfection, biotinylation experiments were performed 48 h after transfection.HEK293 cells were grown in 10 cm tissue culture Petri dishes in Eagle minimal essential medium  supplemented with 10% FBS  and penicillin/streptomycin  at 5% COFor biotinylation, transfected HEK293 cells transiently expressing the GlyR α1 or co-expressing GlyR α1-β were incubated with PKA or PKC modulators for 16 h. Surface proteins were labeled with 1 mg/ml EZ-Link Sulfo-NHS-LC-Biotin . After cell lysis, biotin-labeled protein was removed using streptavidin agarose beads . Total protein, surface protein and intracellular proteins were subjected to SDS-PAGE and Western Blotting.For protein separation, 11% polyacrylamide gels were used. Gels were run at 150 V for 90 min. Proteins were transferred to a nitrocellulose membrane  using a wet blot transfer system . For GlyR protein transfer, 2 h at 200 mA were used. Cadherin transfer required overnight blotting at 100 mA. Membranes were blocked for 1 h with 5% BSA in TBS-T (TBS with 1% Tween 20). Primary antibodies  were incubated overnight at 4°C. Signals were detected using SuperSignal West Pico .To stain internalized GlyRs, GlyR α1 was cotransfected with GlyR β into HEK293 cells grown on glass cover slips. Internalization experiments were performed 24 h post-transfection. Live cells co-expressing GlyR α1-β receptors were labeled with the α1-specific monoclonal antibody Mab2b . Earle’s minimal essential medium was supplemented with 10% fetal calf serum, 200 mM GlutaMAX, 100 mM sodium pyruvate, and 50 U/ml penicillin/streptomycin . Mab2b was purchased from Synaptic Systems, Göttingen, Germany. Following three washing steps in phosphate-buffered saline , cells were incubated with goat-anti-mouse-Cy3 secondary antibody again for 1 h at 4°C . Glass cover slips with pre-labeled GlyRs were transferred into a 24-well plate containing 500 μl of Earle’s minimal essential medium with PKA and PKC modulators . Cells were incubated with modulators for 15 min to allow receptor internalization. Time point zero (0 min) was taken as a negative internalization control to ensure health of HEK293 cells . Following incubation with PKA and PKC modulators, cells were stained with a second secondary antibody  for 30 min at 4°C to label receptor retained at the cellular surface. Cells were fixed with 4% paraformaldehyde, 4% sucrose in PBS for 15 min at 4°C, washed three times in PBS and stained with 4′,6-diamidino-2-phenylindole  to label the cell nuclei for 5 min at 4°C (1:5000 in PBS). Coverslips were washed again with PBS and Aqua Dest and mounted on glass slides using mowiol.The images were obtained using an inverted Olympus IX81 microscope equipped with an Olympus FV1000 confocal laser scanning system, a FVD10 SPD spectral detector and diode lasers of 495 nm (Alexa488) and 550 nm , and an Olympus UPLSAPO 60×  objective. Adobe Photoshop  or ImageJ (1.51)/Fiji2, 1.0 mM MgCl2 and 10 mM Hepes (pH adjusted to 7.4 with NaOH); the internal buffer was 140 mM CsCl, 1.0 mM CaCl2, 2.0 mM MgCl2, 5.0 mM EGTA and 10 mM Hepes (pH adjusted to 7.2 with CsOH). Dose response data was fitted to the Hill equationWhole-cell recording experiments were performed using a HEKA EPC10 amplifier  controlled by Pulse software (HEKA). Recording pipettes were pulled from borosilicate glass  using a Sutter P-97 horizontal puller . Pipettes routinely had an open tip resistance of 2–4 MΩ. HEK293 cells had a membrane capacitance of 3–7 pF. Series resistance, as well as slow and fast capacitance were compensated using the EPC10 circuitry. The different protein kinase modulators were dissolved in intracellular buffer at a concentration of 0.5 μM. For studies of concentration- and time-dependence, kinase modulators were used at concentrations of 0.1 μM and 0.25 μM. Current responses were measured at room temperature (21–23°C) at a holding potential of −50 mV. Solutions were applied using an Octaflow system , where cells were bathed in a laminar flow of buffer, giving a time resolution for solution exchange and re-equilibration of about 100 ms. The external buffer consisted of 135 mM NaCl, 5.5 mM KCl, 2 mM CaClglycine is the current amplitude at a given glycine concentration, Isat is the maximum current amplitude at saturating concentrations of glycine, EC50 is the glycine concentration at half-maximal current responses, and nHill is the Hill coefficient. Currents from each individual cell were normalized to the maximum response at saturating glycine concentrations. Significance of differences between EC50 values were determined using one-way ANOVA with p ≤ 0.05 (*) and p ≤ 0.01 (**) taken as significant. In all experiments, EC50 values were determined for each individual cell from a non-linear fit of dose response data to the logistic equation. An unweighted average ± standard error (SEM) was calculated from all individual EC50 values, without considering the fitting errors.using a nonlinear algorithm in Microcal Origin. Here, I50 data was recorded from each cell. The first concentration-response curve, defined as t = 0, was recorded immediately after establishing the whole-cell configuration and positioning the cell in front of the delivery tube. The dead time did not exceed 2 min. Further recordings were made at t = 3 min, t = 6 min and t = 10 min . Time-independent recordings were taken at t = 0, and values of EC50 and Imax in the presence of PKA- or PKC modulators were normalized to control (no modulator). For time course analysis, responses were normalized to the control value at t = 0. Patch-clamp experiments were carried out on different dates and using different batches of HEK 293 cells. Since control EC50 values varied between different days of plating and between batches of cells, measurements with kinase modulators were always compared to control measurements recorded on the same day and from the same batch of cells. Thus, while individual EC50 values varied between cells, any changes of EC50 induced by kinase modulators could robustly be observed and quantified.For time course analysis, a series of EC50 values were determined for each individual cell and then averaged. EC50 for glycine was 37.4 ± 3.7 μM (n = 11) and 45.4 ± 6.0 μM (n = 8) for α1 and α1-β GlyR, respectively  for α1 and 71.5 ± 4.3 μM (n = 7) for α1-β GlyRs  for α1 GlyRs and 14.9 ± 1.0 μM (n = 7) for heteromeric α1-β receptors, both changes were statistically significant  and 54.0 ± 4.6 μM (n = 8) for α1 GlyRs and α1-β GlyRs, respectively , and α1-β receptors .Presence of intracellular PMA had no effect on receptor desensitization, in contrast to fast desensitization induced by the PKA activator Forskolin. When Staurosporine aglycon was present glycine induced currents showed slow or no desensitization for both, α1 and α1-β receptors. For comparison, unmodified α1 GlyR was non-desensitizing, while α1-β subunits showed 50% desensitizing cells. Thus, significant differences were only observed for α1-β receptors Figure . EC50 vay Figure . The PKCWe noticed that PKA and PKC inhibitors weakened the HEK293 cells more than the activators. When the intracellular concentration of H-89 or Staurosporine aglycon was raised to 1 μM, cells did not survive.50 of GlyR control currents (no modulator) was 20.4 ± 2.7 μM (n = 3), in presence of 0.25 μM Forskolin and 0.25 μM H-89, EC50 was 21.3 ± 1.7 μM (n = 3), while in presence of 0.25 μM PMA and 0.25 μM Staurosporine aglycon, EC50 = 20.3 ± 0.6 μM (n = 3) was found, all changes were not significant . Thus, any shift induced by the PKA-activator Forskolin, or the PKC-activator PMA was counteracted by the corresponding antagonists, H-89 and Staurosporine aglycon, respectively.To test if the observed shifts in GlyR responses were indeed due to changes in PKA- and PKC modulation, we performed a separate experiment where kinase agonist and antagonist were applied simultaneously Figure . To avoi50 values were normalized to control. Shifts at the lowest concentration of modulators (0.1 μM) were not significant, but did show a clear trend, while at 0.25 μM of modulator, significant effects were observed for H-89 and PMA, but not for Forskolin or Staurosporine aglycon , and again after t = 3, 6 and 10 min using 0.1 μM and 0.25 μM of each kinase modulator in the intracellular pipette  a time-dependent change in receptor activation where EC50 values were reduced between 3 min and 6 min after the first measurement. This effect was independent of the absence or presence of kinase modulators; and (ii) distinct effects of the four protein kinase modulators on GlyR EC50 that were reproducibly observed and were additive to the first effect. Presence of the PKA-activator Forskolin and the PKC-activator PMA shifted EC50 towards higher values, while PKA antagonist H-89, and the PKC-antagonist Staurosporine aglycon shifted EC50 towards lower values  for control (no modulator), and also in the presence of 0.25 μM intracellular Forskolin or PMA . In presence of kinase inhibitors H-89 (PKA) and Staurosporine aglycon (PKC), internalization was lower compared to the corresponding PKA and PKC activators. It is noted, that even in presence of the inhibitors, overall internalization was increased relative to control, but these increases were not significant . Prior to protein biotinylation, PKA modulators and PKC modulators were incubated with transfected cells for 16 h. Neither PKA nor PKC modulators had a significant effect on GlyR α1 and GlyR α1-β whole cell or surface expression. Local changes in surface expression might, however, be overlaid by continuous overexpression of GlyRs in transfected HEK293 cells during the incubation period of 16 h. Previously, it has been shown that receptor internalization following PMA treatment starts already within 15 min  in the TM3–4 domain. Here, the first residue of this insert may serve as a phosphorylation site for PKA . GlyR α1 subunits isolated from rat spinal cord were shown to be phosphorylated at Ser391 by PKC p Figure . In contd Lynch, . The Glyd Lynch, . Recentld Lynch, . This phd Lynch, .ins- and β-subunits throughout the central nervous system , which activates adenylyl cyclase, leading to an increase of cAMP levels, which in turn increases PKA activity, and H-89, a competitive inhibitor of the ATP binding site of PKA. PKC activation was done using PMA, a structural analog of the natural PKC activator diacyl-glycerol. For PKC inhibition, we used Staurosporine aglycon, a natural indole antibiotic from Streptomyces staurosporeus, which acts as competitive inhibitor of ATP binding to PKC.We used established, specific modulators of PKA and PKC. For PKA, we used Forskolin, a diterpene from the Indian Coleus plant . The time dependent reduction of Imax was opposed by PKA and PKC inhibitors. A concomitant shift of EC50 towards lower values and increase of Imax would be consistent with increased channel open probability, i.e., more effective gating  changes were all significant after 10 min of application. Changes of ECThe effects of PKA- and PKC-agonists  on GlyR currents could be offset by the addition of the corresponding antagonists, H-89 (PKA) and Staurosporine aglycon (PKC).No changes in receptor surface expression were obvious after long-term (>1 h) incubation with PKA and PKC modulators. In this situation, the effects of a single application of kinase modulators will be counterbalanced by the general turnover of receptor protein. On a shorter time scale, distinct effects of PKA and PKC modulation could be observed. Activation of either PKA or PKC by Forskolin or H-89 treatment induced enhanced receptor internalization after short-term treatment (15 min), while kinase inhibitors H-89 and Staurosporine aglycon showed no significant effects. These findings are in agreement with electrophysiological data: when PKA and PKC activators were present in the intracellular buffer, a modulation of GlyR currents was detected within ~10 min.C receptors (Filippova et al., A receptors (Connolly et al., 32P] revealed that NR2 subunits are highly phosphorylated under basal conditions, whereas basal phosphorylation of NR1 subunits is barely detectable (Hall and Soderling, A receptors in primary cultures of cerebral cortical neurons was increased upon PKC activation, as demonstrated by limited chymotrypsin digestion assays (Herring et al., Evidence for the involvement of protein kinases—especially PKC—in the regulation of ion channel surface expression and clustering was described previously for GlyRs, as well as other members of the ligand-gated ion channel family. Huang et al.  showed tOur findings show that activation of PKA and PKC reduces GlyR-mediated currents in a recombinant system. Inhibition of both kinases has the opposite effect. These effects are concentration-dependent and occur within minutes of application. On the same time scale of GlyR current modification, PKA and PKC activators increase internalization of the receptor, while their corresponding inhibitors have an opposite effect. Thus, GlyR phosphorylation is an important physiological regulatory mechanism that is involved in the fine-tuning of expression, trafficking and ion channel function of GlyRs.UB, LMB, DJ and VR performed experiments. UB, CV and H-GB analyzed data. UB, CV and H-GB wrote the manuscript. UB, C-MB, CV and H-GB devised the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Consequently, conventional rich in vitro culture conditions used widely to evaluate antibacterial agents are often poorly predictive of in vivo activity, especially for agents targeting metabolic pathways. In one such pathway, the methylerythritol phosphate (MEP) pathway, which is essential for production of isoprenoids in bacterial pathogens, relatively little is known about the influence of growth environment on antibacterial properties of inhibitors targeting enzymes in this pathway. The early steps of the MEP pathway are catalyzed by 1-deoxy-d-xylulose 5-phosphate (DXP) synthase and reductoisomerase (IspC). The in vitro antibacterial efficacy of the DXP synthase inhibitor butylacetylphosphonate (BAP) was recently reported to be strongly dependent upon growth medium, with high potency observed under nutrient limitation and exceedingly weak activity in nutrient-rich conditions. In contrast, the well-known IspC inhibitor fosmidomycin has potent antibacterial activity in nutrient-rich conditions, but to date, its efficacy had not been explored under more relevant nutrient-limited conditions. The goal of this work was to thoroughly characterize the effects of BAP and fosmidomycin on bacterial cells under varied growth conditions. In this work, we show that activities of both inhibitors, alone and in combination, are strongly dependent upon growth medium, with differences in cellular uptake contributing to variance in potency of both agents. Fosmidomycin is dissimilar to BAP in that it displays relatively weaker activity in nutrient-limited compared to nutrient-rich conditions. Interestingly, while it has been generally accepted that fosmidomycin activity depends upon expression of the GlpT transporter, our results indicate for the first time that fosmidomycin can enter cells by an alternative mechanism under nutrient limitation. Finally, we show that the potency and relationship of the BAP-fosmidomycin combination also depends upon the growth medium, revealing a striking loss of BAP-fosmidomycin synergy under nutrient limitation. This change in BAP-fosmidomycin relationship suggests a shift in the metabolic and/or regulatory networks surrounding DXP accompanying the change in growth medium, the understanding of which could significantly impact targeting strategies against this pathway. More generally, our findings emphasize the importance of considering physiologically relevant growth conditions for predicting the antibacterial potential MEP pathway inhibitors and for studies of their intracellular targets.The  FICFOS was calculated as the [FOS in the presence of BAP] in the same well, divided by MICFOS. The FIC index is the sum of FICBAP and FICFOS. FIC indices were used to indicate drug synergism (x < 0.5), additivity (0.5 < x < 1.0), indifference (1.0 < x < 2.0), or antagonism (x > 2). Fractional growth was determined relative to the no drug growth control and average values were used.An initial inoculum of 10B medium . Cells wB medium . BrieflyB medium ,86.. After 20 h of growth at 37°C, 1 μL aliquots of cell culture from each well of the 96-well plate were spotted onto agar plates containing the corresponding medium. Colony formation at 1–8 × MIC concentrations indicates a bacteriostatic mechanism of action, while a lack of colony formation indicates some level of bactericidal activity. MBC was defined as the concentration of compound required to kill bacteria, leading to an absence of colonies when spotted on agar plates.As described above, an initial inoculum of 10et. al. and Richter, et. al. was used for cell sample collection and cell lysis [E. coli were prepared as described above, and sub-cultured at a dilution of 1:50 into 200 mL of fresh medium. Cultures were incubated with shaking (250 rpm) at 37°C for approximately 3 hours, until an OD600 of 0.5–0.7 was reached. Cells were isolated via centrifugation in 4 × 50 mL Falcon tubes at 3220 rpm for 20 minutes at 4°C. After discarding the supernatant, the cell pellets were combined and resuspended in fresh media to a final volume of 8.5 mL. Cells were equilibrated to the treatment temperature (0 or 37°C) for 10 minutes, then treated with BAP or fosmidomycin and incubated for 60 minutes, during which no additional cell growth was noted .A protocol modified from Zhou, ll lysis ,87. Expe2O.At the indicated timepoints, 800 μL of culture was gently suspended onto 500 μL of a 3:1 mixture of silicone oil  and dichloromethane in a 1.7 mL microcentrifuge tube. The cells were immediately pelleted through the oil mixture at 12,000 × g to create a density gradient separating cells from the inhibitor-containing medium. After wicking away the aqueous medium with a Kimwipe, tubes were inverted and rested for 5 minutes to allow the oil to drain from the pellet surface. Tubes were cut with a plastic-tubing cutter  at the 500 μL mark, and the cell pellet was transferred to a fresh tube in 2 × 100 μL aliquots of ddHCell lysis was accomplished following three freeze-thaw cycles in which samples were placed in a liquid nitrogen bath for 3 minutes followed by incubation for 3 minutes in a water bath heated to 65°C, and vortexing between each cycle. Cell debris was pelleted at 12,000 × g for 2 minutes, and 180 μL of supernatant was removed into a fresh tube. Methanol (100 μL) was added to the pellet of cell debris, and the tube was agitated and vortexed before pelleting again at 12,000 × g for 2 minutes. The methanol supernatant (100 μL) was added to the aqueous supernatant to a total volume of 280 μL, and the remaining cell debris was discarded. Combined supernatant samples were vortexed and centrifuged at 16,000 × g for 10 minutes to pellet any protein or debris remaining in the sample. An aliquot of this clarified supernatant was then transferred to an LC-MS vial for analysis. LC-MS methods are detailed in Supplementary Information.-1. The autosampler temperature was set at 10 °C. The injection volume was 5 μL. Mass spectra were acquired with negative electrospray ionization at the ion spray voltage of -3,000 V. The capillary temperature was 270°C.Samples were analyzed with an LC-MS/MS system comprised of a Waters NanoAcquity UPLC and a TSQ Vantage Triple Quadripole (Thermo Scientific). The liquid chromatography separation was performed on a Waters Acquity UPLC HSS C18 column  with mobile phase A (0.1% triethylammonium acetate in water) and mobile phase B (acetonitrile). The flow rate was 50 μL minFor BAP, the LC gradient was as follows: 0–1 min, 0% B; 1–7 min, 0–100% B; 7–9 min, 00% B; 9–10.1 min, 100–0% B; 10.1–12 min, 0% B. Selective reaction monitoring followed the transition from parent ion to a metaphosphate fragment (179 to 62.9 m/z) using a collision energy of 38 eV.For fosmidomycin, the LC gradient was as follows: 0–2 min, 0% B; 2–7 min, 0–20% B; 7–7.1 min, 20–100% B; 7.1–9 min, 100% B; 9–10.1 min, 100–0% B; 10.1–12 min, 0% B. Selective reaction monitoring followed the transition from parent ion to a metaphosphate fragment (182 to 79.0 m/z) using a collision energy of 41 eV.-1. The autosampler temperature was set at 20°C. The injection volume was 2 μL for BAP and 1 μL for fosmidomycin. The Jet stream ESI source parameters were as follows: Negative Ion Mode, Drying Gas Temp: 350°C; Sheath Gas Temp: 400°C; Drying and Sheath Gas Flow: 12 L min-1; Nebulizer: 45 psig; VCap: -3000 V, Nozzle: -600 V, Fragmentor; -100 V; Skimmer: -50 V, OCT 1 RF Vpp: -750 V. The acquisition was performed by full scan MS from m/z 50 to m/z 500. Samples were analyzed with an LC-MS system comprised of an Agilent 1290 UHPLC and 6540 Q-TOF mass spectrometer with Jet Stream Electrospray Ionization source. The liquid chromatography separation was performed on a Waters Acquity UPLC HSS C18 column  with mobile phase A (0.1% triethylammonium acetate in water) and mobile phase B (acetonitrile). The flow rate was 200 μL minThe LC gradient for BAP was as follows: 0–3 min, 0–100% B; 3–3.2 min, 100% B; 3.21 min, 0% B. For fosmidomycin, the LC gradient was as follows: 0–3 min, 0–50% B; 3–3.5 min, 50–100% B; 3.5–3.7 min, 100% B; 3.71 min. Analyte peaks areas were determined from extracted ion chromatograms from the full scan data with a ± 20 ppm window for BAP and a ± 10 ppm window for fosmidomycin using Agilent Masshunter Quantitative Analysis Software (B.06.00 SP01).S1 FigStandard curves of BAP  and fosmidomycin  were generated using either the Selective Reaction Monitoring (SRM) method  or the Quadripole Time-of-Flight (Q-TOF) method .(TIF)Click here for additional data file.S2 FigE. coli was treated with BAP (1250 μM) for one hour at either 37°C (black square) or 0°C (gray square) in either CAMHB or M9-glucose medium. Intracellular BAP accumulation was monitored by LC-MS (SRM method). .(TIF)Click here for additional data file.S3 FigE. coli strains lacking bacterial porins (Keio collection: parent BW25113 (dark blue), ΔompR (green), ΔompA (orange), ΔompC (gray), ΔompF (yellow), ΔphoE (light blue)) treated with BAP (a) or Fosmidomycin (b) after growth in M9-glucose minimal medium for 16 h. .MIC values were determined in biological triplicate for (TIF)Click here for additional data file.S4 Fig1H (a) and 31P (b) NMR. The TPPO standard has a chemical shift in between that of the two observable rotomeric species.The synthesis of fosmidomycin was characterized by (TIF)Click here for additional data file.S5 FigE. coli cultures were grown in M9-glucose minimal medium (a) or CAMHB rich medium (b) in 96-well plates for 20 hours. Cell cultures (1 μL per well) were spotted onto agar plates containing the corresponding medium, incubated, and imaged. Red asterisks (*) indicate the MIC  of representative replicates. The BAP-fosmidomycin combination is bacteriostatic (c), indicated by an MBC/MIC ≥ 8.(TIF)Click here for additional data file.S6 FigE. coli was treated with 550 μM (100 μg/mL) fosmidomycin for one hour at either 37°C (■) or 0°C .(TIF)Click here for additional data file.S7 FigE. coli was treated with 550 μM (100 μg/mL) fosmidomycin, 1250 μM (230 μg/mL) BAP, or both for one hour in CAMHB growth medium. Intracellular BAP (a) and fosmidomycin (b) accumulation was monitored by LC-MS (Q-TOF method). .(TIF)Click here for additional data file.S8 FiguhpT BW25113) E. coli strains were treated with fosmidomycin in CAMHB (a), M9-glucose (b), and M9-glycerol (c) growth medium in biological triplicate. Deletion of UhpT does not significantly impact susceptibility to fosmidomycin.UhpT transporter-containing (■ BW25113) and deficient ((TIF)Click here for additional data file.


BRCA1 and BRCA2 identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate BRCA2 exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites  and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14–20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: Δ16p44 , Δ16 , Δ16p55 , Δ16q4 , Δ16q100 , ▾16q20 , as well as minor  and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms Δ16p44, Δ16, Δ16p55, Δ16q4, Δ16q100, and ▾16q20 introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.Genetic testing of  BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) confer up to 87% of risk to develop breast cancer by the age of 70 years  2,495 and 3,454 different variants have been detected in BRCA1 and BRCA2, respectively, where a relevant fraction of them has been classified as variants of uncertain significance (VUS). These pose a challenge in clinical genetics since mutation carriers could benefit from preventive and prophylactic measures as well as new targeted therapies such as the Poly-ADP Ribose Polymerase Inhibitors (Hereditary Breast and Ovarian Cancer (HBOC) represents 5–10% of all breast cancers. Nowadays, more than 25 HBOC susceptibility genes have been identified, most of them involved in DNA repair pathways . Deleter70 years . Apart f70 years , there hhibitors .1]. However, upstream gene expression mechanisms, such as splicing, can be disrupted by DNA changes. In fact, splicing is a critical highly regulated process involved in many cell functions whose disruption has been directly related with disease, being common in cancer  class 5n cancer . Likewisn cancer . This can cancer , as welln cancer . Thus, an cancer .BRCA2. Our previous results showed that more than a half of tested BRCA2 variants impaired splicing  guidelines .BRCA2 introns 15 and 16 and exon 16 variants were collected from the BIC database2 and the BRCA Share Database  (BRCA2 GenBank sequence NM000059.1 and the guidelines of the Human Genome Variation Society (HGVS3)./BRCA2/) . Variant4 (5)  , which i4 (5) .Table 1). They were checked by SANGER sequencing at the Macrogen Spain facility .MGBR2_ex14-20 was assembled as previously described . DNA var5 cells/well) and grown to 90% confluency in 0.5 mL of medium  in four-well plates . Transfections were made with 1 μg of minigene and 2 μL of low toxicity Lipofectamine  in GibcoTM Opti-MemTM . Cells were incubated with 300 μg/mL of cycloheximide  for 4 h to inhibit nonsense-mediated decay (NMD). RNA was purified with the Genematrix Universal RNA Purification Kit  with on-column DNAse I digestion to degrade genomic DNA that could interfere with RT-PCR.MCF-7 cells were plated  and the gene-specific primer RTPSPL3-RV (5′-TGAGGAGTGAATTGGTCGAA-3′). Samples were incubated at 42°C for 1 h, and reactions were inactivated at 70°C for 5 min. Then, 40 ng of cDNA was amplified in 50 μL reaction with pMAD_607FW  and RTBR2_ex17RV2 (5′-GGCTTAGGCATCTATTAGCA-3′) or with RT_ex15FW (5′-CGAATTAAGAAGAAACAAAGG-3′) and pSAD_RT_RV  using Platinum Taq DNA polymerase  . Samples were denatured at 94°C for 2 min, followed by 35 cycles consisting of 94°C for 30 s, Td-2°C for 30 s, and 72°C (1 min/kb), and a final extension step at 72°C for 5 min. Sequencing reactions were performed by the sequencing facility of Macrogen Spain. Semi-quantitative fluorescent 26 cycles PCRs were done in triplicate with primers pMAD_607FW-FAM and RTBR2_ex17RV2 using Platinum Taq DNA polymerase . FAM-labeled products were run with Genescan LIZ-1200 as size standard  at the Macrogen facility and analyzed with the Peak Scanner software V1.0. Only peaks with heights ≥50 relative fluorescence unit (RFU) were considered. Mean peak areas of each transcript of three runs were used to quantify the relative abundance of each transcript.Table 2).Seventy-two variants were collected from the BIC and UMD databases. Among them, 35 had been previously classified as VUS and 34 as pathogenic or likely pathogenic. In order to select possible spliceogenic variants, they were analyzed by the splicing prediction software NNSplice and HSF : six intronic , five missense , and one nonsense (c.7738C>T) variants. Four missense variants  and c.7805+3A>C had been previously classified as VUS. Intronic variants  and c.7805G > C disrupted the canonical ss, whereas variants c.7625C>G and c.7753G>A created new ss. DNA change c.7802A>G was selected because of its proximity to the canonical donor site and the presumable generation of an alternative “gt” donor site 4-nt upstream . Finally, bioinformatics results of c.7738C>T and c.7772A>G suggested the creation of one ESS (hnRNPA1) (Table 2).Twelve variants were selected (Table 2).The minigene MGBR2_ex14-20 had been already shown as a robust tool to assay possible spliceogenic variants contained in any of those exons and flanking introns . The wt Figure 1). However, the high resolution and sensitivity of fluorescent capillary electrophoresis allowed us to identify a total of eight variants, including the three previous ones, that disrupted splicing: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7802A>G, c.7805G>C, c.7805+1G>A, and c.7805+3A>C . Actually, this approach is able to detect rare transcripts with a relative abundance below 1% or can resolve transcripts that differ only in a few nucleotides . A total of at least 10 different aberrant transcripts were characterized by fragment analysis and sequencing: Δ16p44 , Δ16p55 , Δ16 (exon 16 skipping), Δ16q4 , Δ16q100 , ▾16q20 , minor , and uncharacterized transcripts of 893 and 954 nt . On the one hand, fragments analysis and sequences revealed that 3′ss disrupting variants (positions -2 and -1) provoked the use of a cryptic acceptor 44-nt downstream (Δ16p44) within exon 16 . Interestingly, this cryptic 3′ss was not recognized either by NNSplice or MaxtEnt. The loss of 44-nt at 5′ of exon 16 would suppose a frameshift deletion and a premature termination codon (PTC) (p.L2540Qfs∗11). On the other hand, 5′ss variants (positions +1 and +3) produced exon 16 skipping (Δ16), which means a frameshift deletion through the loss of 188-nt from r.7618 to r.7805 . Consequently, BRCA2 would be truncated with a PTC four codons downstream (p.L2540Gfs∗4). Last exon nucleotide variant (c.7805G>C) induced the same outcome (Δ16) highlighting the importance of this position conservation  in exon recognition , provoking r.7706_7805del (p.K2570Lfs∗45) . Finally, missense variant c.7802A>G created a new 5′ss, which resulted in ∼45% of the aberrant transcript Δ16q4 . The loss of four nucleotides would introduce a PTC into the protein (p.Y2601Wfs∗46). Thus, our results showed clearly how these eight variants disrupted splicing. Moreover, seven of them  generated more than ∼92% of frameshift transcripts.Selected variants were introduced into the minigene and functionally assayed in MCF-7 cells. Agarose electrophoresis clearly showed that three of them  induced aberrant splicing patterns . Interestingly, according to the International Agency for Research on Cancer (IARC6), this is a conserved region, since there is ∼22% of ultra-conserved aminoacids from human to sea urchin and ∼54% between mammals. Furthermore, this protein segment belongs to FANCD2- and DSS1-binding domains. Fanconi Anemia group D2 (FANCD2) protein binds to aminoacids from position p.2350 to p.2545 of BRCA2 and it has been suggested to have a role in the repair process (∗4), which would truncate the protein and subsequently loss the C-terminal region that would compromise BRCA2 function. process . DSS1 . Additionally, according to our data Δ16p44 is the main transcript (∼91%) that other authors also identified but described as a minor transcript in agarose gels : Δ16 as the main transcript (∼78%), followed by Δ16q100 (∼14%), and the lack of the full-length transcript. It is also worthy to mention that we detected other minor transcripts due to the high sensitivity of fluorescent capillary electrophoresis  that otherwise could not be easily detected on agarose gels. In any case, the spliceogenic effects of variants c.7618-1G>A and c.7805G>C were supported by our data.This study, based on minigene technology, provides detailed information about the impact on splicing of 12 CA2 gene . Among t16,17p69 . Here weose gels . These danscript . This ou4, so that its interpretation is more complex. The transcript Δ16q4, caused by the use of a new 5′ss, generated a frameshift deletion and the protein truncation by a PTC 46 codons downstream (p.Y2601Wfs∗46). However, it is still unclear if ∼54% of full-length transcript can preserve BRCA2 function, given that, for example, 20–30% of BRCA1 transcript is able to maintain BRCA1 activity . However, patients did not present the typical FA phenotype, which suggested that p.Y2601W BRCA2 maintained at least enough BRCA2 activity to prevent early childhood FA features . However, c.7625C>G only produced the full-length transcript without any splicing anomaly. The protein would even carry the missense variant p.T2542R. However, consistent with PolyPhen, this change might be considered as benign with a score of 0.0, which could be explained by the low conservation of the affected threonine. Anyway, further functional and association studies must be performed to interpret this variant. Other variant that resulted in a normal splicing pattern was the nonsense variant c.7738C>T (p.Q2580X), that a priori had been classified as pathogenic. In this case, the protein would be truncated at codon 2580 losing 839 aminoacids of the C-terminal where the DSS1-binding site, the DNA-binding domain, the RAD51C-binding site, and the cyclin-dependent kinase (CDK) phosphorylation site are located , PM2 , PP3 , and PP5 . On the other hand, variants c.7802A>G, c.7805G>C, and c.7805+3A>C were classified as likely pathogenic as they match criteria PS3, PM2, PP3, and PP5.According to the ACMG guidelines Table 4; , five va9, all variants, except for c.7802A>G, should be reclassified as class 4 (likely pathogenic) because they are “considered extremely likely to alter splicing based on position” and are “predicted bioinformatically to alter the use of the native donor/acceptor site.” Conversely, minigenes are not considered robust approaches to functionally test these variants yet . However, this specific minigene with BRCA2 exons 14–20 was confirmed as a robust tool since it reproduced patient RNA results from eight variants . Moreover, they account for 22% of causal variants of exon 16 and 11% of all recorded variants of this exon at the mutation databases. Taken together this and our previous studies, we have tested 283 BRCA1/2 variants under the splicing perspective, 154 of which induced anomalous patterns and 111 could be classified as pathogenic or likely pathogenic. These data remark the importance of variants of splicing regulatory sequences, which are often underestimated because most of them are placed in non-coding regions of the protein. Until now, genetic family-based studies have set up the impact of some variants on cancer risk. However, because of the exponential increment in the number of variants, their low frequencies and different nature, functional assays are strictly required. In this context, minigene technology constitutes a robust tool which can be used to functionally test spliceogenic candidate variants of any disease-gene without the interference of the counterpart wt allele. Certainly, pSAD-based minigenes represented valuable tools to functionally check variants of the SERPINA1  and CHD7 (Charge Syndrome) genes  genes . RNA assEF-B contributed to the bioinformatics analysis, minigene construction, manuscript writing, and performed most of the splicing functional assays. BD-G and AV-P participated in minigene construction, mutagenesis experiments, and functional assays. AA participated in minigene construction and functional mapping experiments. EV conceived the study and the experimental design, supervised all the experiments, and wrote the manuscript. All authors contributed to data interpretation, revisions of the manuscript, and approved the final version of the manuscript.The authors declare that theresearch was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Excess body burden of uric acid promotes gout. Diminished renal clearance of uric acid causes hyperuricemia in most patients with gout, and the renal urate transporter (URAT)1 is important for regulation of serum uric acid (sUA) levels. The URAT1 inhibitors probenecid and benzbromarone are used as gout therapies; however, their use is limited by drug–drug interactions and off-target toxicity, respectively. Here, we define the mechanism of action of lesinurad , a novel URAT1 inhibitor, recently approved in the USA and Europe for treatment of chronic gout.UA), lesinurad plasma levels, and urinary excretion of lesinurad were measured in healthy volunteers treated with lesinurad. In addition, lesinurad, probenecid, and benzbromarone were compared in vitro for effects on urate transporters and the organic anion transporters (OAT)1 and OAT3, changes in mitochondrial membrane potential, and human peroxisome proliferator-activated receptor gamma (PPARγ) activity.sUA levels, fractional excretion of uric acid (FEUA 3.6-fold (p < 0.001) and reduced sUA levels by 33 % (p < 0.001). At concentrations achieved in the clinic, lesinurad inhibited activity of URAT1 and OAT4 in vitro, did not inhibit GLUT9, and had no effect on ABCG2. Lesinurad also showed a low risk for mitochondrial toxicity and PPARγ induction compared to benzbromarone. Unlike probenecid, lesinurad did not inhibit OAT1 or OAT3 in the clinical setting.After 6 hours, a single 200-mg dose of lesinurad elevated FEThe pharmacodynamic effects and in vitro activity of lesinurad are consistent with inhibition of URAT1 and OAT4, major apical transporters for uric acid. Lesinurad also has a favorable selectivity and safety profile, consistent with an important role in sUA-lowering therapy for patients with gout.The online version of this article (doi:10.1186/s13075-016-1107-x) contains supplementary material, which is available to authorized users. Gout is a urate crystal deposition disease that results from a metabolic disorder, hyperuricemia (elevated serum uric acid (sUA)), and the consequent deposition of monosodium urate crystals in joints, soft tissues, and organs . Gout isSLC22A12) and GLUT9 (SLC2A9) play significant roles in renal reabsorption of urate, as patients with disruptive mutations in these transporters have high fractional excretion of uric acid (FEUA) and hypo-uricemia -genistein substrate at 2 nM was then added for an additional 120 minutes at 37 °C. For basolateral-to-apical (B → A) flux measurements, genistein was placed in the basolateral compartment only (donor well) and a sample was taken after the 120-minute incubation period from the apical compartment (receiver well). For apical-to-basolateral (A → B) flux measurements, genistein was placed in the apical compartment only (donor well) and a sample was taken after the 120-minute incubation period from the basolateral compartment (receiver well). Samples were then subjected to scintillation counting. The inhibition of BCRP/ABCG2 was then calculated by the net B → A flux method /[Em590/Em530 (vehicle only) – Em590/Em530 (100 μM menadione)].This assay was performed using nonhuman mammalian cells engineered to provide a PPARγ-responsive luciferase reporter gene and constitutive, high levels of PPARγ . Cells were incubated with compounds for 24 hours, and then luciferase activity was measured using a luminometer. Agonist activity was reported as fold activation over a vehicle control. A positive control agonist, rosiglitazone, was included in each batch run to verify cell integrity.From a phase 1 clinical trial, RDEA594-109, eight healthy male volunteers were administered single oral doses of 200, 400, or 600 mg lesinurad tablets (with food). In each treatment period, plasma samples were collected within 30 minutes before dosing (pre-dose) and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 30, 36, and 48 hours post-dose. Urine  was collected pretreatment at hours –24 to –18, –18 to –12, and –12 to 0, and post-dose at hours 0 to 6, 6 to 12, 12 to 24, 24 to 30, 30 to 36, and 36 to 48.max) and area under the curve (AUC) were derived using the software WinNonlin Professional, Version 5.2 (Pharsight Corporation). The parameters from individual profiles of lesinurad were determined using noncompartmental methods. Urine samples were analyzed for uric acid and creatinine using enzymatic methods by Covance Clinical Research Unit , and pharmacodynamic (PD) parameters were determined by Covance Clinical Research Unit using SAS, Version 8.2 (SAS Institute Inc). Calculated PD parameters for uric acid included urinary excretion, renal clearance, and FEUA. FEUA was calculated by dividing the uric acid clearance by the creatinine clearance. For analyzing the relationship between plasma lesinurad concentrations and FEUA, the weighted average plasma lesinurad concentration was calculated for plasma samples from each time period that corresponded to the associated urine samples.After protein precipitation of the samples, lesinurad levels in plasma and urine were measured at Ardea Biosciences by quantitative high performance liquid chromatography with tandem mass spectrometry. The lesinurad plasma PK parameters maximum plasma concentration . Lesinurad does not activate either OAT1 or ABCG2 (Fig. In addition to OAT1 and OAT3, the renal proximal tubule expresses a number of additional urate transporters that potentially participate in urate secretion Fig.  18, 54], 5418, 5max of lesinurad. Benzbromarone is a known PPARγ agonist, and although no long-term cardiovascular studies have been conducted with benzbromarone, activity on this nuclear receptor is associated with increased adverse cardiovascular events [Testing benzbromarone-related endpoints, we found that lesinurad and probenecid have no effect on mitochondrial activity, in contrast to benzbromarone, which severely disrupts mitochondrial integrity in human hepatocytes at and above 0.4 μM. Similarly, with respect to PPARγ activity - and in contrast with benzbromarone - lesinurad and probenecid demonstrated no effect at up to 100 μM, thus, likely having no clinical effect based on the Cr events .The action of lesinurad counteracts the inadequate excretion of uric acid commonly found in patients with gout. The potential to combine two mechanisms of treatment, inhibition of uric acid production with XOIs and inhibition of uric acid reabsorption with a selective uric acid reabsorption inhibitor, represents a powerful approach to the reduction of sUA. Lesinurad had significant URAT1 and OAT4 inhibitory activity, likely accounting for its PD effect, as well as a favorable selectivity profile in vitro compared with other drugs that inhibit uric acid reabsorption. The compound is a new treatment option for hyperuricemia associated with gout in combination with XOIs.


Pieris rapae (Lepidoptera:Pieridae) upon ingestion of plant material containing phenylalanine-derived glucosinolates as chemical defenses. As these glucosinolates were widespread within ancient Brassicales, the ability to detoxify cyanide may therefore have been essential for the host plant shift of Pierid species from Fabales to Brassicales species giving rise to the Pierinae subfamily. Previous research identified β-cyanoalanine and thiocyanate as products of cyanide detoxification in P. rapae larvae as well as three cDNAs encoding the β-cyanoalanine synthases PrBSAS1-PrBSAS3. Here, we analyzed a total of eight species of four lepidopteran families to test if their cyanide detoxification capacity correlates with their feeding specialization. We detected β-cyanoalanine synthase activity in gut protein extracts of all six species tested, which included Pierid species with glucosinolate-containing host plants, Pierids with other hosts, and other Lepidoptera with varying food specialization. Rhodanese activity was only scarcely detectable with the highest levels appearing in the two glucosinolate-feeding Pierids. We then amplified by polymerase chain reaction (PCR) 14 cDNAs encoding β-cyanoalanine synthases from seven species. Enzyme characterization and phylogenetic analysis indicated that lepidopterans are generally equipped with one PrBSAS2 homolog with high affinity for cyanide. A second β-cyanoalanine synthase which grouped with PrBSAS3 was restricted to Pierid species, while a third variant , was only present in members of the Pierinae subfamily. These results are in agreement with the hypothesis that the host shift to Brassicales was associated with the requirement for a specialized cyanide detoxification machinery.Cyanide is generated in larvae of the glucosinolate-specialist  Pieris rapae (Lepidoptera:Pieridae). Larvae of Pieris rapae possess a nitrile specifier protein (NSP) in their gut that redirects glucosinolate breakdown yielding simple nitriles instead of isothiocyanates [P. rapae larvae are excreted, aromatic nitriles are further metabolized [P. rapae larvae detoxify cyanide as β-cyanoalanine and thiocyanate indicating the involvement of β-cyanoalanine synthases and rhodaneses [The glucosinolate-myrosinase system serves as an activated chemical defense in plants of the order Brassicales. Upon disruption of the cells, e.g., by herbivores, the glucosinolates, which are amino-acid derived thioglucosides, are hydrolyzed by endogenous thioglucosidases known as myrosinases  Figure A. This ycyanates  , Tu-CAS [Three gut-expressed β-substituted alanine synthases (BSAS) with β-cyanoalanine synthase activity, PrBSAS1-PrBSAS3, have been cloned and characterized from is rapae . In phyl, Tu-CAS . The mit, Tu-CAS . As a li, Tu-CAS ,14. Thus, Tu-CAS .Phenylalanine-derived glucosinolates are among the evolutionarily oldest glucosinolates and were widespread in ancient Brassicales . As the Protein concentrations were determined using the BCA Protein Assay Kit  with bovine serum albumine as a standard. PCR primers were Invitrogen Custom DNA Oligos (Thermo Fisher Scientific). Unless otherwise stated, PCR was carried out in a total volume of 50 µL DreamTaq buffer supplemented with 0.2 mM of each dNTP, 0.2 mM of each primer, 1 µL cDNA, and 0.25 µL DreamTaq Polymerase (Thermo Fisher Scientific) using PCR thermocyclers PeqStar  and TProfessional Gradient  and the following temperature program: 95 °C for 5 min, 35 cycles of 95 °C for 45 s, appropriate annealing temperature for 1 min, and 72 °C for 1 min, final incubation at 72 °C for 10 min. Sequencing was done at Eurofins MWG Operon . β-Cyanoalanine was purchased from Sigma.P. rapae larvae were from a colony maintained on Brassica oleracea ssp. oleracea, cv. Rosella (Brassicaceae) [A. cardamines were collected in Braunschweig, Germany, and kept on Alliaria petiolata (Brassicaceae) until dissection. Larvae of A. crataegi were obtained from M. Wasilewski, Białystok, Poland, and kept on Sorbus aucuparia (Rosaceae). Larvae of G. rhamni were collected in Braunschweig, Germany, and kept on Frangula alnus (Rhamnaceae). Larvae of C. croceus were obtained from G. Gęca, Będzin, Poland, and kept on Lotus corniculatus and Medicago sativa (Fabaceae). Larvae of P. xylostella were obtained from D. G. Heckel, Max Planck Institute for Chemical Ecology, Jena, Germany, and kept on Brussels sprouts . Larvae of Z. filipendulae were obtained from M. Zagrobelny, University of Copenhagen, Denmark. Larvae of S. littoralis were obtained from V. Jeschke, Max Planck Institute for Chemical Ecology, Jena, Germany, and kept on an artificial bean diet [All larvae were kept in a controlled environment chamber at 25 °C and 60% relative humidity with a photoperiod of 16 h with their respective host plants grown in or collected near the Institute of Pharmaceutical Biology. icaceae) . Larvae ean diet ,21.P. xylostella larvae were extracted in toto. Larvae of the other species (late instar) were dissected into front part (“head”), gut tissue, gut content, and remaining parts (“integument”) in 50 mM Tris-HCl, pH 8.5, on ice. All tissues were rinsed with buffer and then ground in 1300 µL ice-cold 50 mM Tris-HCl, pH 8.5, using plastic pestles in 1.5 mL reaction tubes. After centrifugation at 20,000× g for 10 min, the supernatant was used as crude extract for enzyme assays.g, the absorbance of the supernatant was measured at 460 nm and used to determine the SCN–concentration based on calibration with 0–1500 µM KSCN in 100 mM Tris-HCl, pH 8.5.Crude protein extracts  were incubated with 250 µL 125 mM KCN and 250 µL 125 mM sodium thiosulfate  at 30 °C for 120 min ,23. The 3 in 1.2 M HCl were added. After incubation in the dark for 20 min and centrifugation at 22,000× g, the absorbance of the supernatant was determined at 650 nm against a blank  and used to determine the sulfide concentration based on calibration with 0–100 mM Na2S [g. The supernatant was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using Multiple Reaction Monitoring (MRM) as described previously [m/z 112.722 as the mother ion (Q1) and m/z 95.900 as the daughter ion (Q3) [Crude larval protein extract  or purified recombinant protein  was mixed with 250 µL 25 mM KCN and 250 µL 25 mM cysteine  . Assay m mM Na2S . To verieviously  with m/zKm and Vmax were determined as means ± standard error (SE) of n = 3 independent expression experiments.For determination of kinetic parameters, the protein amount was varied to ensure linearity within the incubation time . The protein amounts used for the kinetic characterization were 0.25 µg AcaBSAS1, 2.5 µg AcaBSAS2 and AcaBSAS3, 0.2 µg AcrBSAS1, 2 µg AcrBSAS2, 5 µg AcrBSAS3, 1 µg ZfBSAS2 and PxBSAS2, 5µg GrBSAS2, 3 µg CcBSAS2, 12 µg CcBSAS3, 2.5 µg SlBSAS2a and 0.4 µg SlBSAS2b  for 1 min, and 72 °C for 1 min, 20 cycles of 95 °C for 45 s, 47 °C for 1 min, and 72 °C for 1 min and a final incubation at 72 °C for 10 min). PCR products were cloned into pGEM-T Easy  according to the manufacturer’s instructions and sequenced. To obtain 3′-ends, cDNA obtained using anchor-(dT)18 primer . The ORF was amplified from P. xylostella cDNA using gene-specific primers designed based on the database entry  in a total volume of 25 µL. The PCR products were USER-cloned [To generate expression constructs for recombinant enzymes with an N-terminal Strep-tag, the ORFs were amplified by PCR with 1 µL cDNA, 0.4 µM of each primer P79–106, , and 0.5R-cloned  into pETR-cloned  and sequE. coli BL21 (DE3) pLysS (Thermo Fisher Scientific) was transformed with the expression constructs or the expression vector without insert as a control. A starter culture was used to inoculate 100 mL of TB medium with 100 mg/L ampicillin and 34 mg/L chloramphenicol to an OD600 of 0.1–0.15. This culture was incubated at 18 °C and 220 rpm until the OD600 was 0.4–0.5. Protein expression was then induced by the addition of 1 mM IPTG. After another 15 h incubation at 18 °C and 220 rpm, cells were harvested and resuspended in 1.5 mL 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA per 1 g of cells. After sonication and centrifugation, the supernatant was loaded onto Strep Tactin sepharose resin  for enzyme purification according to the manufacturer’s instructions. The elution fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the fractions with the highest amounts of purified recombinant proteins were pooled and used in enzyme assays to determine kinetics or frozen at −20 °C to be used later in enzyme assays for HPLC-MS (MRM) detection of β-cyanoalanine.Statistics on results of larval extract assays were done with OriginPro 8.0.63.988 SR6. Characterized BSAS were subjected to phylogenetic analysis together with all uncharacterized lepidopteran homologs identified through BLASTp searches against non-redundant protein sequences at NCBI using PrBSAS1 as query. Phylogenetic analysis was performed using MEGA7 after aligning the sequences with the MUSCLE algorithm ,30,31. TG. rhamni produced, on average, more sulfide than extracts of gut content, but this difference was not significant in most comparisons (A. cardamines integument extracts (0.12 µmol/mg), but at levels <0.1 µmol/mg in integument extracts of the other species . Incubations with head and integument extracts resulted in thiocyanate formation close to background levels with highest levels measured for samples from A. cardamines  and ORFs confirmed by independent amplification of the entire sequence in one PCR. Several cDNAs were obtained for most species and designated as putative BSAS cDNAs (BSAS cDNAs encode polypeptides of 322–335 amino acids with a molecular weight of 34.4–35.9 kDa (BSAS1-BSAS3) was based on amino acid sequence similarity to PrBSAS1-PrBSAS3 [In order to obtain cDNAs encoding β-cyanoalanine synthases of  primers . Using gis rapae  had beenome data  and usedAS cDNAs . The BSA35.9 kDa  and a pr-PrBSAS3   was observed in reaction mixtures with all enzymes tested, but not in reactions set up with heat-denatured enzyme or an equal volume of pooled elution fractions obtained from empty vector control . Typically, these enzymes had low turnover rates while the most efficient enzymes had Km values for cyanide in the low millimolar range. Taken together, this indicates that lepidopteran larvae may possess several β-cyanoalanine isoenzymes with different physiological functions.To determine enzyme kinetics, purified recombinant enzymes were subjected to β-cyanoalanine synthase assays with varying substrate concentrations, and sulfide formation was determined in a colorimetric assay. GrBSAS3 could not be obtained in sufficient amounts for kinetic characterization. For all other enzymes, substrate dependence of activity followed Michaelis-Menten-kinetics . Km valu species . Km valu species . For allS. littoralis belonged to the PrBSAS2-group. Uncharacterized homologs from Lepidoptera such as the the three homologs from Heliconius melpomene  also grouped together with members of the PrBSAS2-group, but not with PrBSAS1 or PrBSAS3  have been converted to unstable α-hydroxynitriles, presumably by cytochrome P450 enzymes of the endoplasmic reticulum [The present work was undertaken to test if glucosinolate-feeding Pierinae are equipped with specialized enzymes to detoxify cyanide released upon metabolism of glucosinolates inside the larval digestive system. As expected based on feeding experiments with  cyanide , we dete cyanide . As the  cyanide . The mos cyanide . Tissue  cyanide  could beiewed in ,35,36) aeticulum . Immediaeticulum .BSAS genes in mite and lepidopteran genomes which are likely to account for β-cyanoalanine synthase activity in the gut [T. urticae and PrBSAS1-PrBSAS3 are located within bacterial sequences in phylogenetic trees, but the corresponding genes show signs of amelioration, i.e., loss of bacterial in favour of arthropod signatures [Tu-CAS has been demonstrated to be part of the mite genome [Besides rhodaneses, β-cyanoalanine synthases are likely to be involved in cyanide detoxification in herbivores, and the first arthropod β-cyanoalanine synthases have recently been identified and characterized ,14. In a the gut ,14. In sgnatures ,14. MoreH. melpomene with three BSAS2 homologs encoded in the genome feed exclusively on cyanogenic plants, namely Passiflora species, and are able to sequester and de novo synthesize cyanogenic glycosides [In the present study, we demonstrate that each of the seven analyzed lepidopteran species has one β-cyanoalanine synthase with high affinity for cyanide supporting a role in cyanide detoxification. The enzymes with high affinity for cyanide all belong to the PrBSAS2 group which also includes uncharacterized lepidopteran homologs identified in databases. This suggests that β-cyanoalanine synthases of the PrBSAS2 group provide lepidopterans with a basic protection against cyanide released upon ingestion of cyanogenic plant material or hydrolysis of stored cyanogens. Some lepidopterans possess several homologs within the PrBSAS2 group, maybe due to a high frequency of encountering cyanide-producing food plants. Notably, Heliconius species such as ycosides ,40. As aycosides .P. rapae) and AcaBSAS2 (from A. cardamines), have different properties. PrBSAS2 has an about tenfold lower affinity to cyanide than the other characterized group members including AcaBSAS2  [A. cardamines and rarely encounter plant-produced cyanide. Their interaction with A. petiolata as a host plant depends on species, habitat, and plant chemistry [P. rapae larvae can complete development on A. petiolata [A. petiolata for oviposition [A. petiolata as a host plant for Pieris species is associated with plant cyanide release and insect cyanide detoxification capabilities. While the present study focused on constitutively expressed detoxification enzymes, future studies should test if some of the identified enzymes or additional enzymes are induced upon cyanide exposure.Among the members of the PrBSAS2 (Lepidoptera) group, the two characterized enzymes from glucosinolate-feeding Pierinae, PrBSAS2 (from AcaBSAS2  putting icaceae) , which picaceae) . Thus, Ahemistry ,44,45. Petiolata , but aduposition . Future P. rapae, PrBSAS3 , with deviating kinetic properties. Among these, PrBSAS1 homologs are restricted to glucosinolate-feeding Pierinae and have low cyanide affinity, but may contribute to cyanide detoxification based on their high turnover numbers. Taken together, the results of our study support the hypothesis that Pierinae have been under selection pressure to maintain a broad repertoire of cyanide detoxification enzymes. Whether these enzymes have evolved to also fulfil additional functions remains an interesting subject for future investigations.


Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55β-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55β, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55β in  However, compared to Ser/Thr kinases that are known to play critical roles in numerous cellular processes, Ser/Thr phosphatases are relatively less studied. Ser/Thr phosphatases have been classified into several groups, among which the type 1 (PP1) and type 2A (PP2A) are most abundant2. The catalytic subunits of PP1 and PP2A complex with an array of regulatory targeting subunits, which dictate the substrate recognition of the phosphatase holoenzymes. PP2A exists in the cell predominantly as a heterotrimer composed of a catalytic subunit (C), a scaffold subunit (A) and a targeting subunit (B)3. The A and C subunits of PP2A each contain two possible variants, whereas the B subunits are encoded by at least 15 genes in mammalian cell. The B subunits are highly diverse in structure, and often classified into 4 groups, including B55/PR55, B56/PR61, PR48/PR72/PR130, and PR93/PR110/striatin3.Protein phosphorylation, a major form of post-translational modification, plays a crucial role in regulation of protein functions. The vast majority of protein phosphorylation occurs on specific serine and threonine residues that are oppositely regulated by protein kinases and phosphatases4. The yeast homolog of B55, Cdc55, was shown to regulate cell cycle progression, particularly cell division6. A conserved role of B55 in mitotic regulation was also implicated in vertebrates10. Moreover, emerging evidence linked B55 to regulation of cell signaling, cytoskeleton, and Golgi dynamics12. The B55 subunits are of interests to human cancer, as several genomic studies suggested these subunits as potential tumor suppressors. For example, one study showed depletion of the B55α gene was related to 67% of prostate cancer cases13. Moreover, a large-scale genomic and transcriptomic analysis of 2,000 breast tumors identified B55α as one of the most commonly silenced genes; and the subgroup of breast cancer patients with loss of B55α suffered from poor treatment outcome and survival14. Other studies associated B55α to childhood teratoma15, prostate cancer16, colorectal cancer17, lung cancer18, and leukemia19. Gene deletion of B55β was less implicated in cancer, but epigenetic suppression of B55β expression was functionally characterized in breast and colon cancer21.The B55 group of PP2A regulatory subunits comprises 4 different isoforms  that share high levels of sequence similarity, but may exhibit distinct patterns of expression and subcellular localizationAs a major group of the PP2A targeting subunits, it is expected that B55 directs PP2A to a large number of substrates. However, to date only a few phosphoproteins were defined as direct substrates of PP2A/B55. To fill in this large gap in knowledge, in the current study we characterize the interactomes of B55α and B55β that each contains over 100 proteins. Among these proteins were factors involved in cell division, DNA repair and replication, components of actin, microtubule, Golgi and nucleopore, and regulators of cellular signaling, metabolism and mitochondria function. A fraction of these proteins were previously known as substrates or interactors of B55, but the majority of them are new discoveries of this study.24. Under conditions of replication stress, the RPA2 subunit of RPA is hyperphosphorylated by CDK2, ATM/ATR, DNA-PK and other kinases. In turn, RPA phosphorylation facilitates the stabilization, repair, and recovery of stalled replication forks27. Not surprisingly, RPA and its phosphorylation have been implicated as a valuable marker for cancer progression and drug target for cancer therapy30. Here we show that B55α associates with RPA, and the association is reduced upon replication stress, presumably as a mechanism to allow phosphorylation of RPA2. Consistently, ectopic expression of B55α suppressed RPA phosphorylation, and attenuated checkpoint signaling after replication stress.Interestingly, our proteomic analysis suggested a role of B55 in regulation of multiple DNA replication proteins, particularly replication protein A (RPA). RPA is a crucial single strand DNA-binding protein complex that orchestrates DNA replication and repairXenopus egg extract, a well-established cell-free model system in which protein interaction networks and cellular activities are well preserved. For example, the extracts can biochemically undergo multiple rounds of cell cycle progression. In addition, numerous studies illustrated the value of the extracts in recapitulating DNA replication, repair, microtubule assembly, signaling transduction, and apoptosis35.We sought to reveal the function of B55 in Xenopus egg extracts, affinity purified the protein, and analyzed the eluted protein complexes by mass spectrometry. As expected, a large number of peptides from B55α and B55β themselves were recovered, along with numerous co-purified proteins. The raw results were processed to eliminate non-specific binding proteins, as determined by a parallel pull-down experiment using control beads. Moreover, proteins identified by one single peptide were removed to improve stringency. In turn, 176 and 135 proteins were identified as high confidence candidates of the associated proteins of B55α and B55β, respective 39, DNA replication and repair (such as Mcm and Ruvbl)41, RAS and related small GTPase signaling42, eukaryotic initiation factor 4F (eIF4F) complex43, Golgi and trafficking 45.We incubated the recombinant B55α or B55β protein in Our results showed that, like B55α, B55β associated with a large number of proteins related to cellular metabolism, mitochondria functions, molecular trafficking, cytoskeleton, cell cycle regulation, Golgi regulation, G-protein signaling and nucleic acid metabolism Fig. . The topXenopus.The study identified a number of proteins that are known to bind B55, thus providing a validation for the results. Moreover, as we identified a large number of new proteins associated with B55α and B55β, we sought to confirm some of these candidates by immunoblotting. Importantly, although PP2A was not among the B55-associated proteins identified by mass spectrometry, we confirmed by immunoblotting that both B55α and B55β co-precipitated the catalytic subunit of PP2A Fig. . In addiXenopus egg extracts, PP2A/B55δ was characterized as a phosphatase that dephosphorylates mitotic substrates of CDK17. Thus, other B55 subunits, including B55α and B55β, may also act on substrates of CDKs. In fact, a RNAi-based screen in human cells revealed B55α as a regulator of mitotic exit10. Here we show that upregulation of B55α in metaphase-arrested Xenopus egg extracts accelerated M-phase exit  complex. Thus, B55 may play a role in regulation of these DNA replication factors. As many of these replication factors are substrates of CDK2, we speculate that, like in mitotic regulation, B55 may function in DNA replication by dephosphorylating substrates of CDK2. To this end, many known substrates of CDK2, including replication factors, chromatin and ribosome regulators, and cell signaling proteins, are B55α and B55β-associated Fig. .54. It has been shown that phosphorylation of RPA is largely dispensable for unperturbed DNA replication, but plays a pivotal role in the cellular response to replication stress and DNA damage56. Upon replication stress, RPA2 phosphorylation by CDK2 and other kinases facilitates the stabilization, repair, and recovery of stalled replication forks27. We confirmed the RPA and B55 association in human cells at the endogenous level by reciprocal co-immunoprecipitation .Human cervix carcinoma (HeLa) cells, authenticated by ATCC, were maintained in Dulbecco’s modified Eagle medium  with 10% fetal bovine serum . The HA-B55α expression vector was a gift from Dr. Xuan Liu 62. Antibodies used in immunoblotting include: Chk1 phospho-S317, Mcm2, Ube3a, Incenp, Smc2 antibodies from Bethyl Laboratories ; B55α antibody from Abcam ; β-actin, phospho-Cdk substrate and Plk1 antibodies from Cell Signaling Technology ; Mastl antibody from Millipore , and phospho-RPA S4/8 and S33 antibodies as previously characterized57. Xenopus Cdc25 antibody was a gift from Drs. Kumagai and Dunphy . For immunoprecipitation, anti-mouse or anti-rabbit magnetic beads (New England Biolabs) were conjugated to primary antibodies, and then incubated in cell lysates for 1 h. The beads were collected using a magnet, washed, eluted with Laemmli sample buffer, and analyzed by immunoblotting.Immunoblotting was performed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as previously described63. Briefly, cells were fixed in 3% formaldehyde with 0.1% Triton X-100, washed, and blocked in 10% goat serum in PBS. The primary antibody to RPA2 phospho-S4/8 was diluted in the blocking buffer, and incubated with the cells for 2 h. The cells were then incubated with the Alexa Fluor 555 secondary antibody  for 1 h. Imaging was performed using a Zeiss Axiovert 200M inverted fluorescence microscope at the UNMC Advanced Microscopy Core Facility.Immunofluorescence was performed as previous describedXenopus oocyte cDNA library, and then inserted into pMBP-parallel vector with an N-terminal MBP-tag. The recombinant proteins were expressed in BL21 bacterial cells and purified on amylose beads. For the pull-down assay, approximately 10 μg MBP-B55 proteins conjugated on amylose beads (20 μl) were incubated in interphase Xenopus egg extracts (40 μl). After 30 min incubation at room temperature, the beads were re-isolated, washed, eluted and then resolved by SDS-PAGE for immunoblotting or mass spectrometry . The control pull-down was performed using the same volume of amylose beads that were not conjugated with proteins.B55α and B55β were cloned from a Xenopus egg extracts were prepared as previously described64. For the metaphase-arrested cytostatic factor (CSF) extracts, Eggs were treated with 2% cysteine, washed, and then crushed by centrifugation at 10,000 g. The cytoplasmic layer was collected for further experiments. For the interphase, cycling extracts, eggs were treated with 2% cysteine, and then incubated with Ca2+ ionophore. The eggs were then washed, and crushed by centrifugation at 10,000 g. The cytoplasmic layer was collected and supplemented with energy mix .All data generated or analyzed during this study are included in this published article and its .Supplementary Information