Protein phosphatases 1 and 2A (PP1 and PP2A) are the most ubiquitous and abundant serine/threonine phosphatases in eukaryotic cells. They play fundamental roles in the regulation of various cellular functions. This review focuses on recent advances in the functional studies of these enzymes in the field of smooth muscle physiology. Many naturally occurring protein phosphatase inhibitors with different relative PP1/PP2A affinities have been discovered and are widely used as powerful research tools. Current topics in the chemical biology of PP1/PP2A inhibitors are introduced and discussed, highlighting the identification of the gene cluster responsible for the biosynthesis of calyculin A in a symbiont microorganism of a marine sponge. Protein phosphatases 1 (PP1) and 2A (PP2A) are two of the principal catalytic subunits that dephosphorylate proteins on serine and threonine residues in the cytosol of eukaryotic cells. They are encoded by genes belonging to the PPP family and are abundantly expressed in all cell types. These enzymes achieve the substrate specificity and activity regulation required for their multiple functions through combinatorial interactions with many regulatory subunits i [2+]i has thus been a research focus for understanding the excitation-contraction coupling in each smooth muscle type.Reversible phosphorylation of the 20-kDa myosin regulatory light chain (MLC) governs cell motility, including smooth muscle force development, which is regulated through multiple pathways specific to each cell type. In smooth muscles, the Ca [Ca2+]i , 7 -potentiated inhibitory protein of 17 kDa (CPI-17), a product of the PPP1R14A gene that functions as an endogenous MLCP regulator Fig.  48–50].–50.48–50h muscle , 57. Forh muscle  and asthh muscle , respecth muscle . These lh muscle .MLCP is a multifunctional regulator of various cells, yet our knowledge is incomplete . Cell-peThe physiological parameters of the cardiovascular system, such as blood pressure and heart rate, as well as the occurrence of cardiovascular events, such as ischemic heart attack and myocardial infraction, show diurnal rhythms , 70. The2+-independent manner or indirectly inactivating MLCP activity via MYPT1 phosphorylation. Since the resting level of MLC phosphorylation exhibited no circadian rhythm and ROCK2 has a higher substrate preference for MYPT1 to MLC, the oscillation of the MLCP activity plays a major role in the ROCK2-mediated generation of the circadian rhythm of MLC phosphorylation.Recently, Saito et al.  identifistaggerer mice i. A slight reduction of [Ca2+]i was observed on application of okadaic acid in some types of smooth muscle [2+]i was reduced by only 10–20% even when the contraction was almost completely inhibited by okadaic acid [In most cases reported, the relaxant effect of okadaic acid was not accompanied by a marked reduction of the [Cah muscle , 105. Hoaic acid , 105. Thaic acid  and guinaic acid , MLC remaic acid . FurtherSpecific inhibitors against PP1 and 2A have been discovered from natural product origins, including cantharidin  from bliDiscodermia calyx [Ki values for the interaction of calyculin A with purified native PP1 and PP2A are reported to be 1 and 0.1 nM, respectively [Ki = 15 nM [Calyculin A Fig.  was origia calyx . Ishiharia calyx  showed tectively . Structuectively . This waectively . In cont = 15 nM . It is sIn the case of calyculin biosynthesis, the biosynthetic gene cluster and the producing organism had remained unclear, in contrast to the PP inhibitors derived from microorganisms. This is because marine sponges harbor diverse symbionts, and thus there was speculation that the sponge-derived bioactive molecules are actually biosynthesized by symbiotic microorganisms. To obtain clues about the gene clusters in this complex consortium, the sponge metagenome is a suitable starting point. A PCR-based screening approach was performed on the sponge metagenomic DNA, with degenerate primers targeting the ketosynthase domain of the type I PKS. With a promising amplicon sequence in hand, the fosmid bearing the corresponding sequence was screened from the sponge metagenomic fosmid library. Finally, the PKS-NRPS hybrid gene cluster, ranging over 150 kb, was successfully obtained. The PKS-NRPS modular assembly line is in good agreement with the stepwise biosynthetic pathway of calyculin A .Candidatus Entotheonella’ sp., with a filamentous morphology, is the authentic producer of calyculin A [Theonella, which is closely related to Discodermia. Recently, ‘Candidatus Entotheonella factor’ was identified as the symbiotic producer of almost all bioactive molecules derived from Theonella swinhoei, collected in the waters surrounding Hachijo Island, Japan [T. swinhoei contains another phosphatase inhibitor, motuporin [Using the calyculin biosynthetic gene, the identification of the producer organism was performed. A single cell analysis employing laser microdissection in conjunction with a PCR analysis revealed that the ‘yculin A . The spod, Japan . On the otuporin . Even thotuporin .D. calyx, but it became apparent that the phosphorylation proceeded on the phosphate group to generate a pyrophosphate functionality by the structural elucidation of phosphocalyculin A  as low as 1 nM. Konoki et al. [2+-binding photoprotein from Aequorea aequorea, despite the low sequence homology. Apoaequorin harbors coelenterazine to form active aequorin, which readily emits fluorescence in response to Ca2+. Moreover, OABP2 shares conformational similarity with other marine invertebrate proteins, such as the photoprotein berovin, the nonluminescent coelenterazine-binding protein (CBP) and the GTPase calexcitin, which all bind to small molecules. These findings are indicative of certain ecological functions of these proteins with a common overall fold.Extensive studies have been also conducted on okadaic acid Fig. , which wa okadai . Okadaica okadai  and has calensis , 141. Itaic acid . Okadaicaic acid , 79, 81 aic acid . The affi et al.  successfi et al. , 145. Sii et al. . Remarkai et al. , a Ca2+-2+ sensitization of smooth muscles. According to the current theory, these threonine residues are phosphorylated by RhoA/ROCK activated upon GPCR stimulation; phosphorylation of Thr696 is believed to inhibit MLCP activity to MLC, whereas that of Thr853 is thought to cause dissociation of MLCP from myosin and/or direct inhibition of MLCP activity. An increasing number of experimental observations appear to argue against this theory. However, it should be noted that our present knowledge and opinions totally rely upon the assumed specificity of antibodies. To gain a clearer view, the phosphorylation states of MYPT1 must be carefully re-evaluated using better means of quantification. Another critical factor regulating the MLCP activity is CPI-17, whose phosphorylation, expression and localization synergistically contribute to defining smooth muscle characteristics. CPI-17 may prove to be a very useful research tool as a specific MLCP inhibitor if it can be redesigned by biomolecular engineering for stabilizing the inhibitory potency induced by Thr38 phosphorylation.The two phosphorylation sites, Thr696 and Thr853 (human sequence numbering), of MYPT1 have drawn the most attention in the context of GPCR agonist-induced Castaggerer mutant mice consistently suggest that RORα plays an essential role in the vascular intrinsic clock mechanism that generates the circadian rhythm of vascular contractility. A scheme (Fig. The findings from the experiments with cultured smooth muscle cells and eme Fig.  has beenSince the initial reports in 1989, the relaxant effect of okadaic acid on contractions of intact preparations has been shown in various smooth muscles. The recent findings obtained by the experiments using the highly specific PP2A inhibitors have led to the conclusion that, although there may be some tissue-specific factors in the responses of different smooth muscles, the relaxant effect is attributable to inhibition of PP2A. An important implication is that PP2A, as well as PP1, is somehow involved in the regulation and/or maintenance of the contractility of smooth muscles. Several pieces of evidence suggest that the site of action of PP2A is some rather downstream step of smooth muscle contraction such as cross-bridge cycling. Further studies are expected to elucidate the details of the function of PP2A in smooth muscles.Although the overall picture of the biosynthetic mechanisms of sponge-derived protein phosphatase inhibitors is still largely unclear, recent comprehensive studies have illuminated some intriguing pieces of the complicated processes, such as producer microorganisms, binding proteins and dynamic bioconversions. It is particularly remarkable that the activity of the phosphatase inhibitor calyculin A is controlled by enzymatic phosphorylation and dephosphorylation of itself in the sponge-microbe association.


The percentage of screws requiring repositioning due to drilling error was 85.7% (42/49). Comparisons of preoperative and postoperative function showed significantly improved accuracy. This study showed that inaccurate pedicle screw placement mainly results from errors in preoperative planning, mounting, registration, drilling, and robot assembly. Pedicle screw placement using a bone-mounted miniature robot system requires meticulous preoperative planning to minimize these errors.This article describes factors affecting the accuracy of transpedicle screw placements performed with the Renaissance robot-guided system and reviews the relevant literature. Between January 2013 and January 2015, Renaissance robot-guided spinal surgery was performed in 125 patients at Kaohsiung Medical University Hospital in Kaohsiung, Taiwan. The surgeries included 662 transpedicle screw implants and 49 Kirschner wire (K-wire) reimplants performed by intraoperative repositioning. The lead author evaluated the accuracy of all K-wire insertions and classified their accuracy into 3 categories relative to the preoperative plan for transpedicle screw placement. For cases in which screws required repositioning after the registration step, factors affecting pedicle screw placement were determined according to the consensus of 3 experienced spinal surgeons. According to the scheme developed by Kuo  Nonguided methods of transpedicle screw implantation have a high risk of inaccurate screw positioning, which can cause severe neurovascular complications. Therefore, various guided methods of transpedicle screw implantation have been developed, including fluoroscope-guided placement, navigation-guided placement,,5 and Renaissance robot-guided placement.,7 The accuracy of the Renaissance robot-guided system is considered satisfactory,9 but can still be improved by optimizing certain factors.Transpedicle screw placement, which was first described by Roy-Camille et al,This article describes factors affecting the accuracy of transpedicle screw placements performed with the Renaissance robot-guided system and reviews the relevant literature.22.1We retrospectively analyzed patients who had received robot-assisted transpedicular screw fixation to correct degenerative lumbar spondylosis or spondylolisthesis at Kaohsiung Medical University Hospital between January 2013 and January 2015. Indications for robot-assisted surgery were failure of conservative treatment, ongoing neurological deficit, intractable back pain, and progression of deformity. Inclusion criteria were diagnosis of degenerative lumbar disease, age >20 years at the time of diagnosis, a condition refractory to conservative treatment for at least 6 months, correction by robot-assisted pedicle screw placement, and follow-up >12 months. Patients were excluded if they had any history of spinal trauma, spinal infection, spinal malignancy, or adult degenerative scoliosis. Out of (number) patients who met the inclusion criteria, (number) were excluded. Ten patients were lost to follow up. Therefore, the final analysis included 125 patients.2.2This clinical study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-E(I)-20150167). Written informed consent was obtained from all participants.2.3 and incidental discovery of a slipped K-wire during surgery.The 125 Renaissance robot-guided spinal surgery procedures performed at Kaohsiung Medical University Hospital during the study period included 662 transpedicle screw implants and 49 Kirschner wire (K-wire) reimplants performed through intraoperative repositioning . The Renaissance robot-guided system is described in detail in Lieberman et al,Preoperative planning: Computed tomography (CT) scanning of the spine was performed preoperatively to reconstruct the 3D plane of the spine and to select the best screw placement strategy. Mounting: The mounting system was attached to an appropriate bony structure of the spine to maintain stability during registration. The robotic arm of the guided system was also fixed to the bony structure to provide intraoperative guidance. Registration: The Renaissance robot-guided system automatically registered the intraoperative images with the preoperative CT images by comparing 2 radiographic images (anteroposterior and oblique views). Drilling: The robot was attached to a mounting frame and moved to the position selected in preoperative planning. After directing the guiding tube at the pedicle, the surgeon began drilling. The robot was then used to insert the K-wire.The Renaissance robot-guided system was used to perform transpedicle screw placements in 4 main steps. 2.6 classification scheme.The lead author classified the accuracy of all K-wire insertions relative to the transpedicle screw trajectory selected in preoperative planning as Types I–III in accordance with the Kuo et al2.7In patients who required pedicle screw repositioning after the registration step, the causes of inaccurate K-wire placement were determined according to the consensus of 3 experienced spinal surgeons. Causes of inaccurate K-wire placement were classified into 5 categories of errors: preoperative planning, mounting, registration, drilling, and robot assembly. Preoperative planning error was defined as an entry point at the slope of the bony structure, an insufficiently wide entry angle, or a preoperative in–out–in trajectory. Mounting error was defined as excessive movement of the mount relative to the vertebrae. Registration error was defined as a registration error exceeding 1 mm. Drilling error was defined as relative movement of vertebrae under excessive drilling pressure due to loss of cannula traction in soft tissue. [Robot assembly error was defined as drilling not performed according to standard procedures. Confusing.]2.82.8.1,14Pedicle screw placement accuracy was classified into 4 categories according to postoperative biplanar fluoroscopy results.2.8.2Preoperative and postoperative Oswestry Disability Index (ODI) and visual analog scale (VAS) were used to evaluate functional outcomes.2.8.2.1t tests were used to compare function before and after surgery performed with the bone-mounted miniature robot system. McNemar test was used to compare accuracy before and after repositioning and before and after secondary registration. The analyses were performed with the SPSS 19.0 software package . A P value less than 0.05 was considered statistically significant.Paired 33.1The actual trajectories of K-wires inserted through re-registration were compared with the planned trajectories Table . The res3.2The accuracy of K-wire placement was decreased by errors in preoperative planning, mounting, registration, drilling, and robot assembly. The percentages of screws requiring repositioning were, from highest to lowest, 85.7% for drilling error, 57% for preoperative planning error, 17% for mounting error, 17% for registration error, and 2% for robot assembly error. That is, the most common cause of inaccurate K-wire placement was drilling error; drilling errors were attributable to robot assembly errors Table , Fig. 2.3.3The accuracies of transpedicle screw insertions were as follows: 98.5% in Grade I; 1% in Grade II; 0.5% in Grade III; and 0% in Grade IV.3.4The preoperative and postoperative ODIs were 27.9 ± 6.1 and 13.1 ± 5.2, respectively, and the corresponding VAS scores were 7.2 ± 1.4 and 2.2 ± 1.2, respectively  of the repositioned screws were associated with preoperative planning error . Depending on the operating method and various mounting methods can be employed. A Hover-T or multiuse clamp can be used initially for a long segment, and a bed-mount can be used for a short level. A clamp can then be used for a conventional median incision. To minimize movement of the mounting device relative to the spinal column, our strategy was to select the best mounting method for the individual patient and then enhance fixation during surgery.Deviation of screws from the planned trajectory can also occur when the mounting is not sufficiently stable. Roser et al4.3 reported that transpedicle screws cannot be inserted accurately if registration is inaccurate. Roser et al further reported that registration errors can result from inferior bone quality and from artifacts such as pacemakers or sternal wiring. In our series, the percentage of repositioned screws associated with registration errors was 17% (6/35). Hu et al further reported that registration errors can result from severe deformity, high body mass index, extremely low bone quality, and loose (previously implanted) hardware. Therefore, registration must be performed with extreme care in patients who have received prior surgery and in patients with obesity or osteoporosis.Potential causes of registration error include osteoporosis, various properties of the vertebral column, reoperation using implants, and destruction of bone in previous surgery. Pechlivanis et al4.4soft tissue pressure, bony surface skiving, and incorrect drilling pressure . The drawbridge effect can be avoided by minimizing drilling pressure through the use of a high-speed drill with a sharp bit and the appropriate mounting system.Insufficient drilling pressure can result from using a blunt drill bit, an excessively low drilling speed, or an inappropriate mounting. Pressure may also be insufficient when the drilling site is a hard bony structure Fig. C. In con4.8Human error can lead to unfavorable results. The proportion of intraoperatively repositioned K-wires was 2/35 (5.7%). The improper positioning was attributable to deviations caused by inappropriate robot assembly procedures. Two patients had no problems during the operation; however, a records review showed that the K-wire positions were approximately 1 level higher than the target position. This error resulted from use of the wrong station during robot assembly procedures. Errors in the assembly procedures for the robot-guided system can cause catastrophic neurological damage and massive bleeding.5 also proved to be a feasible method for evaluating K-wire placement accuracy and for using an intraoperative robot grading system to predict postoperative accuracy of pedicle screw placements.The gold standard for evaluating pedicle screw placement accuracy is CT. However, this study evaluated placement accuracy by using an intraoperative robotic classification system. However, postoperative CTFactors that affected the accuracy of transpedicle screw placement were categorized as either robotic or nonrobotic factors. Factors that negatively affected robot-assisted pedicle screw placement were identified according to the consensus of 3 experienced spinal surgeons and divided into 5 categories of errors: preoperative planning, mounting, registration, drilling, and robot assembly. However, this study did not consider nonrobotic factors such as osteoporosis, obesity, and prior spinal surgery. Thus, further studies are needed to evaluate the roles of these factors.Another potential limitation is that, to exclude intraobserver bias, the accuracy of pedicle screw placement using a bone-mounted miniature robot system was only analyzed in robotic spinal surgery procedures performed by a single surgeon. However, differences in surgical skills, experience, and technologies may increase or decrease certain errors. That is specific error types may be associated with specific levels of skill and experience. The factors affecting pedicle screw placement accuracy were only discussed in relation to a single surgeon in this study, which raises the issue of interobserver bias. Further studies are needed to compare factors that affect the accuracy of pedicle screw placements performed by different surgeons (intraobserver bias).6In summary, this study of factors that affect pedicle screw placement accuracy showed that inaccurate pedicle screw placement can result from errors in preoperative planning, mounting, registration, execution, and robot assembly. Pedicle screw placement using a bone-mounted miniature robot system must be performed with extreme care to avoid these errors.


Omalizumab is a human IgG1 antibody against IgE used as a therapy for sever asthmatic patients with asthma. According to the guidelines of the Global Initiative for Asthma, omalizumab is an add-on drug at treatment step 5 that is used for severe asthma patients who are allergic to perennial allergens. The effects of omalizumab for severe asthma therapy have been validated in multiple clinical studies. However, the long-term effects of omalizumab on IgE production and possibility of resetting of administration dose of omalizumab remain unknown.The serum total and free IgE levels were measured over time in a 63-year-old female patient with allergic asthma who was administered 375 mg omalizumab biweekly for 36 months. Her symptoms did not worsen and clinical course remained favorable after reducing the dose to 375 mg per month. The serum free IgE levels temporarily increased following a dose reduction of omalizumab. The serum free IgE trough level temporarily increased at 4 weeks after capable to reduce the dosage; however, thereafter, the serum free IgE level decreased to desired levels (below 30 ng/mL).The present case shows the possibility of reducing the dose following the long-term use of omalizumab. Considering the high medical cost of omalizumab, the dose reduction may be a viable option. It may be useful to measure the serum free IgE level to appropriately identify patients in whom the dose can be reduced, and to carefully monitor the clinical course. Omalizumab is a human IgG1 antibody against IgE used to treat patients with severe allergic asthma with symptoms that are poorly controlled with high doses of inhaled steroids. Numerous clinical studies have demonstrated the effectiveness of omalizumab therapy on intractable atopic asthma, and this has become one of the few available treatment modalities. The goal of omalizumab therapy is to achieve free IgE (unbound IgE that can bind to IgE receptors) of ≤25 ng/mL [To overcome this issue, we previously developed ELISA with solid-phase soluble FcεRI to allow for the measurement of serum free IgE levels in patients undergoing omalizumab treatment . Here weDermatophagoides pteronyssinus, Chironomid midges, and negative for Aspergillus fumigatus, Candida albicans, Penicillium notatum and other common allergens. Despite the treatment with the combination of budesonide 1280 μg and formoterol 36 μg per day, a leukotriene receptor antagonist, and low-dose theophylline, she experienced exacerbations several times. The asthma control test (ACT) score was 18, and %FVC 86.7%, FEV1.0 0.88 mL, %FEV1 46.1% at 2 month before omalizumab therapy which initiated the year at in September 2013. Table A 63-year-old female patient developed bronchial asthma at approximately 5 years of age. Her asthma did not spontaneously resolve and she subsequently suffered from regular asthma attacks. Specific IgE testing was positive for house dust, At present, free IgE and IgE-omalizumab complexes cannot be distinguished using current clinical methods for measuring serum total IgE levels, like ImmunoCAP ; thus, serum total IgE levels appear to increase following the administration of omalizumab , 4. ThisLowe et al.  have useWhile the mechanism underlying the decrease in the serum total IgE level due to the continuation of omalizumab therapy remains unknown, there are several possible explanations. First, decrease of free IgE levels by omalizumab can lead to down regulation of FcεRI on mast cell and dendritic cells . Second,The serum free IgE levels would be the most reliable marker reflect the IgE production status in the patients treated with omalizumab. One of the benefits of the long-term use of omalizumab is the improvement of patients’ atopic status by reducing IgE productivity. Moreover, if IgE production can be suppressed, it is possible to re-adjust and reduce the dosage.The present case shows the possibility of reducing the dose of omalizumab following its. Considering the high medical cost of omalizumab, reduction of the dose may be a viable option. In such a case, it is useful to measure the serum free IgE level to appropriately identify patients in whom the dose can be reduced. Because there are patients similar to the present study in whom serum free IgE levels temporarily increase following a dose reduction of omalizumab, it is recommended that their clinical courses be carefully monitored by observing their serum free IgE levels.


Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA. Proliferating cells are characterized by elevated production of cellular components needed for the survival of new born cells. Protein synthesis is necessary not only to ensure cellular functions but also to achieve the proper outcome for cell division. Therefore, regulation of ribosome biogenesis and cell cycle must be tuned to guarantee the survival of doubling cells . DiffereIn particular, it has been demonstrated that 5S rRNA is present in a 60S preribosomal complex with RPL5 and RPL11, defined as 5S RNP, which is presently considered as the key regulator of MDM2 inhibition , 23, 24.Previous works have been characterized the involvement of 5S RNP in p53 stabilization after ribosome biogenesis inhibition. In particular, it has been demonstrated that RPL11, RPL5 and 5S rRNA act as a ternary complex in a mutually dependent manner . On the In a preliminary set of experiments, we set up the conditions to down-regulate the expression of 5S rRNA, according to previous studies , 23. In In order to verify how 5S rRNA neosynthesis inhibition affects p53 stabilization, we treated TFIIIA-silenced HCT116 cells with Actinomycin D  for 4 and 8 hours. Western blot analysis indicated that p53 levels are in general much lower in TFIIIA silenced samples, in comparison with control cells. However, by treating cells with ACTD, the amount of p53 progressively increased, despite the inhibition of 5S rRNA transcription Figure . In contIn fact, in HCT116 cells, RPL11 RNA interference Figure  abrogateThis data was confirmed by using a different set of siRNA against TFIIIA and RPL11 mRNAs .Moreover, a slighter difference between TFIIIA and RPL11 or RPL5 depleted samples was observed in terms of p53 stabilization after the selective inhibition of POLI transcription by treatment with the non-intercalating agent CX-5461 .These data suggest that the suppression of 5S rRNA synthesis do not abrogate p53 response to inhibition of ribosome biogenesis, in contrast to RPL11 and RPL5 reduction.The above reported data indicated that during ACTD treatment, p53 stabilization occurred also in absence of 5S rRNA neosynthesis. In order to ascertain whether in this case the 5S rRNA no longer constitute the 5S RNP complex, we performed TFIIIA RNA interference in HCT116 cells and we evaluated the amount of 5S rRNA co-immunoprecipitated with MDM2 at 4 and 8 hours of ACTD treatment.The data obtained showed that, in control cells, ACTD treatment led to a progressive time dependent increase of co-immunoprecipitated 5S rRNA and MDM2 Figure . InteresThe data reported above suggest that a non-ribosomal fraction of 5S rRNA, stored in the cell, take part in the assembly of the 5S RNP complex during a prolonged inhibition of rRNA transcription. To verify this hypothesis, we measured changes in different populations of 5S rRNA bound to MDM2 under ribosome biogenesis inhibition.®, Life technologies, Eugene, Oregon, USA) and collected the unlabeled fraction from the supernatant of the capturing phase (pre-existing fraction).In particular, we labeled HCT116 cells with 5-Ethynyl Uridine (5-EU) in a dose which did not stabilize p53  for 8 hoFollowing this approach we observed that after ACTD treatment, the increase in the total amount of MDM2 immunoprecipitated 5S rRNA is mainly dependent by the pre-existing population of 5S rRNA rather than the neo-synthesized captured fraction, even under 5S rRNA neosynthesis inhibition Figure  and 4C.These results demonstrate that under ribosome biogenesis inhibition, in addition to neo-synthesized 5S rRNA, a pre-existing fraction of 5S rRNA is recruited in 5S RNP, ensuring the efficient inhibition of all the increasing MDM2 molecules.Here we reported that 5S rRNA synthesis did not abrogate p53 accumulation due to ActD treatment, in contrast to RPL5 or RPL11 neosynthesis inhibition.Therefore, this data might suggest that the stabilization of p53 after inhibition of ribosome biogenesis can occur without the contribution of 5S rRNA, being exclusively due to RPL11 and RPL5 MDM2 binding.This appears to be in contrast with the observation that under a prolonged down-regulation of rRNA synthesis by POLR1A RNA interference, p53 stabilization was induced as a consequence of MDM2 inhibition by the 5S RNP ternary complex which contains RPL11, RPL5 and 5S rRNA .In order to clarify this apparent discrepancy, we measured the total amount of 5S rRNA bound to MDM2 during a progressive hindering of ribosome biogenesis by ActD treatment. We found that, despite the inhibition of 5S rRNA synthesis, a progressive increase of 5S rRNA co-immunoprecipitated with MDM2 occurred. These results confirmed that during ribosome biogenesis inhibition the 5S rRNA is necessary for the formation of the 5S RNP, thus suggesting that a pre-existing population of 5SrRNA is involved in 5S RNP assembly.To verify this point, we measured the amount of total, neo-synthesized and pre-existing 5S rRNA from MDM2 immunoprecipitated samples after ActD treatment.The data obtained showed that under inhibition of ribosome biogenesis, the increase in total 5S rRNA co-immunoprecipitated with MDM2 observed is mainly dependent by the pre-existing population, even after TFIIIA interference. Since RPL5 and RPL11 silencing led to a strong inhibition of p53 induction after ACTD treatment, it was very likely that the newly synthesized RPL5 and RPL11 are responsible for stored 5S rRNA recruitment and accumulation in cells. On the other hand, data indicated that RPL5 and RPL11 exert different dynamics relative to 5S RNP assembly. In fact, It has been shown that RPL11 is localized principally in the nucleolus and cytoplasm and significantly increases in nucleoplasm only after ACTD treatment, in contrast to RPL5 and 5S rRNA which are accumulated between nucleolus, nucleoplasm and cytoplasm.Moreover, RPL5 is required in 5S rRNA maturation  and accuIn order to better characterize the role of RPL11 and RPL5 in 5S RNP assembly during a progressive inhibition of ribosome biogenesis, we compared the amount of RPL11 and RPL5 in cytoplasmic and nuclear extracts from HCT116 cells after ACTD treatment. Our data demonstrated that the amount of RPL11 in the nucleus was increased after 8 hours of ACTD administration. In contrast, we found that the amount of RPL5 in the nuclear extract remained constant during ACTD treatment . While tHuman cancer-derived cell lines were cultured in monolayer at 37°C in humidified atmosphere containing 5% CO2. HCT116, and A549 were cultured in Dulbecco's modified Eagle's medium , while MCF7 in RPMI medium  supplemented with 10% fetal bovine serum . Actinomycin D  was used at a final concentration of 8 nM for the indicated times. siRNA transfection was performed by Lipofectamine RNAi Max and OPTIMEM medium  as manufacturer's protocol. Each siRNA was transfected at a concentration of 50 nM and cells are always treated for the indicated times after 72 h from the beginning of the transfection procedure. Two set of siRNAs were used. The sequences of the first set, targeting TFIIIA, RPL11 and RPL5 mRNA are listed in supplemental materials and methods section, while non silencing siRNA , was used as transfection control. For the second set, non silencing siRNA, or sequences targeting TFIIIA or RPL11 were purchased from Life technologies, Eugene, Oregon, USA.T-Test on three experimental replicates by considering significative data with a P value < 0,05.RNA extraction from whole cell lysate or from immunoprecipitated samples was performed using Tri Reagent  according to the manufacturer's protocol. Reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit  following the manufacturer's protocol. The relative amounts of TFIIIA mRNA, RPL11 mRNA, RPL5 mRNA, 5s rRNA was evaluated by Sybr Green method  using primer's sequences listed in supplemental materials and methods section, while b-glucoronidase was used as standard control and quantified with TaqMan Gene expression assay . Real time qPCR was performed on an ABI prism 7000 Sequence Detection system  with the 2E−ΔΔCT method. The mean ΔCT value of the control sample was used in each experiment to calculate the ΔΔCT value of sample replicates. Statistical analysis on the data obtained was performed by using the paired 3H-Uridine for 1 hour (indicated as Pulse). Cells were then changed to medium containing non-radioactive Uridine at the concentration of 1 mM and harvested after 4 h (here described as Chase). Total RNA was isolated with TRIreagent  and the extracted RNA was quantified by spectrophotometric analysis and the radioactivity incorporation was evaluated with β-Counter analysis. For 18S and 28S rRNA processing analysis, we collected samples both after 1h of Pulse labeling or after a chase time of 4 h and equal amount of 3H-Uridine labeled RNAs were resolved in Formammide loading Dye  by 1% agarose–formaldehyde gel . For small RNA analysis, samples were collected after a chase time of 4h and electrophoresed in Formammide Loading Dye on a 10% polyacrylamide (19:1)/TBE/7M Urea Gel in TBE 0.5X running buffer at 200V constant for 1h. At the end of the run, the gel was stained with etBr at 0.5 mg/ml in TBE 0.5X for 10 min at room temperature while shaking and destained for 10 min in TBE 0.5X at room temperature while shaking. For both RNA electrophoresis, relative images were collected on UV transillumiator. Labeled RNA was then transferred on Hybond N+ membrane  by semidry transfer in TBE 0.5X for small rRNAs or by backward capillarity transfer in 20X SSC for 18S and 28S evaluation. RNAs were UV-crosslinked to the membrane by Stratalinker® UV Crosslinker and the membranes was treated with EN3HANCE™ Spray Surface Autoradiography Enhancer . Finally, for labeled RNAs autoradiographic detection, the membranes were exposed for 6 days at −80°C on ECL Amersham Hyperfilms .For 18S and 28S rRNA processing analysis or evaluation of neo-synthesized 5S rRNA and tRNAs, pulse-chase labeling was performed. In brief, 72 hours after transfection with siRNA against TFIIIA or NS siRNA, HCT116 cells were incubated in medium containing 2,5 μCi/mL of HCT116 cells were cultured in 150 mm dishes and treated with 100 μg/ml Cycloheximide for 15 min, 72 hours after the beginning of the transfection procedure. Cells were then washed twice on ice in PBS + 100 μg/ml Cycloheximide (CHX); resuspendend in LSB buffer  and lysate on ice for 10 min. by adding detergent buffer  The lysate was cleared by centrifugation at 14000 RCF for 10 min. at 4°C. Ribosomes was then separated on chilled 15%–50% Sucrose Gradient (in LSB buffer) by ultracentrifugation at 160000 G for 2 hours at 4°C. Polysome Profile was monitored at 254 nm  and fractionated  using an ISCO gradient fractionator system interfaced to an UA-6 absorbance detector . Collected data were digitally converted by using Minilab 1008  and TracedDaq software , by acquiring data in differential mode at +/− 4 V and 4 Hz.Whole cell protein extraction was performed in lysis buffer  and 0.1mM b-glycerolphosphate for 20 min. and cleared by centrifugation at 14000 RCF for 20 min. Nucelar/cytoplamic fractionation was performed by cell pellets were lysis in Hypotonic buffer  supplemented with protease inhibitors cocktail , and centrifuged to separate the cytoplasmic fraction from the nuclear pellet. Nuclei were lysed in a 1:1 mixture of Low Salt Buffer  and High Salt Buffer , both supplemented with protease inhibitors cocktail , with nuclear extracts obtained by high speed centrifugation at 12,000 RPM at 4°C for 30 minutes.Protein extract was quantified spectrophotometrically with the Bio-RadProtein Assay .The same amount of proteins was separated in Laemmli loading Dye  by SDS PAGE in a polyacrilammide gel in Running Buffer  at constant 30 mA for 1 h. Proteins were then transferred on a PVDF membrane (millipore) by a semydry transfer system with Transfer Buffer  for 1,30 h. Employed antibodies are listed in supplemental materials and methods section.Immunoprecipitation assay was performed by cell lysis on ice in immunoprecipitation buffer , as indicated after.10% glycerol, 0.8% Igepal/NP40, 0,4 U/μl RNAsi OUT and protease inhibitors cocktail from Roche Diagnostics, Basel, Switzerland). The lysates were cleared by centrifugation and quantified by Bradford protein assay . Equivalent amounts (200 μg) of protein were incubated at 4°C with rotation overnight in immunoprecipitation buffer with 3 μg of anti-MDM2  Protein A/G-coated agarose beads  were added to the extracts and mixed by rotation for an additional 3 hours at 4°C. The beads were washed four times with immunoprecipitation buffer. At the end of the final wash, beads were resuspended in Laemmli loading buffer for western blot analysis or in TRIreagent  for RNA extraction. The extracted RNA was divided in two fractions and used directly for total 5S rRNA analysis or to retrive the 5-EU labeled and the unlabeled RNA fraction by using the nascent RNA capture kit®  following manufacturer's instructions. In particular, HCT116 cells were seeded in 100 mm culture dishes and were labeled by 5-Ethynyl Uridine (5-EU) at 50 μM for the indicated times after 72 hours from the beginning of RNA interference. Cells were then collected after a pulse labeling of 1 hour and a chase time of 2 hours with 1 mM Uridine or after a continuous 5-EU labeling (8 hours) in presence or absence of 8 nM ACTD. At the end of the treatments, RNA was extracted with TRIreagent  from whole cells  or from MDM2 immunoprecipitated samples (for the measurement of neo-synthesized and pre-existing 5S rRNA bound to MDM2). The same amount of RNA (200 ng) was used directly for total fraction evaluation or was modified via click chemistry. Click chemistry based biotinylation was carried out in click-it reaction buffer  for 30 min at room temperature. Biotinylated RNA was precipitated by ammonium acetate for 10 minutes. After 10 minutes of centrifugation at 14000 RCF the pellet was washed twice with ETOH at 75%. All the retrieved RNA was then bound to 15 ul of streptavidin magnetic beads  for 30 minutes at room temperature while gently shaking. The unlabeled RNA fraction (Pre-existing) was obtained by precipitation of the supernatant of streptavidin beads binding reaction. Beads was washed 5X 150 ul of Click it wash buffer 1 and 5 × 150 ul of Click it wash buffer 2, resuspended in 15 μl of wash buffer 2 and in bead reverse-transcribed for labeled RNA evaluation. The captured labeled RNA (neo-synthesized), the unlabeled RNA (pre-existing) and the total RNA were reverse-transcribed in cDNA by using High capacity reverse transcription kit /Δct TOT;% Pre = 2E−ΔCT Pre * (2E−ΔCT Neo + 2E−ΔCT Pre)/ 2E−ΔCT TOT. Δct TOT was relative to the maximum 5S rRNA amount measured (ACTD 8 h), while ΔctNeo and ΔctPre were relative to the background measured in the NS IGG immunoprecipitated sample.Neo-synthesized and pre-existing RNA amounts were evaluated by Nascent RNA Capture KitT-Test on three experimental replicates by considering significative data with a P value < 0,05.Statistical analysis on the data obtained was performed by using the paired


Amorphous silica comprises >95% of this flux and is highly soluble in sea water, as indicated by a significant increase in dissolved silica across a fjord salinity gradient. Retreating palaeo ice sheets were therefore likely responsible for high dissolved and amorphous silica fluxes into the ocean during the last deglaciation, reaching values of ∼5.5 Tmol year−1, similar to the estimated export from palaeo rivers. These elevated silica fluxes may explain high diatom productivity observed during the last glacial–interglacial period.Ice sheets play a more important role in the global silicon cycle than previously appreciated. Input of dissolved and amorphous particulate silica into natural waters stimulates the growth of diatoms. Here we measure dissolved and amorphous silica in Greenland Ice Sheet meltwaters and icebergs, demonstrating the potential for high ice sheet export. Our dissolved and amorphous silica flux is 0.20 (0.06–0.79) Tmol year Glacial runoff often has relatively low dissolved silica concentrations and therefore ice sheets have been thought insignificant in the global silicon cycle. Here, the authors show that ice sheets likely play an important role in the production and export of dissolved and dissolvable amorphous silica downstream. Silicon (Si) plays a crucial role in global biogeochemical cycles, acting as an essential nutrient for a number of marine organisms, particularly for diatoms, who use it to build their cell frustules and which account for up to 50% of oceanic carbon fixation12Over 70% of oceanic dissolved silica (DSi) is derived from riverine input, with the majority of this >80%) believed to be delivered as DSi70% believ911121314151617820−1 , and by extension former northern hemisphere ice sheets on broadly similar lithologies, for the global Si cycle. We present DSi and easily dissolvable amorphous particulate silica (ASi) concentrations from subglacial meltwaters exciting a glacial catchment in western Greenland. The GrIS provides an accessible ice sheet system, with large, land-terminating glaciers allowing direct sampling of waters of subglacial origin at the ice margin. We highlight the importance of a labile solid-phase amorphous silica phase in meltwaters, with evidence of its dissolution across a glaciatied fjord mixing zone. We also document measurements of iceberg-rafted amorphous silica, and calculate potential fluxes from both icebergs and meltwaters discharged from the GrIS to the ocean. We propose that ice sheets deliver a large amount of dissolved and labile amorphous silica downstream. They therefore have a more important role in the silicon cycle, both now and during past glacial-interglacial periods, than previously apprechiated.2 hydraulically active catchment, ∼80 km long)Meltwater samples were collected at least daily during the 2012 melt season from the subglacial channel draining Leverett Glacier, south-west Greenland  in glacial meltwaters. ASi comprised 0.91 (0.51–1.21) wt.% of SPM equating to very high mean ASi concentrations of 392 (120–627) μM . High-re2 year−1  exceeds Qtz=−0.13 to 0.09; ASi=−1.41 to −1.63). Catchment DSi denudation rates of ∼980 kg Si km−2 year−1 are comparable to other glaciersDSi concentrations in bulk meltwater runoff were generally low, with a discharge weighted mean of ∼10 μM (0.8–41.4 μM) similar to runoff from other glaciers23−1 for icebergsIceberg ASi concentrations were lower than those found in glacial meltwaters. ASi SPM content at our two sites was 0.27 0.16–0.46) wt.% for Sermilik fjord icebergs (east Greenland) and 0.28 (0.16–0.47) wt.% for Tunulliarfik fjord icebergs (south Greenland), giving mean ASi concentrations of 47.9 (28.2–81.1) and 50.5 (28.2–83.2) μM for Sermilik and Tunulliarfik fjord icebergs, respectively (assuming an estimated sediment load of 0.25–0.75 g l–0.46 wt.−1 of DSi and 0.16 (0.05–0.75) Tmol year−1 of ASi to the surrounding fjords and oceans (−1 of ASi assuming that our samples are representative of other Greenlandic icebergs. Iceberg DSi fluxes are not included because of the low concentrations measured in clean ice (<1 μM). These calculations give a total GrIS Si flux of 0.2 (0.06–0.79) Tmol year−1. This is 1.8% (0.7–8.4%) of the estimated global Si input to the oceans (9.4 Tmol year−1)−1)We estimate GrIS meltwaters currently deliver 0.01 (<0.01–0.02)  Tmol yeard oceans , based od oceans . IcebergQtz and SIASi track the evolution of subglacial drainage .DSi values are an order of magnitude lower than the discharge weighted global mean riverine value of 158 μMdrainage  and 4. Hdrainage , with wa−1)41Glacial silica export is dominated by the ASi fraction . The ASi−1).It has previously been demonstrated that iceberg-rafted debris is likely a large source of reactive nanoparticulate iron to the euphotic zone+ and K+) from the mineral surface leave an amorphous crust rich in more insoluble ions such as silica. Both mechanisms invoke higher chemical weathering rates in subglacial environments than previously realizedRecent studies have exploited the use of modern nano-observation technologies to study ASi formation on mineral surfaces from aqueous weathering processeset al.The comminution of bedrock by glaciers and ice sheets is also likely to be important in producing structural change to mineral surfaces4142+) and alkali earth metals 4949There is considerable uncertainty around the lability of ASi before long-term burial in fjords and near coastal regions. We believe ASi associated with glacial sediments will be highly labile downstream for three main reasons. First, glacial rock flour is potentially highly reactive because of a disturbed surface layer and large surface area per unit mass35332CO3), showed only minor Si dissolution over a period of 672 h . This demonstrated rapid release of DSi from ASi over a period of days to weeks . Treated28 days;  compared28 days; . Under t28 days; . Under tWe found further evidence of rapid ASi dissolution from Greenlandic meltwaters, with more than an order of magnitude increase in DSi concentrations across a buoyant SPM-rich glacial meltwater plume mixing with saline fjord waters downstream of Leverett Glacier 53. Our d1955The glacial impact on the marine Si cycle and associated budgets will depend on the magnitude of the glacial flux and the lability of the exported Si. High rates of physical weathering−1), groundwater (0.6 Tmol year−1) and hydrothermal sources (0.6 Tmol year−1)et al.33−1 , but substantially less than the GrIS (0.2 Tmol year−1). Iceberg calving fluxes are significantly higher from the AIS than the GrIS. Depoorter et al.3 year−1 from the AIS. If we assume a similar sediment loading (0.5 g l−1) and ASi wt.% to our GrIS iceberg estimates −1, ∼3% of the global Si budget (The Antarctic Ice Sheet (AIS) may also be a significant source of dissolved and amorphous silica to the Southern Ocean. Previous published estimates indicate the AIS DSi flux is in the region of ∼0.1 Tmol year−1 ref. , but it r−1 ref. . This giDSi flux . AIS melstimates , this gii budget . However3 year−1, equivalent to sea level rise of >4 cm year−1. A crude calculation indicates ice sheets would have delivered on the order of 5.7 Tmol year−1 of DSi+ASi to the oceans, assuming a similar SPM, ASi and DSi concentration to modern-day Leverett Glacier . The impact of the palaeo ice sheet Si flux will be felt for an extended period after inputStudies postulate a link between the supply of Si to the ocean and the efficiency of the biological carbon pump12656667 Glacier . Nonglac−2 measured at a large ice sheet catchment. Our flux estimates of dissolved and amorphous silica for the GrIS demonstrate that meltwater and iceberg discharge are significant and may provide similar amounts to the oceans as dust deposition, groundwater discharge and hydrothermal input. Hence, the GrIS likely contributes a large proportion of the dissolvable silica in the productive fjord and near coastal regions, where diatoms make up a large proportion of the phytoplankton community. These results indicate that glaciated regions play a more important role in the Si cycle than previously appreciated, and should be considered in future marine dissolved and amorphous silica budgets. Our findings have significant implications for the understanding of the Si cycle in the past, with globally significant fluxes of silica into the oceans likely during catastrophic melting of the large palaeo ice sheets that covered nearly 30% of land surface area. Large ice sheet pulses of dissolved and amorphous silica during these periods are a viable driver of deglacial diatom-dominated phytoplankton communities as observed in core records, in turn potentially enhancing the efficiency of the biological pump.Our findings indicate that ice sheets play a more significant role in the global Si cycle than previously recognized, mainly via export of large quantities of potentially labile amorphous silica. This phase dominates the glacial dissolved and amorphous silica meltwater flux, with ASi concentrations up to 627 μM and yields of >36,000 kg Si km2  in 2012. Leverett Glacier is a large land-terminating outlet of the GrIS. It is ∼80 km long, and has a hydrologically active catchment area of ∼600 km2 refs , 71. Mea1 ref. 36.Iceberg samples were collected from Tunulliarfik Fjord (61.1°N 45.4°W) in July 2013 and Sermilik Fjord (65.7°N 37.9°W) in July 2014. Both fjords receive ice discharged from local marine-terminating glaciers and were sampled at least ∼18 km (Tunulliarfik Fjord) and ∼40 km (Sermilik Fjord) downstream of where they calved. The shield bedrock geology from these catchments is broadly similar to Leverett Glacier n=25) were collected from the retained sediment on the cellulose nitrate filter. These were stored air dried and refrigerated until analysis.Bulk meltwater samples were collected at least once daily  throughout the main melt period 11Fjord water was collected (17 June 2012) using a 0.45 μm Whatman GD/XP PES syringe filter using a PP/PE syringe. Surface water samples were collected in 2 L HDPE bottle. Bottles were rinsed three times with sample water and then fully immersed ∼0.3 m below the surface to collect the final sample. Salinity and pH were taken at each sampling site.n=12), southern Greenland and Sermilik fjord (n=5), eastern Greenland, using a clean ice axe. Excavated blocks of iceberg were placed in new, clean Whirl-Pak bags. The outer layer of ice was allowed to melt and was discarded to minimize potential contamination from the sampling process. The remaining ice was transferred to a new Whirl-Pak bag and allowed to melt completely. Iceberg-entrained sediments were collected by filtration of the melted ice through a 47 mm 0.4 μm Whatman Cyclopore PC membrane filter mounted on a Nalgene PS filtration unit. The filtrate was retained for analysis of DSi.Iceberg samples were retrieved from a boat in Tunulliarfik Fjord (−1 (0.36–71.43 μM) Si. The methodological limit of detection was 0.3 μM, precision ±0.5% and accuracy −1.2%.DSi was determined using a LaChat QuikChem 8500 series 2 flow injection analyser (QuikChem Method 31-114-27-1-D). This method uses the well-established molybdic acid colourimetric method. Seven standards (matrix matched for fjord samples) were used, ranging from 10 to 2,000 μg l2CO3 solution added. Bottles were placed in an 85 °C hot water bath, and 1 ml aliquots of samples were taken from the sample bottle after 2, 3 and 5 h, using a precalibrated 1 ml automatic pipette. Aliquots were stored in 2 ml PP microcentrifuge tubes at 4 °C until analysis <24 h later. Before analysis, 0.5 ml of sample was neutralized with 4.5 ml of 0.021 M HCl in a 10 ml plastic centrifuge tube. Samples were analysed using the dissolved silica method described above. Total ASi was determined by calculating the intercept of a linear regression line through collected aliquots at 2, 3 and 5 h, assuming amorphous Si phases dissolve completely within the first hour of extraction and clays/more crystalline material release DSi at a constant rate over the experimental time frame4774ASi was determined using an alkaline digestion47−1) at the sampling time point and the wt.% ASi of that sample. The ASi concentration was then converted from mg l−1 to μM.ASi is expressed as both % dry weight and as concentration in μM. The ASi wt.% was calculated using the weight of sediment added for each extraction, the amount of extraction solution at each sampling time point  and the amount of dissolved silica in the solution at that time point. The final ASi % was the value of intercept of the linear regression as described above. The concentration of ASi was derived from the SPM concentration . Four 15 ml centrifuge tubes  were filled with 10–15 mg of accurately weighted sediment and 10 ml of sea water (1–1.5 g l−1) for each of the four time points  when measurements were taken. Tubes were incubated at lab temperature (18 °C) in the dark, and gently agitated on an orbital shaker (at 50 r.p.m.). Leaches were terminated by filtration of 5 ml aliquots through a 0.22 μm syringe filter  into a clean 15 ml centrifuge tube. Aliquots were stored refrigerated before analyses for DSi within 24 h of sampling, following the procedure above. Results are expressed as the percentage of ASi that has dissolved. The ASi content of the all sediment used was determined according the analytical procedure above, to calculate this.A simple leach on SPM from Leverett Glacier was conducted using natural low Si sea water. The seawater DSi concentration was 1.6 μM, and the salinity 35.49 PSU. All sea water was sterilized by filtration through a 0.22 μm PES Stericup filtration unit. Two types of Leverett Glacier SPM were used. Both were derived from the same bulk SPM sample . The first, ‘treated' sediment, was extracted using the 0.1 M NaThe morphology and structure of silica phases was determined using high-resolution field emission gun transmission electron microscopy  operating at 200 kV. Particulate material were removed from the filters and dispersed in ethanol using an ultrasonic bath for ∼1 min. A drop of this solution was pipetted onto a standard holey carbon support cupper grid. High-resolution images were complemented by  to determine the elemental characteristics of material. Amorphous silica was characterized by the lack of any crystallinity and the large Si and O peaks in the EDS, although other cations were also often present.n=41) over a range of water levels. Errors associated with discharge readings are estimated to be <15% . Filters were subsequently oven dried overnight and re-weighed. Uncertainty in suspended sediment measurements is estimated to be ±6% ref. .3 year−1 from Tedesco et al.−1. An iceberg mass flux of 612 km3 year−1 (2000–2010) is derived from the solid ice discharge data of Bamber et al.−1 as sediment content of icebergs is poorly constrained. These values may be conservative as higher concentrations of 0.6–1.2 g l−1, based on excess 224Ra activity in the vicinity of icebergs in the Weddell Sea, have been estimated by others−1.GrIS freshwater fluxes were derived using mean modelled meltwater runoff from 2000 to 2012 of 437 kmet al.3 year−1 (contribution to sea level rise), but glacial runoff may have been ∼25,000 km3 year−1 with ∼10,000 km3 year−1 of mass accumulation in the ice sheet interior.Paleo ice sheet fluxes were calculated using modelled ice sheet reconstruction data (ICE-6G_C) from Peltier The data used in this article are available from the corresponding author on request.How to cite this article: Hawkings, J. R. et al. Ice sheets as a missing source of silica to the polar oceans. Nat. Commun.8, 14198 doi: 10.1038/ncomms14198 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Parietobalaena yamaokai, from the lower part of Itahashi Formation  in Shobara, Hiroshima, Japan. A critical and previously unnoticed feature, the open suture between the supraoccipital and exoccipital, in one specimen indicates the preservation of a very young individual–under six months old and even close to a new-born calf. Given the occurrence of a new-born whale and relatively abundant assemblage of Parietobalaena yamaokai, I propose a previously hidden and unknown breeding ground for the extinct baleen whale, P. yamaokai, in the Middle Miocene of Shobara (16.1–15.6 Ma), Hiroshima. Discovery of paleo-breeding sites of extinct populations/species should further help us to understand biological extinctions from a long-term perspective as conservation paleobiology aims to offer new insights into policy making for conserving endangered populations/species.Locating breeding sites is definitely a key to understanding the ecological requirements and maintaining the sustainability of populations/species. Here I re-examined published specimens of an extinct baleen whale,  Eschrichtius robustus, is currently large and stable, and even becomes an attraction for whale watching. In fact, this population used to be close to the brink of extinction in the early 20th century, but the population recovered, thanks to the well-known breeding sites so that it facilitated the recovery and protection of a nearly extinct population. On the contrary, the status of the western North Pacific population is more uncertain, with their population down to 100 individuals, it could face the similar as their North Atlantic counterparts  from Shobara, Hiroshima, Japan . The finding of a previously unknown breeding ground of an extinct baleen whale should give us an opportunity for a new look at extinction event and further understand the ecological and evolutionary response of species to environmental changes from a long-term perspective as the conservation paleobiology aims to offer a new aspect for policy making when concerning the conservation of endangered populations/species.Here, I report a Miocene breeding site for an extinct baleen whale, Parietobalaena yamaokai were re-examined, including HMN-F00023, HMN-F00024 , HMN-F00042, HMN-F00044, HMN-F00054, HMN-F00127 , Balaenoptera acutorostrata , B. physalus , and Megaptera novaeangliae  , to show the open suture between the supraoccipital and exoccipital and facilitate the comparison with a very young individual of Parietobalaena yamaokai  in the P. yamaokai.Determination of ontogenetic ages follows the results of studying early juvenile specimens and calves of extant k whale) ; here, Iyamaokai . The phyopteroid  or a “censu lato . Given tParietobalaena yamaokai remains unknown. I use two equations, both of which rely on the bizygomatic width of the skull to reconstruct the body size specifically for baleen whales (y (body length) = 8.209*x (bizygomatic width) + 66.69; F00127. Given the preserved condition, the exact bizygomatic width of HMN-F00127 is uncertain, but likely approximates 30 cm. In order to have a better understanding of their body size, a larger individual, HMN-F00042, whose bizygomatic width approaches but slightly less than 50 cm, was also estimated.In addition, the body size could be an alternative approach to estimate the ontogenetic age, although the growth curve or full-grown size of Parietobalaena yamaokai show that several reported fossils are juvenile specimens, although it remains problematic for clearly identifying exact ontogenetic ages of each fossil specimen. However, one specimen (HMN-F00127) appears to be a very young individual, under six months old, judging from the open suture between the supraoccipital and exoccipital , the body size for HMN-etic age , but canm 477 cm  equationm 477 cm  equationF00127 represents a rare baleen whale fossil whose ontogenetic age was previously unrecognized and actually can be genuinely determined the ontogenetic age to some extent–under six months old, given the open suture between the supraoccipital and exoccipital. Furthermore, the supraoccipital is virtually floating, lacking a firm contact with surrounding bones  and in turn suggesting a much younger developmental stage , comparable to a fetal specimen of blue whale, Balaenoptera musculus  in the Middle Miocene , substantiating the early origin of baleen whale gigantism , together with isotopic analyses of other eomysticetid species  for baleen whale breeding in the northern hemisphere . Howevernus orca . Accordi


IDO1) catalyses the initial, rate‐limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1‐dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine‐based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP‐1 and SHP‐2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine‐phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half‐life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM‐associated tyrosines with phospho‐mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 – but not ITIM2 mutant – did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen‐specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1‐dependent events will occur in a local microenvironment.The enzyme indoleamine 2,3‐dioxygenase 1 ( SOCS3 versus SHPs). By using murine IDO1 mutants mimicking stable and specific tyrosine phosphorylation of ITIM1, ITIM2, or both, we identified distinct roles for ITIM1 and ITIM2 in the selective association with their protein partners and the resulting opposite outcome. The identification of mechanisms that diametrically control the functions and fate of IDO1 opens up new perspectives for pharmacologic control of IDO1‐related events.We here examined the specific contribution of each ITIM to the association of pIDO1 with either type of protein partner (Ido1−/−) C57BL/6 mice were purchased from Jackson Laboratories and bred at Charles River Breeding Laboratories. All animals were housed and fed under specific pathogen‐free conditions. All in vivo studies were in compliance with National  and Perugia University Animal Care and Use Committee guidelines. P1.HTR, a highly transfectable clonal variant of mouse mastocytoma P815 H‐2Db‐restricted HY peptide (WMHHNMDLI) – containing the immunodominant epitope of the male mouse‐specific minor transplantation antigen – was synthesized by BioFab Research .Female wild‐type C57BL/6 mice were purchased from Charles River Breeding Laboratories  and female IDO1‐deficient (In silico models of l‐glutamate (l‐Glu), l‐tyrosine (l‐Tyr) and l‐phosphotyrosine (l‐pTyr) were generated using Maestro (version 9.5), as implemented in Schrödinger software modules. l‐Glu and l‐pTyr were considered as bearing a formal charge of −1. Geometry optimizations were carried out using quantum mechanics methodology with density functional theory (DFT) B3LYP/6‐31G** and diffuse++ function. All DFT calculations were carried out using Jaguar software (version 7.9), with a medium grid density and a fully analytic accuracy level. Atomic electrostatic potential charges (ESP) were calculated by fitting the electrostatic potential on atom centres. Geometry optimizations were performed with the standard Poisson–Boltzmann solvent model. The electrostatic potential surface was generated by solving the Poisson–Boltzmann equations and using ESP charges of all atoms in the molecule. Values for each compound were mapped on the solvent accessible molecular surface of l‐Glu, l‐Tyr and l‐pTyr respectively.SASA) Crystal structures of IDO1 were obtained from the Protein Data Bank Ido1 were generated from cDNA of conventional dendritic cells (DCs) stimulated with IFN‐γ, as previously described Ido1 amplification, primers containing SpeI (sense) and NotI (antisense) restriction sites were used  Ido1  and Gapdh . Values were expressed as the ratio of Gapdh‐normalized transcript expression of Ido1 mutated forms to Gapdh‐normalized transcript expression of WT Ido1.Real‐time PCR analyses were performed as described 5 cells/sample) or after immunoprecipitation  – were lysed on ice in RIPA buffer . Lysates were first immunoprecipitated by means of rabbit polyclonal antibody specifically recognizing murine IDO1, raised in our laboratory Stably transfected cells – to be immunoblotted as such  in combination with appropriate horseradish peroxidase‐conjugated antibody , followed by enhanced chemiluminescence (ECL). Anti‐β‐tubulin (Sigma‐Aldrich) was used as a normalizer.Densitometric analysis of specific signals was performed within a linear range of blot exposure, in each experiment selecting the two lowest‐exposure times required for detecting signals.in vitro in terms of the ability of stably transfected cells – incubated for 6 or 16 hr in a medium supplemented with 100 μM Trp – to metabolize Trp to Kyn, whose concentrations were measured by HPLC in culture supernatants. The detection limit of the assay was 0.05 μM. WT and mutated IDO1 proteins were immunoprecipitated and assayed by means of a phosphatase assay kit according to manufacturer's instructions (Sigma‐Aldrich).The enzymatic activity of IDO1 was measured Ido1−/− pDCs and CD8− DCs  were as described in vitro transcription of mRNA, using the mMESSAGE mMACHINE T7 Ultra Kit . In vitro transcribed‐mRNA (2 μg) was transfected into Ido1−/− pDCs by means of DOTAP , as described Ido1−/− pDCs transfected with a control mRNA supplied by the manufacturer (Ambion). For silencing Ptpn6 and Ptpn11 (coding for SHP‐1 and SHP‐2 phosphatases respectively), gene‐specific small interfering RNA (siRNA) was designed on the basis of the target sequence and synthesized by Ambion Life Technologies, which also supplied the negative control siRNA (NC siRNA). A one‐to‐one mix of Ptpn6‐ and Ptpn11‐specific siRNA was transfected into Ido1−/− pDCs for gene silencing. Transfected cells were incubated at 37°C in medium alone and, after 24 hr, recovered, washed and immediately used for in vitro and in vivo experiments.All purification procedures of splenic Ido1−/− pDCs, transfected with WT and ITIM‐mutated Ido1 mRNA and incubated for 48 hr at 37°C, were prepared according the manufacturer's protocol for the TransAM Flexi NF‐κB Family Kit (Active Motif). Analysis of p52 and RelB expression was performed on normalized samples of pDCs using rabbit polyclonal anti‐p100/p52 , anti‐RelB (Santa Cruz Biotechnology) antibodies. Anti–β‐tubulin and anti‐Lamin B1  were used as normalizers of the cytoplasmic and nuclear extracts respectively. TGF‐β production in culture supernatants from those cells was measured by means of TGF beta 1 Emax Immuno Assay System .Nuclear and cytoplasmic extracts from in vivo immunization, 3 × 105 peptide‐loaded cDCs, combined with a minority fraction (5%) of peptide‐loaded transfected Ido1−/− pDCs, were injected subcutaneously into recipient mice. Two weeks later, a DTH response was measured to intrafootpad challenge with the eliciting peptide, and results were expressed as the increase in footpad weight of peptide‐injected footpads over that of vehicle‐injected  counterparts A skin test assay was used for measurements of major histocompatibility complex class I‐restricted delayed‐type hypersensitivity (DTH) responses to the HY peptide s t‐test was used to analyse the results of in vitro studies in which data were mean values (±S.D.). All experiments were performed at least three times using triplicate samples. In the in vivo skin test assay, statistical analysis was performed with two‐tailed paired t‐test, by comparing the mean weight of experimental footpads with that of control, vehicle‐injected counterparts Student’l‐pTyr and l‐Glu amino acidic residues relative to the l‐Tyr neutral aromatic residue  and tyrosine‐253 (Y253) residues, present in the respective ITIM1 and ITIM2 motifs Ido1, by incorporating nucleotide changes for the substitution of either one or both phosphorylable ITIM tyrosines with a glutamate residue , Y253E (ITIM2) or Y115_253E (ITIM1 and 2) mutated forms of the enzyme were obtained by PCR‐based site‐directed mutagenesis from WT Ido1 constructs were transfected into P1.HTR cells to obtain stable cell lines expressing IDO1 mutants, mimicking the phosphorylation of one or both ITIMs. As the mock control cells, P1.HTR cells were transfected with the empty plasmid. We first quantified Ido1 transcripts in Y115E, Y253E and Y115_253E transfectants by real‐time PCR. Nucleotide changes did not affect the transcriptional activity of Ido1 mutants, as compared to WT Ido1 used as a calibrator , indicates a poor solvent exposure of tyrosine residues in ITIMs occurring in the small domain of the enzyme   are constitutively expressed in P1.HTR cells and, differently from IDO1 expression, showed a comparable expression level in WT, Y115E, Y253E and Y115_253E transfectants  associated with the enzyme , particularly in the expression of the Y253E mutant – mimicking tyrosine phosphorylation in ITIM2 – relative to WT IDO1. Although less significantly (P < 0.05), the protein fold change in the double Y115_253E mutant also decreased over time .Although it has previously been shown that IDO1's half‐life depends on phosphorylated ITIMs ime Fig. B.), indicating that the degradation of both WT and ITIM‐mutated IDO1 protein is indeed proteasome‐mediated. Moreover, quantitative analysis of IDO1 fold changes in MG132‐treated cells revealed a significant increase in the expression of the Y253E mutant  significantly decreased TGF‐β level  and the combined silencing of SHP‐1/SHP‐2 would negate the immunosuppressive phenotype of ITIM1‐transfected pDCs  to engage in novel activities, to meet any newly emerged environmental needs. Of note, solvent exposure of the phosphorylation sites – namely, Y115 and Y253 – is very low in all of the available crystal structures of IDO1 (solvent accessible surface area <0.5 Å2), confirming that major conformational changes may occur in the non‐catalytic small domain in specific microenvironment conditions, so to favour exposure and/or selective phosphorylation of ITIMs.However, in a different cytokine milieu, tyrosine phosphorylation can provide IDO1's ITIMs with an ability to interact with other SH2‐containing proteins – namely, phosphatases – thus, triggering downstream signalling effects via replacement of phosphorylable tyrosines with glutamate residues Because the dynamic identification of distinct phosphorylated forms of IDO1 under specific conditions could be difficult to observe in living cells, owing in part to the short half‐life of phospho‐(Y)‐ITIMs, we reasoned that an alternative approach could be the use of stable recombinant phospho‐(Y)‐mimetic IDO1 mutants, Ido1 in transfected cells. However, despite detectable translation into IDO1 protein , we observed complete loss of Trp‐degrading activity in cells transfected with all mutants. These data therefore suggest that the negative charge(s) carried by the glutamate residue(s) – or, physiologically, by phosphorylated tyrosine(s) – greatly affects IDO1's folding conformation and thus modify the shape of the catalytic site. This also suggests that ITIM phosphorylation occurs within an IDO1 conformation different from that hosting a ligand at the catalytic site. A folding conformation not compatible with the one required for the signalling function of IDO1 might be required for the enzymatically active form of IDO1, implying that the catalytic and ITIM‐dependent activities of IDO1 are not only functionally segregated but also mutually exclusive, as dictated by the local environmental needs. The available crystal structures of ligand‐bound IDO1 point indeed to ITIMs with poor solvent exposures of Y115 and Y253, which are not amenable to post‐transcriptional modifications, including phosphorylation.Transcript expression of those mutants did not differ from that of WT via SOCS3 and molecular signalling via SHPs – are associated with distinct phospho‐mimetic IDO1 mutants. This observation confirmed the non‐overlapping function of the two ITIMs and the requirement of a cellular milieu for switching on a specific ITIM function. Although the double mutant could be expected to behave as the single ITIM1 mutant, these data suggested that the copresence of a negatively charged ITIM2 might either confer a distinct conformation on IDO1 – making it incapable of transducing signals – or divert IDO1 functioning and/or drive the direct proteasomal degradation of the enzyme. Phosphorylation of either ITIM1 or ITIM2 will thus provide the enzyme with a different ITIM‐related fate/function, depending on the needs of the local microenvironment. Specifically, the ITIM‐mediated IDO1 plasticity would translate in DCs into opposite functional phenotypes, that is, immunoadjuvant versus immunoregulatory in nature vice versa in response to inflammatory versus anti‐inflammatory signals versus anti‐inflammatory milieu sensed by pDC may promote not only a differential expression of the IDO1's molecular partner (i.e. SOCS3 versus SHP1/2 phosphatases) Src tyrosine kinases In spite of the loss of catalytic activity, the IDO1 mutants maintained their ability to associate with SOCS3 or SHP‐1/SHP‐2 phosphatases, allowing triggering of ITIM‐related functions. We observed a faster dropdown of Y253E and Y115_253E relative to Y115E and more so WT proteins, which was mediated by a proteasomal mechanism. These results highlight a pivotal role of Y253E, mimicking pITIM2, in the proteasome‐mediated degradation of IDO1, in spite of the lack of a specific binding ability in associating SOCS3. To further clarify the apparently disparate functions of phosphorylated IDO1's ITIMs, we analysed the signalling activity resulting from the association of the mutants with the tyrosine protein phosphatases, SHP1 and SHP2. The phosphatase activity co‐immunoprecipitated with the different forms of IDO1 was significantly increased in Y115E, mimicking pITIM1, and it was lost in the other mutants that can associate with SHP1/2 phosphatases yet. Although all the pITIM‐mimic mutants could associate whichever molecular partners of IDO1, the resulting ITIM‐dependent activities of the enzyme – namely, proteasome degradation versus tolerance in several disease states The identification of distinct roles for the two pITIMs of IDO1 may pave the way to innovative strategies in treating autoimmune, chronic inflammatory and neoplastic diseases by either enhancing or terminating IDO1's activity, as appropriate. The finding that the enzymic and signalling functions of IDO1 appear to be mutually exclusive is important in that it may be key to a better understanding of dysfunctional IDO1‐dependent regulation of immunity The authors declare that they have no conflicts of interest with the contents of this article.in vitro experiments and analysed the data. GM, RB and CaVa performed in vivo experiments. AM performed computational analysis of IDO1's ITIMs. All authors reviewed results and approved the final version of the manuscript.CO designed the study and wrote the paper. UG and PP supervised the experiments. MG, MLB and FF cloned IDO1 mutants. EA,VR, MTP, CA and ClVo designed and performed


Excessive nutrition promotes the pathogenesis of non-alcoholic steatohepatitis (NASH), characterized by the accumulation of pro-inflammation mediators in the liver. In the present study we investigated the regulation of pro-inflammatory transcription in hepatocytes by protein inhibitor of activated STAT 4 (PIAS4) in this process and the underlying mechanisms. We report that expression of the class III deacetylase SIRT1 was down-regulated in the livers of NASH mice accompanied by a simultaneous increase in the expression and binding activity of PIAS4. Exposure to high glucose stimulated the expression PIAS4 in cultured hepatocytes paralleling SIRT1 repression. Estrogen, a known NASH-protective hormone, ameliorated SIRT1 trans-repression by targeting PIAS4. Over-expression of PIAS4 enhanced, while PIAS4 knockdown alleviated, repression of SIRT1 transcription by high glucose. Lentiviral delivery of short hairpin RNA (shRNA) targeting PIAS4 attenuated hepatic inflammation in NASH mice by restoring SIRT1 expression. Mechanistically, PIAS4 promoted NF-κB-mediated pro-inflammatory transcription in a SIRT1 dependent manner. In conclusion, our study indicates that PIAS4 mediated SIRT1 repression in response to nutrient surplus contributes to the pathogenesis of NASH. Therefore, targeting PIAS4 might provide novel therapeutic strategies in the intervention of NASH. Nutrient surplus induces a pro-inflammatory niche and inflicts insults in different types of cells and organs. This scenario constitutes the major mechanism underlying the pathogenesis of non-alcoholic steatohepatitis (NASH) characterized by numerous inflammatory infiltrates in the liver eventually transitioning into cirrhosis and hepatocellular carcinoma . In patiIn mammals, expression of pro-inflammatory is programmed by a handful of evolutionarily conserved transcription factors such as NF-κB . The actMounting evidence suggests that SIRT1, a class III NAD-dependent deacetylase, plays diverse roles in a number of cardiovascular and metabolic diseases including NASH . SIRT1 ain vitro and in vivo in response to nutrient surplus. As such, our data provide novel insights into the development of interventional strategies for NASH.Previously, our laboratory has identified a SUMOylation dependent pathway wherein the SUMO E3 ligase PIASy (PIAS4) represses SIRT1 expression in response to hypoxia in cancer cells , 14. HerApoe−/− mice were fed a western diet (HFD) for 8 weeks  diet plus fructose-containing drinking water . Compares Figure . TherefoNext, we asked whether our model, i.e., PIAS4 represses SIRT1 transcription in the liver, was operative in cultured hepatocyte in response to nutrient surplus. Indeed, exposure to high glucose (35mM) led to a decrease in SIRT1 expression while simultaneously stimulating PIAS4 expression in both primary mouse hepatocytes Figure  and immoin vivo in the pathogenesis of NASH in mice. Injection of lentivirus carrying interfering RNA against PIAS4 (shPias4) into the NASH mice induced by the HFHC diet resulted in ~80% decrease in PIAS4 expression in the liver compared to the NASH mice injected with an empty vector (SCR) as measured by qPCR . Sirtuins, particularly SIRT1, have been shown to afford protective effects in the context of NASH by limiting hepatic inflammation , 18, 19.NASH is primarily a hepatic pathology in which excessive energy input or deficient energy expenditure helps build up a pro-inflammatory microenvironment leading to disease progression. We demonstrate for the first time that PIAS4 expression and activity alter in response to energy input in mouse livers and in cultured hepatocytes. This is consistent with the notion that protein post-translational modification (PTM) enzymes participate in cellular metabolic processes by sensing energy influx/efflux –22. Of nSIRT1 mediated suppression of NF-κB dependent pro-inflammatory transcription, via lysine deacetylation of p65, is considered a central mechanism underlying its protective roles in a range of human diseases including atherosclerosis, stroke, and diabetes –32. We hA key issue is left unaddressed by the current report. Although we have demonstrated a role of PIAS4 in hepatocyte, it remains elusive whether there is a communication between different liver cells fueled by PIAS4-mediated transcriptional program. Recently, it has been reported that within the liver, different cell types  may forge extensive dialogues to maintain homeostasis; once this communication is disrupted, liver pathologies ensue , 36. Tisdb/db mice compared to wild type littermates. In addition, adenovirus-mediated PIAS1 over-expression alleviated inflammation in adipocytes although it remained untested whether PIAS4 depletion would have a similar effect. Because PIAS proteins all rely on the same ring-finger domain (RFD) to regulate pathobiological processes  that carry short hairpin RNA (shRNA) targeting PIAS4 (GTGCTGTACGGGAAGTACTT) or scrambled shRNA (SCR) every 10 days for the duration of the experiments.All animal protocols were approved by the NJMU Intramural Ethic Committee on Animal Studies. To induce steatohepatitis, 8 week-old male C57/BL6 mice were fed a high fat high carbohydrate (HFHC) diet  for 16 consecutive weeks . AlternaTissue and cell lysates were obtained as previously described . WesternChIP assays were performed essentially as described before  with antHistological analyses were performed essentially as described before . BrieflySupernatants containing pro-inflammatory mediators were collected from cultured hepatocyte or liver homogenates and ELISA was performed to measure IL-1, IL-6, and MCP-1 using commercially available kits (R&D).p values smaller than .05 are considered significant. For the in vivo experiments, specific p values are spelled out. All the in vitro experiments were repeated at least three times with a power analysis showing β>80.Data are presented as mean±SD. For experiments concerning multiple groups, one-way ANOVA with post-hoc Scheffe analyses were performed to evaluate the differences. The differences between two  groups were determined by two-sided, unpaired Student's t-test.