Mechanical forces play fundamental roles in cellular processes related to movement. The major generator of mechanical forces in living systems is myosin II, which lies inside cells. Myosin II propels actin filament sliding, converting chemical energy into movement in various scales, from stress fibre formation and sliding to muscle contraction. In non-muscle cells, such forces are exerted at a cellular/subcellular scale and drive motility processes, e.g. cell migration, division, etc.There are several isoforms of myosin II, divided into muscle, smooth muscle and non-muscle. They all consist of a hexamer made of two heavy chains (MHCII) and four light chains, two structural (ELC) and two regulatory (RLC). The heavy chain displays three domains: a globular, actin- and ATP-binding domain, a supercoiled long domain that mediates dimerization of the MHCII; and a non-helical tail domain. Non-muscle myosin II (NMII) appears as three isoforms, II-A, II-B and II-C depending on the MHCII. Their ability to generate mechanical forces is context-dependent, thereby each isoform regulates specific cellular functions. In addition, every cell type displays a specific pattern of expression that may evolve over time, e.g. during embryogenesis, being crucial for proper development.Of the three isoforms, NMII-A is the fastest moving, although it can bear considerably less load than the other two. It displays fast assembly immediately behind the leading edge of migrating cells. Conversely, NMII-B and II-C are slower, but bear more load . NMII-B When not forming filaments, NMII is folded in a compact, assembly-incompetent conformation (10S) that extends by phosphorylation of the RLC on Ser19. The same phosphorylation triggers the conformational movement of the head of the heavy chain that enables actin filament movement . Anotherde novo mini-filament formation). S1935 is much closer to the domain-breaking Pro in NMII-B than S1943 is in NMII-A, suggesting different mechanisms that nevertheless have a similar outcome, i.e. decreased filament stability. In the case of NMII-A, S1943 phosphorylation regulates its interaction with S100A4/Mts1, which may control NMII-A assembly dynamics and stability [A regulatory hotspot of the stability of the oligomers is the phosphorylation of the heavy chain. Unlike RLC phosphorylation, this is an isoform-specific mechanism. For example, the stability of the NMII-A filaments is controlled by phosphorylation of MHCII-A on S1943. Phospho-S1943 NMII-Amini-filaments are unstable, thus forming very small and weak filaments, and overall loss of cellular contraction . This motability .The physiological implications of these mechanisms of regulation are just beginning to be elucidated. Phosphorylation of S1943 controls CD34+ hematopoietic stem cell fate . Also, a


Infectious bronchitis (IB) is one of the major economically important poultry diseases distributed worldwide. It is caused by infectious bronchitis virus (IBV) and affects both galliform and nongalliform birds. Its economic impact includes decreased egg production and poor egg quality in layers, stunted growth, poor carcass weight, and mortality in broiler chickens. Although primarily affecting the respiratory tract, IBV demonstrates a wide range of tissues tropism, including the renal and reproductive systems. Thus, disease outcome may be influenced by the organ or tissue involved as well as pathotypes or strain of the infecting virus. Knowledge on the epidemiology of the prevalent IBV strains in a particular region is therefore important to guide control and preventions. Meanwhile previous diagnostic methods such as serology and virus isolations are less sensitive and time consuming, respectively; current methods, such as reverse transcription polymerase chain reaction (RT-PCR), Restriction Fragment Length Polymorphism (RFLP), and sequencing, offer highly sensitive, rapid, and accurate diagnostic results, thus enabling the genotyping of new viral strains within the shortest possible time. This review discusses aspects on pathogenesis and diagnostic methods for IBV infection. Infectious bronchitis (IB) causes significant economic losses to the poultry industry worldwide , 2. The  Gammacoronavirus genus in the family Coronaviridae, order Nidovirales. Like other members of coronavirus family, the IBV genome is composed of structural and nonstructural proteins. Structural proteins include the spike [S] glycoprotein, envelope [E], matrix [M], and nucleocapsid [N]. These proteins together play different roles in viral attachment, replication, and inducing clinical disease. Of major structural proteins, the M protein is the most abundant transmembrane protein, which play vital role in coronavirus assembly through interaction with viral ribonucleocapsid and spike glycoprotein , 3938, 3Histopathological changes include loss of cilia, oedema, rounding and sloughing of epithelial cells, and infiltration by lymphocytes . Presenc E. coli may lead to a more complex outcome, usually associated with high morbidity and mortality. Similarly, infection with nephropathogenic IBV strains may result in pale, swollen, and mottled kidneys [Nephropathogenic IBV strains cause nephritis characterized by swelling and congestion of the kidney , sometim kidneys , 41. His kidneys . Experim kidneys , 43.When the reproductive system is affected, there may be nonpatent and hypoglandular oviduct, especially in severely affected chickens , 44. LarE. coli) or coinfection with immunosuppressive viruses such as Marek's disease virus, infectious bursal disease virus (IBDV) [Morbidity due to IBV infection can reach up to 100%. Mortality rate may range from 25 to 30% in young chicks but may increase to 80% as a result of factors that are host-associated , virus-associated , or environmental . Secondary bacterial infections (s (IBDV) , 47, 48,s (IBDV) .Conventional and more advanced methods have been used for the diagnosis of IBV infection. The choice of one test over another is guided by type of sample, availability of test materials and facilities, test reporting time, purpose of the test, and whether the test is carried out in the field or at the laboratory. Selected testing procedures are discussed below.In the past, serological assays such as virus neutralization (VN) and haemagglutination inhibition (HI) were used widely for detecting and serotyping IBV strains. These tests also have been used to measure flock protection following vaccination , 51. SerVirus isolation has been the gold standard for the diagnosis of IBV , 55. TakMost IBV strains grow well when inoculated into the allantoic cavity of a 9–11-day-old chicken embryo. Clinical samples from tracheal swab, broth, or tissue homogenate (10% w/v) are inoculated into the allantoic cavity of specific pathogen-free eggs and incubated at 34–37°C, after inoculation. Eggs are candled daily to monitor embryo viability; death within 24 hrs is considered nonspecific. After 48–72 hrs, allantoic fluid (AF) is harvested from representative eggs that were chilled overnight and tested for the presence of IBV using serological tests or RT-PCR assay. Sometimes the allantoic fluid needs to be subjected to several passages to allow the virus to adapt and replicate to high titre, thus increasing the period that is needed to obtain results. The latter may vary among viral strains . After 5Isolation of IBV has been attempted in various primary and secondary cells, such as chicken embryo kidney fibroblast and Vero cells, respectively , 59. InfTracheal organ culture (TOC) can be used to propagate both embryo-adapted and non-embryo-adapted IBV strains. TOC is prepared from tracheal rings of 20-day-old chicken embryo. The tracheal rings are maintained in a roller bottle and infected with IBV-suspected samples. The culture is observed microscopically for evidence of ciliostasis under light microscope. Complete impairment of ciliary activity usually is considered as a positive culture . SuccessElectron microscopy provides a direct means of detecting and identifying IBV in biological samples based on morphological characteristics of coronavirus. Positive cultures are confirmed based on the presence of coronavirus-like pleomorphic structures with spike projections, following negative staining with phosphotungstic acid . ImportaImmunoperoxidase and immunofluorescence are two important histochemistry methods for detection and confirmation of IBV antigen from infected tissue and/or cells. These methods work based on antigen-antibody reactions , 65. ImmIn view of their increased sensitivity and reduced reporting time, molecular methods, such as Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), real-time PCR, Restriction Fragment Length Polymorphism (RFLP), and genome sequencing, have nearly replaced conventional serology and virus cultivation methods of IBV diagnosis , 69.This approach uses viral RNA, amplified either directly (one-step RT-PCR) or following cDNA synthesis (two-step RT-PCR). An RT-PCR assay was designed and introduced in 1991 for detecting the IBV-S2 gene . SubsequThis is an IBV genotyping method carried out to differentiate different known strains of IBV and to identify new variants following RT-PCR amplification . Full-leFor increased test sensitivity and specificity, real-time RT-PCR assays , 79 haveFor genotyping, S1 gene usually is amplified using RT-PCR, sequenced, and subjected to bioinformatics analyses , 89. FolSeveral respiratory diseases, such as Newcastle disease (ND), infectious laryngotracheitis, infectious coryza, avian metapneumovirus (aMPV), and avian influenza (AI), may produce clinical signs similar to avian infectious bronchitis. However, certain clinical features, including neurological signs and diarrhoea in ND, high mortality in AI, and pronounced head swelling in coryza, are uncommon in IBV infection and thus may be used in ruling out or arriving at narrowed tentative differential list , 90.Ever since the first identification of IBV in 1930s, the poultry industry has suffered a growing number of emerging IBV serotypes. Importantly, the newly evolved strains have been favoured by selection pressure, mutation, and/or recombinations, thus allowing them to avoid detection, evade host immune response, and cause diverse pathological outcomes. Lack of effective diagnostic methods and vaccines that could easily tackle the menace caused by multiple IBV serotypes is partly blamed for the serious economic losses as results of infectious bronchitis disease. Conventional detection assays such as virus neutralization and virus isolation have been used extensively, but, due to lack of sensitivity and specificity of serological assays and laborious nature of virus isolation methods, these assays have gradually been replaced by the new sensitive and specific assays such as RT-PCR, RFLP, and qRT-PCR that enable rapid genotyping and identification of new IBV strains. However, there is a need for standardization across laboratories with respect to the type and length of target gene to be considered for genotyping so as to ensure common understanding of genotype distributions in order to guide vaccine selection for prevention and control.


Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC. PKA signaling is important for the post-translational modification of proteins , 2. PKA Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC) as the target of miR-483-5p. The cells over-expressing RAI16 showed resistant to apoptosis to some extent [We performed a proteomic screen for interaction partners of RAI16. Lysates from HEK293T cells expressing Flag-tagged RAI16 were incubated with anti-Flag antibodies, immunoprecipitated complexes were isolated and associated proteins were identified by tandem (MS/MS) mass spectrometry. Detection of peptides from RAI16 was used as quality controls. Peptides from PKA-RIIα, HSP70 and 14-3-3θ were also identified .The interaction of RAI16 and PKA-RIIα was validated in HEK293T cells when recombinant PKA-RIIα was detected in recombinant RAI16 immunoprecipitated complexes Figure . Next, mHSP70 was initiated for RAI16-binding partners that may also be PKA substrates. We hypothesized that RAI16 targets PKA to phosphorylate HSP70. Lysates from HEK293T cells expressing Flag-tagged RAI16 were incubated with anti-PKA substrate antibodies, immunoprecipitated complexes were isolated and separated by SDS-PAGE. Robust phosphorylation of HSP70 was detected in the presence of RAI16 Figure , lane 2.HSP70 contains several phosphorylation sites for basophilic kinases, including a well-conserved consensus PKA site surrounding Ser 486 . TreatmeHSP70 are known to have special anti-apoptotic properties , we inve14-3-3θ was also detected in RAI16 immunoprecipitated complexes. The interaction of RAI16 and 14-3-3θ was validated in HEK293T cells when recombinant 14-3-3θ was detected in recombinant RAI16 immunoprecipitated complexes Figure . In addiMembers of the 14-3-3 family interact with several cellular targets through the association with phosphor-serine or phosphor-threonine containing motifs . Phosphomyr (FSK+PKImyr) strongly inhibited this interaction  strongly inhibited this interaction  enhanced the association of 14-3-3θ to Flag-RAI16 by 3.25–fold . Accordingly, treatment of 10% serum strongly stimulated RAI16 phosphorylation of HSP70  . 14-3-3 Conclusively, the implication of our study is that we firstly demonstrate the functional role of RAI16 as an AKAP. Our findings suggested RAI16 might play a role in HSP70 related anti-apoptosis pathway. We also demonstrate that binding of 14-3-3θ to RAI16 maintains the anchoring protein in an inactive state under basal conditions.Human embryonic kidney cell line HEK293T was cultured and performed transfection, cell viability, cell apoptosis assays and immunoblotting as described previously .The full length gene of PKA-RIIα (GeneBank: NM_004157.2), HSP70 (GeneBank: NM_005345.5) and 14-3-3θ (GeneBank: NM_006826.3) were PCR amplified from total RNA of HEK293T cell as described previously  and clonFragments of 1-45 and full length gene of PKA-RIIα were PCR amplified from the p18T- PKA-RIIα plasmid and subcloned into pcDNA3.1 with a Ha tag. Fragments of 1-250, 251-500 and 501-743 and full length gene of RAI16 were PCR amplified from the p18T-RAI16 plasmid [The S325A and Δ352-370 mutants of RAI16, S486A mutant of HSP70, Δ1-45 mutant of PKA-RIIα were introduced into the corresponding plasmids by standard PCR directed mutagenesis using the Hot Star DNA polymerase (Qiagen), respectively.in vitro phosphorylation assay was performed as reported previously [Immunoprecipitation and eviously . All proeviously .His-tagged fusion proteins of RAI16 and PKA-RIIα were expressed using pET30 in BL21DE3 bacteria and purified with Nickel-NTA chelating resin (Amersham Pharmacia Biotech) according to manufactures' protocol. The direct interaction of RAI16 and PKA-RIIα were studied by ELISA. In brief, 2ug purified PKA-RIIα protein was coated overnight at 4°C. Following a blocking with 3% BSA in PBST, purified RAI16 protein alone or combinated with stH31 or sYF were added to microplate and incubated for 1h at 37°C, and then washing 5 times with PBST, anti-RAI16 antibody was added to detect the interaction, HRP-anti-rabbit IgG and TMB (HRP substrate) were used for signal development. The values were recorded using a multiplate reader .myr, sHt31 were from Promega. Peptide sYF (Aa348-373: YCDHLITEAHTVVADALAKAVAENF) was synthesized by ABclone .RAI16 and PKA-RIIα antibodies were from Abcam; 14-3-3θ, Caspase-3, Flag and Myc epitope antibodies were from ABclone; HA and His epitope antibodies were from MBL; HSP70 and GAPDH antibodies were from Santa Cruz Biotechnology; Anti-PKA substrate antibody were from Cell Signaling. RAI16-siRNA, HSP70-siRNA and nonrelative control (NC) siRNA were synthesized by GenePharma ; FSK were purchased from Sigma; PKIhttp://scansite.mit.edu/motifscan_seq.phtml).14-3-3 binding site in RAI16 and phosphorylation sites in RAI16 and HSP70 were predicted by Scansite Motif Scanner (All experiments were performed at least three times, and data are presented as mean ± SD. Comparisons were made by using a two-tailed t test or one-way ANOVA for experiments with more than two subgroups. P<0.05 was considered to be statistically significant.


We investigated the efficacy of targeting the PIM kinase pathway in Philadelphia chromosome-positive (Ph+) leukemias. We provide evidence that inhibition of PIM, with the pan-PIM inhibitor SGI-1776, results in suppression of classic PIM effectors and also elements of the mTOR pathway, suggesting interplay between PIM and mTOR signals. Our data demonstrate that PIM inhibition enhances the effects of imatinib mesylate on Ph+ leukemia cells. We also found that PIM inhibition results in suppression of leukemic cell proliferation and induction of apoptosis of Ph+ leukemia cells, including those resistant to imatinib mesylate. Importantly, inhibition of PIM results in enhanced suppression of primary leukemic progenitors from patients with CML. Altogether these findings suggest that pharmacological PIM targeting may provide a unique therapeutic approach for the treatment of Ph+ leukemias. Chronic myelogenous leukemia (CML) evolves from abnormal hematopoietic stem cells of myeloid origin. Its signature characteristic is the genetic translocation, t9;22) q34;q11) BCR-ABL1, also known as the Philadelphia chromosome (Ph+) [1 BCR-ABL2 q34;q11PIM genes, as PIM1−/− mice do not have a significant phenotype [PIM1/2/3 −/− mice [PIM1 in a lymphoma mouse model resulted in upregulation of PIM2 [One pathway that has attracted attention for anticancer drug development is the proviral insertion site in Moloney murine leukemia virus (PIM) kinase pathway , 11. To henotype  compared−/− mice . Further of PIM2 . Thus, tMYC-dependent transcription of genes such as MCL-1 [Although not yet well understood, the PIM kinase pathway is involved in the regulation or transmission of many signals that lead to leukemogenesis. For example, PIM-mediated phosphorylation of histone 3 at serine 10 increases as MCL-1 , 16. Celas MCL-1 –20. PIM as MCL-1 –23. Anotas MCL-1 , while tas MCL-1 .In the present study, we sought to test the efficacy of PIM inhibition alone or in combination with imatinib mesylate on Ph+ leukemia cells. Our data shows that inhibition of PIM, with the pan-PIM inhibitor SGI-1776, results in suppression of the mTOR pathway as well as other downstream effectors. We also found reduced leukemic cell proliferation, induction of apoptosis, and inhibition of colony formation in Ph+ cell lines including those resistant to imatinib. In imatinib-sensitive cell lines, an enhanced effect was seen when combining inhibition of PIM with imatinib. Moreover, we establish that PIM inhibition results in suppressive effects on primary leukemic progenitors from CML patients, further suggesting a potential role for PIM targeting as a novel therapeutic approach for Ph+ leukemias.In initial experiments, we assessed the expression of all 3 PIM kinases in K562, KT1, BV173, and BV173R cell lines, by immunoblotting. As shown in Figure We subsequently examined the effects of SGI-1776 on downstream components of the PIM kinase pathway. Treatment with SGI-1776 inhibited the phosphorylation of histone 3 on serine 10 and as well as Mcl-1 expression Figure . When thleft panel). Furthermore, the area under the curve  indicated over two-fold reduction in polysomal fractions . We also examined whether SGI-1776 modulates polysomal mRNA levels for cyclin D1 (CCND1), a gene encoding for a protein that promotes cell proliferation and is highly expressed in different malignancies [CCND1 transcript levels in KT1 cells  Figure  or AZD-1The PIM kinase pathway involves many signaling elements that play roles in the regulation of leukemogenesis , 28–31. Both mTORC1 and mTORC2 have been shown to be activated in CML , 35. PreCyclin D1 gene, providing a mechanism or generation of growth inhibitory responses by PIM inhibition. Moreover, our data establish that PIM activity plays an important role in survival of BCR-ABL1 transformed cells, as shown by the induction of leukemic cell apoptosis in response to PIM pharmacological targeting. A possible mechanism for the induction of apoptosis may involve modulating phosphorylation of histone H3 on serine 10 that leads to effects on MYC-dependent transcription [Importantly, our studies demonstrate that PIM kinase inhibition of mTORC1 signals results in negative regulatory effects on mRNA translation of genes encoding for mitogenic proteins in BCR-ABL transformed cells, as evidenced by the effects on leukemic polysomal profiles and the inhibition of mRNA translation of the cription . Consistcription  but not cription , our fincription , 37. Notcription , 28, 29.in vitro. Notably, pharmacological PIM inhibition appears to exhibit activity against T315I-BCR-ABL1-expressing cells, suggesting that targeting the PIM kinase pathway may provide an approach to overcome resistance to tyrosine kinase inhibitors in CML. Future studies to test PIM inhibitors in in vivo mouse models of CML or Ph+ ALL, especially imatinib resistant models, is warranted and may have important clinical-translational implications for the treatment of CML and Ph+ ALL.Finally, we provide direct evidence that PIM inhibition results in potent suppression of primitive leukemic progenitors from patients with CML and enhances antileukemic responses to imatinib mesylate Ba/F3 p210 WT and kinase domain mutations including T315I, E255K, H396R, and Y253F were kindly provided by Dr. Brian J. Druker . K562, KT1, and Ba/F3 p210 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). BV173, BV173R  and LAMAImmunoblotting experiments were performed as previously described . All antThese assays were performed essentially as previously described . BrieflyThese assays were performed essentially as in our previous studies . PeripheBriefly, cells were washed twice with Dulbecco's phosphate buffered saline (DPBS) with 100 μg/ml cycloheximide and then lysed in lysis buffer . Lysates were then centrifuged at 12000g for 5 minutes at 4°C and supernatants were collected and snap frozen in liquid nitrogen. To isolate ribosomal fractions, lysates were layered on a sucrose gradient of 5 to 50%. Samples were centrifuged at 4°C for 110 minutes at 35000 rpm in a Beckman SW41-Ti rotor. The absorbance was measured at 254 nm continuously in an ISCO density gradient fractionator with the following settings: pump speed, 0.80 ml/min; fraction size, 10 drops per fraction; chart speed, 300 cm per hour; sensitivity, 1; peak separator, off; noise filter, 0.5 seconds. A 5% sucrose solution was used to set the baseline in an UA-6 detector for all experiments. Polysomal RNA from the fractions were isolated using a Qiagen AllPrep RNA/Protein kit. RNA was reverse transcribed using oligodT primers and the Qiagen Omniscript RT PCR kit, according to the manufacturer's instructions. Quantitative real-time (RT) PCR was carried out by using validated Taqman FAM-labeled probes, and Taqman PCR master mix (Life Technologies) according to the manufacturer's instructions. The probes used were as follows: GAPDH, Hs99999905_m1; Cyclin D1, Hs00765553_m1. GAPDH was used for normalization as indicated, and results were plotted as a ratio of Cyclin D1/GAPDH.5 of K562 or KT1 cells were plated per well and treated for 24 hours with SGI-1776, imatinib mesylate, or SGI-1776 plus imatinib mesylate. Samples were stained with Annexin V and 4′-6-diamidino-2-phenylindol (DAPI)-DNA stain according to the manufacturer's instructions (BD Biosciences). Fluorescence was measured on stained cells using a LSRFortessa Analyzer (BD Biosciences). Data was analyzed using FlowJo software .Briefly, either 1 or 2 × 10All statistical analyses were performed using Prism GraphPad 6.0.


SHANK-associated RH domain interactor (SHARPIN) inhibits integrins through interaction with the integrin α-subunit. In addition, SHARPIN enhances nuclear factor-kappaB (NF-κB) activity as a component of the linear ubiquitin chain assembly complex (LUBAC). However, it is currently unclear how regulation of these seemingly different roles is coordinated. Here, we show that SHARPIN binds integrin and LUBAC in a mutually exclusive manner. We map the integrin binding site on SHARPIN to the ubiquitin-like (UBL) domain, the same domain implicated in SHARPIN interaction with LUBAC component RNF31 (ring finger protein 31), and identify two SHARPIN residues  required for both integrin and RNF31 regulation. Accordingly, the integrin α-tail is capable of competing with RNF31 for SHARPIN binding in vitro. Importantly, the full SHARPIN RNF31-binding site contains residues (F263A/I272A) that are dispensable for SHARPIN-integrin interaction. Importantly, disrupting SHARPIN interaction with integrin or RNF31 abolishes SHARPIN-mediated regulation of integrin or NF-κB activity, respectively. Altogether these data suggest that the roles of SHARPIN in inhibiting integrin activity and supporting linear ubiquitination are (molecularly) distinct. Integrins are heterodimeric transmembrane receptors composed of an α- and a β-subunit that mediate cell interaction with the extracellular matrix and neighboring cells. Binding of extracellular ligands or intracellular integrin activators, such as TALINs or FERMTs , induces a pronounced conformational change in the integrin heterodimer. This conformational switch results in integrin activation and the formation of a large macromolecular complex at integrin cytoplasmic tails that connects the adhesion site to the actin cytoskeleton and activates a plethora of intracellular signaling pathways . DeregulNuclear factor-kappaB (NF-κB) is an oncogenic and pro-inflammatory transcription factor that mediates the cellular response to a wide array of stimuli. SHARPIN was identified as an essential component of the Linear Ubiquitination Assembly Complex (LUBAC) –8, whichThese studies demonstrate that SHARPIN regulates at least two important, yet seemingly distinct cellular pathways: integrin activation  and canoDirect interaction between SHARPIN and integrin α-tails or RNF31 is essential for SHARPIN-mediated integrin inhibition  and LUBA181-310 was not included due to very low expression levels. Interestingly, the ITGAL tail was found to interact with all SHARPIN fragments containing the central UBL domain, but not the N- or C-terminal fragments, suggesting that the UBL domain contains the integrin-binding site  superfold that mediates homomultimerization ,11, the ing site . Far-WesAL-ITGB2  confirmeV240A/L242A, SHARPINE260A/L261A, SHARPINL261A/F263A, SHARPINV267A, SHARPINI272A and SHARPINL276A. SHARPINI272A was shown previously to abolish RNF31 binding  were pre-incubated with 250 mM membrane-permeable SHARPIN binding-tail peptide (α1-TAT) or scrambled peptide (ScrTAT)  were plated in serum free, antibiotic free medium on wells coated with 5 μg/ml fibronectin (Sigma) or 10 μg/ml poly-L-lysine (Sigma) for 16 h. Subsequently, cells were incubated for 5 h with or without 50 ng/ml TNF in serum free, antibiotic free medium, before being processed as described above (n = 2 with 5 replicates each).2 and 1 mM DTT in Tris-buffered saline (TBS)), wells were incubated 3 h at room temperature with 0.1 mM of recombinant WT or mutant GST-SHARPIN in blocking buffer. After washing with blocking buffer, samples were incubated for 1 h with anti-GST antibody in blocking buffer, followed by 1 h incubation with anti-Rabbit horseradish peroxidase (HRP)-conjugated secondary antibody in blocking buffer. HRP activity was detected with the TMB substrate kit and the Multiscan Ascent plate reader (both Thermo Scientific).Recombinant RNF31 was coated on 96-well plates  overnight at +4°C. After blocking with blocking buffer . The Student's t-test was used for normally distributed data . For all other experiments the Mann-Whitney test was used. A p < 0.05 was considered significant.All statistical analyses were performed using GraphPad Prism version 5.03 for Windows  Western blot analysis of WT or mutant GFP-SHARPIN in HeLa cells. (B) Western blot analysis of GFP alone or WT or mutant GFP-SHARPIN in CHO cells. Also the levels of RFP-TALIN head were determined. Non-transfected CHO cells were used as control.(TIF)Click here for additional data file.


Mitochondrial RNA polymerase produces long polycistronic precursors that contain the mRNAs, rRNAs and tRNAs needed for mitochondrial translation. Mitochondrial RNase P (mt-RNase P) initiates the maturation of the precursors by cleaving at the 5′ ends of the tRNAs. Human mt-RNase P is only active as a tripartite complex , whereas plant and trypanosomal RNase Ps (PRORPs)—albeit homologous to MRPP3—are active as single proteins. The reason for this discrepancy has so far remained obscure. Here, we present the crystal structure of human MRPP3, which features a remarkably distorted and hence non-productive active site that we propose will switch to a fully productive state only upon association with MRPP1, MRPP2 and pre-tRNA substrate. We suggest a mechanism in which MRPP1 and MRPP2 both deliver the pre-tRNA substrate and activate MRPP3 through an induced-fit process. MRPP1 (MRPP1-3)  and perfmt-tRNAs –9. This mt-tRNAs ; in fact or m1G9 . The metproteins . Furtherproteins ,12.Arabidopsis thaliana) that function as single-subunit mt-RNase Ps  family, with a typical and functional two-metal-ion catalytic site that has conserved aspartate residues . Single and double mutants were generated by site-directed mutagenesis. Longer sequence replacements  were created by In-Fusion HD cloning (Clontech), using gBlocks (IDT) for the longer replacements. All of the constructs were verified by DNA sequencing.Human MRPP3 and different N-terminal truncations of MRPP3 were cloned from the Mammalian Gene Collection (gi:21595090) into the pNIC28-Bsa4 (gi:124015065) vector for expression of N-terminally His6-tag were expressed in Escherichia coli strain KRX (Promega) harboring a pRARE2 plasmid (Novagen). The proteins were purified using nickel-affinity and size exclusion chromatography. The N-terminal His6-tag was removed by TEV cleavage. The protein, which was in 20 mM HEPES pH 7.5, 300 mM sodium chloride, 10%(v/v) glycerol and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP), was concentrated by ultrafiltration to 5–25 mg/ml and stored at –80°C. MRPP1  and MRPP2  were produced as described previously  at a concentration of 25 mg/ml was crystallized using the vapor diffusion method at 298 K. Crystals were obtained in 0.28 M sodium malonate pH 7.0 and 16%(w/v) PEG 3350 ; in 30 mM potassium citrate pH 6.0, 16%(w/v) PEG 3350 and 20 mM magnesium chloride ; or in 100 mM HEPES pH 8.0, 30%(w/v) PEG 6000 and 20 mM magnesium chloride . Crystal 3 was further soaked with 100 mM magnesium chloride in the reservoir solution. All of the crystals were cryoprotected using the reservoir solution supplemented with 17%(v/v) glycerol and all data collections were performed at 100 K. Anomalous X-ray diffraction data from crystal 1 were collected at beam line P14 at PETRA-III, EMBL/DESY, Germany, using a wavelength of 1.2777 Å. For crystals 2 and 3, X-ray diffraction data were collected at a wavelength of 0.97625 Å at beam line ID29 at ESRF , France,www.pymol.org).The zinc ion in the anomalous data set of crystal 1 was located using PHENIX.AUTOSOLVE , which ain vitro using an MS2- and GlmS-based affinity purification setup  was gene synthesized (IDT). The final pre-mt-tRNATyr substrate was 141 ribonucleotides long and contained a 5′-leader of 43 nucleotides, the tRNA sequence of 65 nucleotides and a 3′-trailer of 33 nucleotides.The human pre-mt-tRNA was produced on setup . The DNATyr, 400 nM MRPP1/2 and 200 nM MRPP3 or its mutants. As a negative control (NC), 100 mM ethylenediaminetetraacetic acid (EDTA) were added to the reaction mix prior to the start of the cleavage reaction. The reactions were incubated at 30°C for 1.5 h and then quenched with 100 mM EDTA. The reaction products were resolved on 7M urea 8% polyacrylamide gels in TBE buffer. After electrophoresis, the reaction products were visualized by staining with SYBR Green II (Invitrogen).The mitochondrial RNase P activity was analyzed as described previously ,9. For aMature, full-length human MRPP3 (residues 45–583) persistently resisted crystallization. Activity assays showed that MRPP3 lacking part of the N-terminal flexible region  was fully active, while removal of the N-terminal flexible region and of PPR motif 1–2  gives raise to inactive enzyme  intercalates between the metallonuclease and PPR domains. The lariat loop winds through a network of water-mediated hydrogen bonds , which comprises a four-stranded, anti-parallel β-sheet (β1 and β8–10) and harbors the conserved zinc-binding site. The zinc-binding site is flanked on one side by an extended loop region that succeeds strand β1 Figure , while ts Figure . Specifis Figure . FurtherThe overall structure of the metallonuclease domain (residues 361–541) belongs to the NYN family  and compP, D474P, D475P and D493P coordinate two magnesium or manganese ions (where P denotes PRORP1 numbering). Furthermore, N492P, which precedes the active-site residue D493P in PRORP1, forms a bulge in the backbone and interacts with the backbone atoms of I519P, located in PRORP1 β6. This interaction is likely to stabilize the active-site area. In human Δ206-MRPP3, however, the equivalent residue to N492P is R498, which occupies the metal-binding site, and forms a salt bridge with D478. At the same time, D499 is flipped out of the active site to hydrogen bond with the S569 of the lariat loop. In addition, the PRORP1 and Δ206-MRPP3 active-site areas differ around D478 and D479 as their relative positions are swapped in comparison to those of D475P and D474P in PRORP1  rendered the enzyme inactive, while D478N and D409N (D475P and D399P) retained very low residual activity and partial activity, respectively.The lack of metal ions bound in the active site was puzzling; therefore, in order to eliminate the possibility that this was due to the presence of metal-chelating citrate or malonate in the initial crystallization conditions, Δ206-MRPP3 was crystallized in alternative buffers  in the presence of excess MgP) was produced in which the complete MRPP3 metallonuclease domain was replaced by the PRORP1 metallonuclease domain. Cleavage tests showed that MRPP3-MNDP had very weak cleavage activity in the presence of MRPP1/2 and was still completely inactive on its own . However, the mt-RNase P activity tests clearly showed that all of the mutants were able to perform the 5′-cleavage reaction in the presence of MRPP1/2 but not in their absence Figure . In a mon Figure .A. thaliana PRORP1 . Interestingly, the lariat loop sequence is strongly conserved in higher vertebrates . To test whether removing the lariat loop from MRPP3 would result in MRPP1/2-independent activity, we replaced the MRPP3 lariat loop with a short linker (GSG). However, yet again, the MRPP3 mutant was only active in the presence of MRPP1/2 .The second key difference is the organization of the lariat loop. In Δ206-MRPP3, the lariat loop forms an extended hydrogen bond network between the PPR and metallonuclease domain Figure . In contΔ206-MRPP3 has the same overall structure as its plant homolog PRORP1; however, there are significant structural differences between the two proteins. The most striking difference is that the active site of Δ206-MRPP3 is in a non-productive conformation, in which no divalent metal ions are present. Furthermore, D499, which was shown to be essential for enzymatic activity, is rotated away while the preceding R498 occupies the proposed metal-binding site. Despite multiple attempts, it was not possible to load divalent metal ions into the active-site area. This supports the conclusion that the presence of a non-productive active site is physiologically authentic and not a crystallization artifact.The non-productive active site observed in our MRPP3 structure raised the question as to whether MRPP3 is really the metallonuclease that is responsible for the endonucleolytic activity of mt-RNase P. To test this, we mutated the proposed metal-binding aspartic acid residues, which resulted in partial to complete inhibition of the enzymatic activity. This indicates that a proper metal-binding site, probably very similar to the PRORP1 active site, indeed must form in MRPP3 in order to perform the cleavage reaction. Similar active-site mutants have been created for PRORP family members in plants ,15,17,29A. thaliana PRORP1 show only a few differences around the active-site area. Therefore, we anticipated that we could rebuild the MRPP3 active-site area following the architecture of A. thaliana PRORP1. This included replacing individual residues up to replacing the whole metallonuclease domain. However, we were not able to generate a human MRPP3 variant that was enzymatically active as a single protein. Hence, MRPP3 activity without MRPP1/2 does not hinge on just a few key sequence differences in the active site—or even differences in the metallonuclease domain as a whole—but on larger differences in the overall structure. We propose that MRPP3 is kept in a non-productive conformation by a dual-control mechanism, namely by the presence of a non-productive active site and by the orientation of the individual domains toward each other. However, structural information about MRPP1/2/3 in complex with the pre-tRNA substrate is required to fully elucidate how MRPP3 is transformed into a productive state in the tertiary complex.Sequence and structural comparisons of human MRPP3 and A. thaliana PRORP1 points toward large differences in the localization of the individual domains. While Δ206-MRPP3 has a relaxed, twisted, L-shaped conformation, PRORP1 adopts a compact, V-shaped conformation in which all domains are located in one plane . Unfortunately, we could not achieve soluble expression of MRPP3 variants lacking the complete N-terminal flexible region while keeping all five PPR domains intact. However, similar experiments performed on ectively . Since tectively , a simil1A9 or m1G9 methylation for proper cloverleaf folding of the majority of the human mitochondrial tRNAs (Given the role of mt-RNase P in the early stages of mitochondrial RNA processing –5 and thal tRNAs ,10, it iThe atomic coordinates and structure factors for MRPP3 crystals 2 and 3 have been deposited in the Protein Data Bank with the accession numbers 4XGL and 4XGM, respectively.Supplementary Data are available at NAR Online.


In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. The mTORC1 complex integrates growth factor signals with the nutrients signals to control cell growth and proliferation2The mTORC1 is a major anabolic regulator that controls an array of macromolecule biosynthetic processes, such as protein translation, mRNA transcription, ribosome biogenesis, lipid biogenesis, autophagy, mitochondrial function and the immune response591012Pyruvate kinase (E.C. 2.7.1.40) is a rate-limiting glycolysis enzyme that catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, resulting in the formation of pyruvate and ATP15Proposed underlying tumorigenic mechanisms of PKM2 include facilitating anabolic metabolism by diverting glycolytic intermediary metabolites to anabolic pathways202025Dimeric PKM2 was initially found to phosphorylate histone H3 at T11 upon EGF receptor activation333CN), respectively. Phospho-peptides were enriched by Ti4+ -IMAC microspheres and the relative abundance between PKM2-untreated and -treated phosphor-peptides were compared by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS)  .K270M mutantin vitro substrates identification, as this ratio was well beyond the range of inherent variability.Consistent with that wild-type PKM2, not its inactive PKM2Among the positively identified substrates of PKM2, 876 are serines, 81 are threonines and 17 are tyrosines, confirmed that PKM2 is a multi-specific protein kinase. WebLogo analysisCellular compartment analysis by WoLF PSORT2728Gene ontology annotationsTyrosine substrates of PKM2 were not subject to bioinformatics analysis because the number of identifications was insufficient for statistical analysis, due to that insufficient extraction of membrane fraction, where most phospho-tyrosine proteins are located. In line with this expectation, we did see increase of phosphor-tyrosine in the membrane fraction by PKM2 overexpression .Y105FK270M mutant with AKT1S1 augmented the P-Ser level of AKT1S1 (The phosphorylation levels of serine 202 (S202) and serine 203 (S203) of mTORC1 inhibitor AKT1S1 were increased by 177.0 fold and 137.7 fold, respectively, by PKM2 treatment , suggestf AKT1S1 , confirmK270M, treatment , phospho-S203 (P-S203) and phospho-S202/203 (P-S202/203) of AKT1S1 by employing synthetic phosphorylated AKT1S1 peptides as antigens . The levreatment , confirmS202/203 . Convers3T cells . These rK270M, decreased the interaction between AKT1S1 and raptor in HEK293T cells  or AKT1S1S202E,S203E (2SE)  . These r3E (2SE) , suggestIn vitro treatment of purified AKT1S1 by recombinant PKM2 increased its interaction with 14–3–3; however, the same treatment failed to increase the interaction between the 2SA mutant and 14–3–3  on raptor25/46–4EBP . Interes/46–4EBP . These r of PKM2 . Moreovet AKT1S1 , but nottant 2SE , into thActivation of mTOR signaling usually requires nutritional and hormonal signalsp53+/+ and HCT116 p53−/− cells cultured under serum starvation  and breast cancer , PKM2 was overexpressed in cancer tissues, alongside with elevated phosphorylation of S202/203, phosphorylation of mTOR and phosphorylation of 4EBP that signaled activation of mTOR signaling, and elevated p62 that signaled inhibited autophagy. These results provided evidence that PKM2 activates mTOR signaling and inhibits autophagy in vivo.To confirm that PKM2 expression activates mTOR signaling and inhibits autophagy, we analyzed markers of each molecular events in renal cell carcinoma (RCC) and breast cancer samples. Immunohistochemistry (IHC) analysis was performed to 10 samples of each tumor that contained both normal and cancer tissues to show differential expression of each marker in normal and cancer tissues. In both RCC , S6A andt cancer , S6B, PKIn the current study, we report as far as we acknowledged the first phosphorylome of PKM2, shed lights on the plethora functions of PKM23353The finding that PKM2 phosphorylates S202/203 of AKT1S1 revealed a linkage between PKM2 overexpression and mTOR activation in cancer cells. Previous phosphorylomic studies had repeatedly found that Ser202 and Ser203 are phosphorylated by unidentified upstream kinases5556575961in vivo. Given that PKM2 expression and its protein kinase activity is involved in cell cycle regulationIn RCC and breast cancer samples, strong correlations had been found among PKM2 overexpression, S202/203 phosphorylation, mTOR activation and autophagy inhibition , consist564+/+ and HCT116−/− were cultured in McCoy′5A containing 10% FBS. Cells were transfected with plasmids using polyethylenimine . AKT1S1 S202A, S202E, S203A and S203E were cloned into PRK7-Flag, PKM2 and K270M was cloned into pcDNA3.1-Flag, pcDNA3.1-Myc and pcDNA3.1-HA, respectively.HEK293T and HeLa cells were cultured in DMEM containing 10% NCS. HCT116Chloroquine diphosphate salt (c6628-25 g) was from Sigma-aldrich, TEPP46 was from Cayman. Antibodies were either home-made or commercially purchased.E. Coli BL21(DE3)plysS overexpressing pET22b-His-PKM2, respectively. Proteins or proteome of HEK293T were incubated with 1 mg/ml PKM2 in kinase buffer contains 100 mM KCl, 50 mM MgCl2, 1 mM DTT, 1 mM NaVO4, 5% glycerol, 30 mM HEPES (pH 7.6) at 30 °C for 30 minutes. For in vitro dephosphorylation, proteins or proteome were incubated in dephosphorylation buffer that contains 50U alkaline phosphatase (Sigma), 1 mM MgCl2, 1 mM ZnCl2, 50 mM HEPES (pH 7.5) at 30 °C for 120 minutes.The recombinant Flag-PKM2, His-PKM2 and its mutant were affinity purified from HEK293T cells overexpressing pcDNA3.1-Flag-PKM2 and from 4+ -IMAC microspheres were used to enrich the phosphopeptides following the reported protocol. For mass spectrometric analysis, a quaternary surveyor MS pump  coupled with a LTQ Orbitrap XL mass spectrometer  was used. Data analysis and detailed parameters are described in the Tryptic peptides of PKM2 treated and untreated proteome of HEK293T cells were heavy and light labeled, respectively, as described in text. The TiStandard procedures were followed for western blot, dot blot and Immunofluorescence analysis. Western blot signals, dot blot signals were obtained by detecting chemiluminescence on Typhoon FLA 9500 . Scoring of Immunofluorescence of LC3B was analyzed using ANOVA with Turkey’s post-test .http://had.co.nz/ggplot2/) in the R package (http://www.r-project.org/). The amino acid preferences were visualized with WebLogo, while the comparisons of amino acid preferences were visualized with Two Sample Logo.All analysis was performed using published software. The gene ontology annotations were downloaded from QuickGO database, while the enrichment analyses were performed with a hypergeometric distribution. The enrichment analyses of KEGG pathways were carried out using DAVID. All the heatmaps were visualized with the ggplot2 program  at 99 °C for 60 minutes. The endogenous peroxidase activity was inactivated in solution of methanol with 3% H2O2. The slides were incubated with primary antibody for 60 minutes and secondary antibody for 8 minutes, followed by DAB Chromagen stainning for 8 minutes. All procedures were performed using stainer  and the slides were scanned by scanner (Ventana iScan Coreo). The quantification of IHC results were performed by an experienced pathologist. The intensity was calculated according to positive areas and positive degree. Sections were staining with PKM2 (1:100), P-S202/203-AKT1S1 (1:30), P-S2448-mTOR (1:100), P-T37/46-4EBP1 (1:500), p62 (1:200) and LC3B (1:100) antibody using an Ultraview Detection Kit.Significant differences between groups were determined using Student’s T test. The significance level for statistical testing was set at p < 0.05.How to cite this article: He, C.-L. et al. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1. Sci. Rep.6, 21524; doi: 10.1038/srep21524 (2016).


AD) patients mount IgE antibody responses to a variety of environmental allergens and also to autoantigens. We analyzed serum samples from four AD patients who had received oral cyclosporine A (CyA) treatment for up to 17 months regarding IgE autoreactivity to nitrocellulose‐blotted human epithelial cell extracts and IgE levels to environmental allergens by quantitative ImmunoCap measurements. Skin inflammation was assessed by SCORAD. During full‐dose treatment, a strong reduction in T‐cell‐mediated skin symptoms was observed which reappeared when CyA treatment was reduced or stopped. The intensity of IgE autoreactivity seemed to follow skin inflammation as it was reduced during full‐dose treatment and increased upon inflammation. Interestingly, IgE levels to exogenous allergens were boosted by allergen exposure, declined thereafter, and seemed to be unaffected by CyA. Our data thus indicate that allergen‐specific IgE production is boosted by allergen contact and cannot be reduced by CyA‐mediated T‐cell suppression.Atopic dermatitis ( Type I allergy is an IgE‐mediated hypersensitivity disease affecting almost 25% of the population in industrialized countries In this study, we had the opportunity to investigate the effects of treatment with systemic cyclosporine A, a T‐cell‐targeting drug on allergen‐specific IgE production. Sera from patients with atopic dermatitis who had received systemic CyA treatment for up to 17 months were studied regarding IgE reactivity to exogenous, respiratory allergens and autoantigens.Dermatophagoides pteronyssinus) were performed using the CAP FEIA system . Clinical staging was done according to the criteria of Hanifin and Rajka Residual serum samples from four atopic dermatitis patients, three males, one female, aged between 31 and 54, fulfilling the clinical and morphological criteria of AD 125I‐labeled antihuman IgE antibodies  as described The human epithelial cell line A431 was obtained from the American Type Culture Collection . Cells were cultured and proteins extracted as described A strong improvement of skin symptoms as reflected by a reduction in the SCORAD was observed when patients received full‐dose CyA treatment during the first 4 months Figs B, C. SimStrong increases of Bet v 1‐specific IgE levels were observed in the birch pollen allergic patient 2 shortly after the birch pollen seasons during the observation period  grants P23350‐B11 and F4605.Acquisition of data was done by SL. PSG, DK, BW, RV, and BL contributed to conception and design of the study, analysis and interpretation of data, and drafting of the manuscript.RV and BL have received research grants from the Austrian Science Fund (FWF). RV is consultant to Thermo Fisher Scientific, Uppsala, Sweden, Biomay AG, Vienna, Austria,and Fresenius Medical Care, Bad Homburg, Germany.Figures S1 and S2. Time courses of cyclosporin A treatment, skin symptoms, and IgE antibody reactivities to autoallergens and exogenous allergens in two AD patients. (A) Cyclosporin A dose (y‐axes: mg/kg bodyweight/day), (B) SCORAD documentation of skin manifestations (y‐axes: SCORAD indices), (C) IgE auto‐reactivity to nitrocellulose‐blotted human epithelial cell extracts are displayed for different time points of blood sampling. Molecular weights (kDa) are shown at the left margin and (D) time course of specific serum IgE levels (y‐axes: kUA/L) to exogenous allergens . An arrow (“>“) indicates the time point of tissue damage due to massive sun exposure in patient 3.Click here for additional data file. Click here for additional data file.


Surface ponding and ice layers such as the one we report are likely to form on a wider range of Antarctic ice shelves in response to climatic warming in forthcoming decades.Surface melt ponds form intermittently on several Antarctic ice shelves. Although implicated in ice-shelf break up, the consequences of such ponding for ice formation and ice-shelf structure have not been evaluated. Here we report the discovery of a massive subsurface ice layer, at least 16 km across, several kilometres long and tens of metres deep, located in an area of intense melting and intermittent ponding on Larsen C Ice Shelf, Antarctica. We combine borehole optical televiewer logging and radar measurements with remote sensing and firn modelling to investigate the layer, found to be ∼10 °C warmer and ∼170 kg m The influence of surface ponding on the interior of ice shelves is currently unknown. Here, the authors combine surface and borehole geophysics on the Larsen C Ice Shelf, Antarctica, with remote sensing and modelling and show how pond refreezing increases ice shelf density and temperature. The implications of the layer's presence for ice-shelf thickness estimates, flow and stability are explored.In this study, we report the presence of a massive ice layer, at least 16 km across, several kilometres long and tens of metres deep, present beneath an area of intermittent pond formation on LCIS, Antarctica. We combine field data with firn-modelling and remotely sensed data to investigate the layer's properties and formation. The layer is found to be composed of two units: an upper, solid ice unit formed largely from the continual refreezing of ponded water; and a lower, infiltration ice unit formed largely from the refreezing of meltwater that has percolated into very dense firn. The layer is found to be ∼10 °C warmer and ∼170 kg m−3. −3) than the underlying ice layer , which is principally composed of bubbly host ice containing coarse, irregularly dipping bubble-free ice layers. The proximity of this generally massive ice layer to the shelf surface, and the sharpness of its contact with the overlying snowpack, preclude formation by the usual process of compaction–metamorphism. Instead, we interpret this Unit 1 ice as having formed as a consequence of refreezing, following periods of intense surface melting and intermittent pond formation. The characteristics revealed by the OPTV image are consistent with this unit (2.9 to ∼45 m) being largely bubble-free pond ice formed from the refreezing of surface water ponded during extended periods of intense, presumably summer, melting. Here the fine-scale horizontal layering apparent in In early austral summer 2014/2015, we drilled a ∼100-m long borehole into the flank of a Cabinet Inlet trough indicated by satellite imagery to have repeatedly hosted melt ponds over the past 15 years, but not since 2008/2009. Although the shelf was snow-covered at the time of drilling, inspection of the wall of a 2-m deep pit revealed the presence of both numerous ice layers within the snowpack and an unusually thick ice layer at a depth of 2.0 m that prevented continued excavation. The borehole was logged to a depth of ∼97 m by optical televiewer (OPTV)15We evaluate this hypothesis for the recent past through a one-dimensional firn densification and hydrology modelAn approximation of the lateral extent of the massive subsurface ice layer we report is provided by the area known from satellite images to host melt ponds: ∼60-km across-flow and ∼20-km along-flow . However−3 for the shelf's uppermost ∼100 m, based on inverting seismic data recorded in the shelf's southern sector. In contrast, our OPTV-derived densities indicate a measured density of ∼870 kg m−3 over this depth range in the Cabinet Inlet area, 24% higher. Such a density enhancement influences calculations of the shelf's thickness based on the surface elevation data−3, a sea water density of 1,026 kg m−3, a surface elevation of 63.5 m  and material density (D) on the basis of samples recovered from a core retrieved from a borehole logged by OPTV on the Roi Baudouin Ice Shelf, East Antarctica. However, due to equipment loss, the light-emitting diode brightness of the OPTV probe used in the current study was different from that of Hubbard et al.D=950–40.1 eL)(0.0101 (R2=0.82), with root mean square values of the residuals of 40.4, 35.2 and 21.7 kg m−3 for the density ranges 600–700, 700–800 and 800–900 kg m−3, respectively. While these values provide an error range for absolute densities derived from OPTV luminosity, the relative changes in density reported herein, that is, along a single borehole log, reduce to the precision of the method. This is approximated by the density range (910 kg m−3) divided by the luminosity range (256), or ∼3.5 kg m−3.The borehole was drilled by pressurized hot water and logged by OPTV13Borehole temperatures  were recThe firn model used is IMAU-FDM v1.0, which takes into account firn compaction, meltwater percolation and refreezing17st December and 30th April from 2000 (Aqua) or 2002 (Terra) to 2015 were classified to generate the time series shown in MODIS Terra and Aqua level 1 (250 m) data were ordered via the level 1 and atmosphere archive and distribution system, and used to check for the presence of melt ponds in Cabinet Inlet. A total of 577 cloud-free band 2 (848 nm) images between 1−1 with an eight stack trace recorded every 3 m. A Leica VIVA GS10 GNSS rover unit was connected directly to the GPR console; the base station was located at the drill site. Surface elevation was referenced to the Earth Gravitational Model 1996 geoid, taken to represent sea level, and no correction was made for dynamic topography. GPR data were processed in Reflex-W. Processing steps included de-wow of 25-ns filter length, spherical divergence compensation, spectral whitening between 70 and 200 MHz, and a two-dimensional mean-averaging filter. The static correction was based on Leica Geo Office post-processed GNSS data, resulting in a vertical accuracy of ±0.5 m. Radar wave propagation velocities used to convert travel times to the depths shown in −1 for the uppermost 3 m  and a typical value for glacial ice−1 below that.GPR data  were colhttp://www.projectmidas.org/data/hubbard2016/ and from the corresponding author upon request.The data that support the findings of this study are available from How to cite this article: Hubbard, B. et al. Massive subsurface ice formed by refreezing of ice-shelf melt ponds. Nat. Commun. 7:11897 doi: 10.1038/ncomms11897 (2016).


Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear.in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks).Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation.Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation  Su. Su35]. A DNA construct of mNgn2, codon optimised for bacterial expression, was produced by Genecust (Luxembourg) in a pUC vector. This was placed into the pGEX  vector In vitro translation was carried out using the TNT® SP6 quick coupled transcription/translation system (Promega), in the presence of 35S-methionine , according to the manufacturer¿s instructions.in situ hybridization was transcribed in vitro using constructs with a linearised pCS2+ vector template. Transcription was performed as described previously ,,..In situ Xenopus embryos were injected with 20 pg of mRNA at the 1 cell-stage and embryos were allowed to develop to Nieuwkoop and Faber stage 15  HSQC 2D spectra were recorded at 277 K on a Bruker 600 spectrometer equipped with a triple resonance cryogenic probehead . Assignment was performed on a sample of doubly labelled protein at 150 ?M, using classical pairs of 3D experiments. Spectra were processed using Bruker TOPSPIN 2.1 . Peak picking was performed using Sparky .1 mM DThe data sets supporting the results of this article are included within the article and in its additional files. NMR datasets for 2D and 3D experiments are labelled as ¿2DNMR¿ and ¿3DNMR¿ respectively.Ngn2: Neurogenin2Xenopus laevisx: Mus musculusm: Homo sapiensh: bHLH: Basic Helix-Loop-HelixID: Intrinsic disorderin vitro translationIVT: pI: Isoelectric pointThe authors declare that they have no competing interests.in situ hybridizations and qPCR analysis. GM and IL purified protein and carried out the NMR studies, and IL analysed the data. AP and GL conceived the study, and participated in its design and coordination. GM, CH, IL and AP wrote the manuscript. All authors have read and approved the final manuscript.GM carried out degradation assays and bioinformatic analysis. CH carried out SP sites are conserved in the C-terminal domain. Ngn2 protein sequences from the NCBI database were aligned using ClustalW2. The bHLH domain is indicated by the green box and SP sites conserved in over half of all species are indicated by the red boxes.Click here for fileSP site xNgn2 mutants schematic. Illustration of the various SP site mutants of xNgn2 used in this study.Click here for file2D NMR dataset. Raw data for 2D NMR experiments.Click here for filemNgn2 CA and CB chemical shifts. Available CA and CB chemical shifts for visible residues.Click here for file3D NMR dataset. Raw data for 3D NMR experiments.Click here for file


The pressure–temperature phase diagram of ice displays a perplexing variety ofstructurally distinct phases. In the century-long history of scientific research onice, the proton-ordered ice phases numbered XIII through XV were discovered onlyrecently. Despite considerable effort, none of the transitions leading from thelow-temperature ordered ices VIII, IX, XI, XIII, XIV and XV to theirhigh-temperature disordered counterparts were experimentally found to display thefull Pauling entropy. Here we report calorimetric measurements on suitablyhigh-pressure-treated, hydrogen chloride-doped ice XIV that demonstrate at the maximum 60% ofthe Pauling entropy is released at the transition to ice XII. Dielectric spectroscopy on undoped and on variously doped ice XII crystals reveals that addition of hydrogen chloride, the agent triggeringcomplete proton order in ice XIV, enhances the precursor dynamics strongest. Thesediscoveries provide new insights into the puzzling observation that differentdopants trigger the formation of different proton-ordered ice phases. The preparation of fully proton-ordered ice phases remains challengingand residual entropy usually remains even at the absolute zero. Here, the authorsachieve a transition from disordered ice XII to fully ordered ice XIV triggered by HCldoping, which enhances proton transfer dynamics by five orders of magnitude. Theentropy change, ΔS=ΔH/Tc, at theorder/disorder transition can be quantified and compared with its theoreticalmaximum—given (approximately) by the Pauling entropy,ΔSP=R ln(3/2)  refs , 7, 8, 92O7 ref. .However2O7 ref. , 13, ice2O7 ref.  or ice Xet al.et al.Tc=273 K is rather high. For ices withTc<150 K, dopants are always required to facilitateproton ordering and thus to prevent immobilization of slow local motions. Orderparameters exceeding 82% of the theoretical maximum were apparently neverestablished for ice XI, despite considerable experimental efforth, doping with alkali hydroxide,notably KOH, is required26h cantransform into a proton ordered state, see ref. 28The proton-ordered phases displaying a near-maximum order parameter seem to be the onesmost abundantly investigated. Dielectric studies indicate that 83% of the maximumproton order was achieved  in ice VIII ref., whereas2019This work shows by means of calorimetry that HCl doping and suitable sample preparationallows one to achieve a transition from completely disordered ice XII to 60% orderedice XIV, the highest known proton order in ice XIV. Our dielectric spectroscopy studies reveal that the transition is triggered bya five orders of magnitude enhancement of the proton-transfer dynamics induced by HCldoping. The proton-ordering process is shown to be composed of a fast component,yielding about 20% of the Pauling entropy, and a slow component achieving 60%order.3or KOH as dopants (see Methods). For HCl and HF doping, this results in an iceXII→XIV transition. After recovery of the samples we use dielectricspectroscopy to explore potentially enhanced proton dynamics and calorimetry toexamine the degree of order achievable in ice XIV. These experiments are carriedout at ambient pressure on heating and they probe the XIV→XII transition,demonstrating that ice XIV can exhibit 60% proton order under suitablehigh-pressure preparation conditions. Only for HCl-doped ice XIV a pronouncedendotherm is detectable at an onset temperatureTc=102±3 K, which indicates conversionfrom ice XIV to ice XII, see Tc in protonated ice XIV and isin very good agreement with the onset (near 98 K) of lattice constantchanges at the transition of deuterated ice XIV/XII obtained by Salzmann etal.S,depends on the rate q with which HCl-doped ice XII was cooled at0.81 GPa to temperatures below Tc. Forq≤15 K min−1, we obtain valuesscattering between about 57% and 63% ofΔSP=3.4 J K−1 mol−1,whereas for25 K min−1<q<70 K min−1only ∼20% of the Pauling entropy are recovered. Thisdemonstrates that 60% proton order in ice XIV was achieved by coolingHCl-doped ice XII at 0.81 GPa andq≤15 K min−1 to 80 K. Bycontrast, for DCl-doped D2O ice XIV only partial ordering was foundfrom powder neutron diffraction experiments after cooling the sample at0.8 K min−1 from 180 to 80 K at1.2 GPaWith the goal to establish conditions enabling one to achieve maximum protonorder, it is advisable to start from an entirely proton-disordered phase such asice XII ref. . We cool−1) in the calorimeter, weregained only ∼12% of the Pauling entropy for the XII→XIVtransition (not shown). This finding is consistent with earlier Ramanq>30 K min−1, employed inour study resulted in a gain of ∼20% of the Paulingentropy, see q from ∼34 to 68 K min−1 doesnot reduce the entropy gain. It thus seems that the process of proton orderingin ice XII involves two stages: first, a rapid stage proceeding easily both atambient and at high-pressure conditions, which yields about20% of the Pauling entropy, and a second much slowerstage that takes place only at high pressures and yields about40% of the Pauling entropy. It appears plausible thatthe two processes are related to sets of crystallographically distinct hydrogenatoms, some of which do not order easily. For deuterated ice XIV, these wereidentified to be the deuteron pairs D4-12 and D5-13 , recordedevery 3 K on heating from 90 to 126 K are presented in νmax, which defines a motional correlation timeτmax=(2πνmax)−1,is seen to shift through the experimentally accessible frequency window. Allspectra display essentially the same shape. At the lowest temperatures, thedielectric loss maxima of ice XIV are out of the frequency range. Assuming thatsimilar to that for ice XII, for ice XIV the shape of the loss peak is virtuallytemperature invariant as well, cf. cf. Tc the static dielectric relaxation strength drops asexpected for  dipolar ordering. Reassuringly, the transitiontemperature, Tc=104±2 K, resulting fromthe dielectric experiments, confirms the calorimetrically determinedTc. Near Tc the activation energyE increases by about 70% (from 21 to36 kJ mol−1) as E (from 14.6 to32.6 kJ mol−1) was found near the iceIh→XI transitionLet us now demonstrate that the ice XIV→XII transition can be resolved notonly in thermodynamic but also in dielectric relaxation experiments for samplesprepared using a high-pressure cooling rate of3 and KOH, cf. h, IV,V, VI and undoped XII. Orientational glass transitions were observed for iceIh near 110 K , in which KOHpromotes proton ordering, whereas HCl does not. Thus, the role theCl− ion plays for the high-pressure ices XIII, XIV and XVis obviously taken over by the K+ ion  latticeRb+ ref. ) for the3 and KOH impurities are ineffective inaltering the motion in ice XII, whereas for HF doping its speed-up is moderate. Onlyaddition of HCl generates a dramatic dynamic enhancement. In combination with slowcooling at high pressures, this allows us to recover 60% of the Paulingentropy. Our calorimetry data imply that ∼25–35% of allprotons can readily be ordered in the presence of HCl dopants, both at high-pressureand at ambient-pressure conditions. The remaining 65–75% of all protonsin ice XII can solely be ordered using HCl doping together with slow cooling at highpressure. This suggests that the stresses associated with the change in space groupcan be released only at high pressures and with a significantly distorted localgeometry. Under these conditions, the four to five orders of magnitude enhancementof relaxation dynamics in HCl-doped ice XII that we discovered with dielectricspectroscopy is crucial for establishing the ice XIV phase. Hence, our experimentaldata show novel routes to an understanding of proton-ordering transitions andthereby paves the way for future computer simulation work directed at ultimatelysolving the riddle regarding the mechanism driving doping-induced proton-orderingtransitions.In summary, we show that NHet al.−1 to 185 K. At suchhigh heating rates the competing crystallization channel to ice IV iseffectively suppressed and pure ice XII forms. This was shown in earlier workfor pure water samplesIce XII can be obtained using different routes3041Kα,1,λ=1.5406 Å) inθ–θ geometry. Samples were cold loaded tothe sample holder through ambient air, which results in condensation of a tinyamount of hexagonal ice on the sample surface. All powder patterns, both fromdoped and undoped ices, show the pattern well known for ice XII , see the vertical dashed lines in Ice XII and ice XIV can be distinguished by X-ray diffraction on the basis of theorthorhombic splitting of the Bragg peak at 2Tc≈102 Kindicates the proton order–disorder transition from ice XIV to ice XII measurements were seen to fluctuate by upto ±10% .


Improvement of material property of spinal instrument has brought better deformity correction in scoliosis surgery in recent years. The increase of mechanical strength of instrument directly means the increase of force acts on bone-implant interface during scoliosis surgery. However, the actual correction force during correction maneuver and safety margin of pull out force on each screw were not well known. In the present study, estimated corrective forces and pullout forces were analyzed using a novel method based on Finite Element Analysis (FEA).Twenty Adolescent idiopathic scoliosis patients (1 boy and 19 girls) who underwent reconstructive scoliosis surgery between June 2009 and Jun 2011 were included in this study. Scoliosis correction was performed with 6mm of diameter titanium rod (Ti6Al7Nb) using the simultaneous double rod rotation technique (SDRRT) in all cases. Material properties of this rod are Young's Modulus, yield stress, yield strain and hardening coefficient equal to 105 GPa, 900 MPa, 8.57x10-3 and 2.41 GPa, respectively. The pre-maneuver and post-maneuver rod geometry were collected from intraoperative tracing and postoperative 3D-CT images, and 3D-FEA was performed with ANSYS. Cobb angle of major curve, correction rate and thoracic kyphosis were measured on X-ray images.Averaged age at surgery was 14.8 and averaged fusion length was 8.9 segments. Major curve was corrected from 63.1 to 18.1 degrees in average and correction rate was 71.4%. Rod geometry showed significant change on the concave side, and curvature of the rod on concave and convex side reduced from 33.6 to 17.8 degrees, from 25.9 to 23.8 degrees, respectively. Estimated pull-out forces at apical vertebrae were 160.0N in the concave side screw and 35.6N in the convex side screw. Estimated push-inn force at LIV and UIV were 305.1N in the concave side screw and 86.4N in the convex side screw.Corrective force during scoliosis surgery was demonstrated to be greater in concave side about 4 times than in convex side. Averaged pull out and push in force fell below previously reported safety margin (400 to 600N/screw), therefore SDRRT maneuver was safe for correcting moderate magnitude curve. For prevent implant breakage or pedicle fracture during maneuver in severe curve correction, mobilization of spinal segment by releasing soft tissue or facet joint could be more important rather than the use of stronger correction maneuver with rigid implant.


We demonstrate that expansion and retreat of sea ice varies consistently in pace with the rapid climate changes 90 kyr ago to present. Sea ice retreats abruptly at the start of warm interstadials, but spreads rapidly during cooling phases of the interstadials and becomes near perennial and perennial during cold stadials and Heinrich events, respectively. Low-salinity surface water and the sea ice edge spreads to the Greenland–Scotland Ridge, and during the largest Heinrich events, probably far into the Atlantic Ocean.In the light of rapidly diminishing sea ice cover in the Arctic during the present atmospheric warming, it is imperative to study the distribution of sea ice in the past in relation to rapid climate change. Here we focus on glacial millennial-scale climatic events (Dansgaard/Oeschger events) using the sea ice proxy IP The response of Arctic sea-ice to rapid climatic change in the past remains uncertain. Here, the authors use biomarkers and microfossils to reconstruct Arctic sea-ice changes over the past 90,000 years, and demonstrate millennial-scale variability. Dansgaard/Oeschger (D/O) events in Greenland ice cores consist of warm interstadial (IS) and cold stadial events24The Nordic seas are characterized by northward inflow of warm, saline Atlantic surface Water  and southward outflow of cold Polar surface Water (East Greenland Current and East Icelandic Current)628The Atlantic surface Water is ice-free throughout the year, while the East Greenland Current is covered by drifting near-perennial sea ice. In the central parts of the Nordic seas, mixing of Atlantic Water with Polar Water forms the zone of Arctic surface Water, which is located between the Arctic and Polar fronts1125 isoprenoid lipid (IP25) synthesized mainly by diatoms have shown its potential as a valuable new proxy for the reconstruction of the presence of seasonal sea ice141516171825 reportedly is produced by a few sea ice diatom species within the genera Haslea and Pleurosigma25 is a stable organic compound preserved in sediments as old as Late Miocene and Pliocene212214152525 to brassicasterol and dinosterol, the so-called PBIP25 and PDIP25, respectively, can be related to sea ice coverage on a scale from permanent sea ice to year-round open water. In both ends of the scale, the IP25 proxy is practically absent151626Previous studies of a C25, brassicasterol, dinosterol, total organic carbon (%TOC) and δ13Corg  Greenland ice core δ18O signal3The age model for the last 90 kyr interval of sediment core JM11-FI-19PC is based on well-known tephra layers , 11 AMS-4C dates  and 20 ml onsets . It has et al.The age control of the core JM11-FI-19PC younger than 65 kyr b2k has been presented by Ezat 25 record are similar to core MSM5/5-712-2 from the western Svalbard margin  and related short-term changes in surface-water productivity (reflected in the brassicasterol and dinosterol records and in the flux patterns of diatoms) seem to follow very clearly the D/O events , the interstadials of D/O events 6–2 are short-lasting, and because of the relatively low sampling resolution in JM-FI-19PC in this time interval (>300 years per sample) not all the interstadials are equally well resolved . A total25, maximum content of brassicasterol and dinosterol, and low PBIP25 and PDIP25 values  Hasle, an indicator of relatively warm temperatures323 in support of the interpretation based on diatom floras and organic biomarkers. Furthermore, the δ13Corg values are higher in interstadials, which point to a (relative) decrease of terrigeneous organic matter supply and/or an increase in marine organic matter inputInterstadials are characterized by absence or near absence of IP5 values . The , an abrupt decrease in the concentration of brassicasterol, dinosterol and high PBIP25 and PDIP25 values (Fragilariopsis oceanica (Cleve) Hasle3334163613Corg values might indicate decreased marine organic carbon flux due to the low productivity and/or an increased influence of terrigenous organic matter, probably being ice-rafted to the core site. The presence of near-perennial sea ice prevented heat exchange and the atmosphere was probably very cold. These last phases of the D/O events represent the stadial conditions of the Greenland ice cores.The cooling phase terminated in a phase of extended sea ice cover. This is associated with a maximum in IP25, respectively, absent or minimal brassicasterol and dinosterol, and maximum PBIP25 and PDIP25 values). In these intervals, the IP25 values of zero cannot be interpreted as absence of sea ice. Instead, the combined signals likely represent a thick permanent sea ice cover and very cold temperatures1525 can grow attached beneath the ice1416BIP25 and PDIP25 can occur  and cold polar conditions  were performed at the Department of Geology, UiT, Arctic University of Norway, Tromsø, Norway. Before opening the magnetic susceptibility (MS) was measured with a Bartington MS meter MS2 . After opening of the core, but before sampling, it was scanned for light and heavy elements, such as potassium (K) or titanium (Ti) with an Avaatech XRF core scanner in a resolution of 0.5–1 cm ref. .Samples taken at 5-cm intervals in 1-cm thick slices were weighed, freeze-dried and weighed again before wet sieving over 63 and 100-μm sieves. Tephra particles were counted in the size fraction >100 μm. The method for counting tephra particles followed the procedures described in Wastegård and RasmussenSubsamples at 5-cm intervals from the presented core section (801–2 cm) were used to determine TOC. For %TOC dried and powdered aliquots of the samples were treated with 10% hydrochloric acid (HCl), for carbonate removal, before being measured with a Leco CS-200.13Corg were measured every 5 cm in a powdered and carbonate-free aliquot of the samples, using a Finnigan MAT Delta-S mass spectrometer equipped with a FLASH elemental analyser and a CONFLO III gas mixing system for the online determination of the carbon isotopic composition. Measurements were conducted at the Alfred Wegener Institute Helmholz Centre for Polar and Marine Research in Potsdam, Germany. The s.d. (1σ) is generally better than δ13C=±0.15‰.Carbon isotopes δN. pachyderma s. For this, about 30 specimens in the size fraction 150–250 μm were analysed with a Finnigan MAT 251 mass spectrometer with an automated carbonate preparation device. Measurements were performed at the Department of Geosciences, at the University of Bremen, Germany. The external standard error of the oxygen isotope analyses is δ18O=±0.07‰. Values are reported relative to the Vienna Pee Dee Belemnite, and calibrated by using the National Bureau of Standards NBS18, 19 and 20.Oxygen isotopes were measured on specimens of the planktic foraminiferal species et al.2O2 37%; 10 drops) instead of nitric acid (HNO3 65%) to remove existing organic matter around the diatom valves. Quantitative diatom slides were produced as described in Koç Karpuz and Schraderet al.Chaetoceros spp. free basis444850The preparation method for diatom samples given in Koç For the interval between 78 and ∼15 kyr b2k numerous fragmented frustules were found. This might indicate that this interval was influenced by dissolution processes14166 (cholest-5-en-3β-ol-d6) were added as internal standards. Hydrocarbons and sterols were separated via open column chromatography (SiO2) using n-hexane (5 ml) and methyl-acetate:n-hexane , respectively. Sterols were silylated with 500 μl BSTFA 25 and sterols see Fahl and Stein25 is 0.01 ng on column. PBIP25 and PDIP25 were calculated from IP25, brassicasterol and dinosterol concentrations, respectively, according to Müller et al.The lipid biomarker analyses were performed at the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany. Freeze-dried and homogenized sediments (4–5 g) were extracted with an Accelerated Solvent Extractor  using a dichloromethane:methanol mixture (2:1 v/v). Before this step, 7-hexylnonadecane, squalane and cholesterol-d25 index to distinguish between different sea ice conditions, that coevally high amounts of both biomarkers (suggesting ice-edge conditions) as well as coevally low contents (suggesting permanent-like ice conditions) would give a similar or even the same PIP25 value. Especially, for the latter situation of permanent sea ice conditions both biomarker concentrations may approach values around zero and the PIP25 index may become indeterminable (or misleading). For a correct interpretation of the PIP25 data the individual IP25 and phytoplankton biomarker concentrations must be considered15et al.25 calculation. This modified PIP25 approach is based on biomarkers from the same group of compounds  with more similar diagenetic sensitivity, which is important for palaeo-sea ice reconstructions and comparison of records from different Arctic areas.It is important, when using the PIP14C dates of piston core JM11-FI-19PC have previously been presented by Ezat et al.N. pachyderma s 64Eleven AMS-yderma s . Measureet al.et al.18O) measured in the planktic foraminiferal species N. pachyderma s .Supplementary Figure 1, Supplementary Tables 1-4 and Supplementary References.


In human adherent monocytes matrix metalloproteinase-9 (MMP-9) expression and secretion is upregulated by the lipid moiety of natural haemozoin . A role for 15--hydroxy-6, 8, 11, 13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, has been suggested. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Either early or late p38 MAPK phosphorylation was induced by nHz, which did not modify basal phosphorylation and expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK. Lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Both nHZ and 15-HETE promoted the phosphorylation of MAPK-activated protein kinase-2, a known substrate of p38 MAPK. Such an effect was abolished by SB203580 (synthetic p38 MAPK inhibitor). SB203580 also abrogated the nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein levels in cell lysates and supernatants. These data suggest that nHZ and 15-HETE upregulate MMP-9 expression and secretion through the activation of p38 MAPK pathway in human adherent monocytes. This work provides new evidence on the mechanisms underlying MMP-9 deregulation in malaria. These data might help to design new specific drugs for adjuvant therapy in complicated malaria.


Draxin is an important axon guidance cue necessary for the formation of forebrain commissures including the corpus callosum, but the molecular details of draxin signaling are unknown. To unravel how draxin signals are propagated we used murine cortical neurons and genetic and pharmacological approaches. We found that draxin-induced growth cone collapse critically depends on draxin receptors , inhibition of protein kinase B/Akt, activation of GSK-3β (glycogen synthase kinase-3β) and the presence of microtubule-associated protein MAP1B. This study, for the first time elucidates molecular events in draxin repulsion, links draxin and DCC to MAP1B and identifies a novel MAP1B-depenent GSK-3β pathway essential for chemo-repulsive axon guidance cue signaling. During development of the nervous system growth cones at the tip of extending axons are guided to their targets with the help of guidance cues which can either attract or deflect axon growth . Growth A key target of guidance cue signaling is the growth cone cytoskeleton consisting of F-actin and microtubules . MicrotuIn the present study we set out to identify signal transduction pathways involved in draxin signaling. Given the similarity of phenotypes of draxin- and MAP1B-deficient mice we also investigated whether MAP1B mediates draxin-induced growth cone collapse and inhibition of axon extension. Using genetic and pharmacological approaches we were able to show that the PI3K/Akt/GSK-3β is necessary to transduce draxin signaling and that MAP1B is indeed involved.The current study does not contain in vivo experiments using live animals. Newborn mice were sacrificed by decapitation in compliance with the Austrian law regulating the use of animals in biomedical research, Tierversuchsgesetz, BGBl. Nr. 501/1989 and BGBl. I Nr. 162/2005. Since no experiments on live animals were performed, approval of the experiments by the Institutional Animal Care and Use Committee was not required according to the above cited law . Cells were co-transfected with 0.75 μg of an expression vector encoding GFP  to mark transfected cells and 2.25 μg of either pcDNA3 (empty vector control) or a construct encoding constitutively active Akt kinase  or a coIn general, treatments were carried out 60 h after plating except for experiments with transfected neurons where treatments were performed 26 h after transfection and plating to avoid the potential induction of apoptosis due to prolonged expression of Akt-DN ) at 1 μgCortical neurons were grown on glass coverslips and treated as described above. Following treatment cells were fixed by addition to the medium of an equal volume of 8% paraformaldehyde, 22% sucrose and incubated for 20 min. Fixed cells were washed twice with PBS and permeabilized with 0.1% of Triton X-100 in PBS for 10 min. Cells were blocked with 2% BSA in PBS containing 0.01%Tween 20 for 1 h, followed by staining with Texas red-conjugated phalloidin  for 1 h for visualization of F-actin. Images were acquired on an Axio-observer microscope. Quantification was carried out as described . Growth Pelleted cells were lysed in RIPA buffer (Sigma-Aldrich) as described  in the pAll values obtained in individual experiments are available in Draxin-induced growth cone collapse and inhibition of neurite outgrowth are suppressed in MAP1B-deficient neuronsThe similarity of corpus callosum and hippocampal commissure defects in draxin  and MAP1MAP1B is subject to posttranslational modification by GSK-3β which phosphorylates MAP1B at Ser1260 . To testWe next analyzed whether the draxin-induced GSK-3-dependent increase in MAP1B phosphorylation correlates with an increase in GSK-3β activity. GSK-3β is negatively regulated by Ser9 phosphorylation ,32. We fIn axon growth and guidance GSK-3β is a major downstream effector of PI3K and Akt with Akt negatively regulating GSK-3β by phosphorylating it on Ser9 ,32. ThusTo test whether GSK-3β activation is necessary for draxin-induced growth cone collapse we analyzed the effect of draxin in the presence of the specific GSK-3 inhibitor SB216763. Treatment with SB216763 had no effect on its own, but completely blocked the response of cortical neurons to draxin . These r3) in the plasma membrane which leads to translocation and activation of Akt. Thus, inhibition of PI3K will result in inhibition of Akt. Wortmannin induced growth cone collapse in cortical neurons to about the same extent as draxin and simultaneous presence of draxin and wortmannin had no additional effect  and expression vectors encoding constitutively active (Akt-CA) or dominant negative Akt (Akt-DN). Cells co-transfected with the GFP construct and empty vector served as controls. Neurons were grown for 26 h, treated with draxin and analyzed in parallel for growth cone collapse and expression of the Akt mutant proteins. We found that in cells ectopically expressing constitutively active Akt draxin failed to induce growth cone collapse . In contDraxin has been shown to exert its effects on axon growth through the netrin receptor DCC . To testDraxin has obtained significant interest as a novel chemo-repulsive guidance cue involved in development of the central nervous system, in particular the formation of forebrain commissures including the corpus callosum and the hippocampal commissure –10. Howe3 in the plasma membrane. PIP3 levels in turn reflect the relative activities of PI3K which synthesizes PIP3 and PTEN which hydrolyses it [The role of the PI3K/Akt pathway in neuronal morphogenesis and axon guidance signaling is well established . For exalyses it ,42. We slyses it  and neurlyses it . These flyses it –41.Xenopus laevis spinal neurons [Caenorhabditis elegans motor neurons [Our results raise the question as to how draxin might modulate PI3K/Akt signaling. One possibility is that draxin acts by binding to DCC. For one, we confirm a previous report  that dra neurons  and in C neurons . HoweverMAP1B is essential for axon guidance by cues as diverse as netrin-1, reelin, lysophosphatidic acid and nitric oxide –20. We sRemarkably, both attractive (netrin-1) and repulsive (draxin) guidance cues trigger phosphorylation of MAP1B on Ser1260. This modification by itself does not change MAP1B interaction with microtubules , but corThe essential role of GSK-3β in repulsive axon guidance has previously been characterized for semaphorin 3A . CrucialS1 ARRIVE Checklist(PDF)Click here for additional data file.S1 FigUncropped immunoblots for (TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.