Toll-like receptor 4 (TLR4) plays an important role in innate immunity by eliciting inflammation. Upon receptor engagement, transforming growth factor β-activated kinase 1 (TAK1) is an essential mediator that transmits a signal from the receptor to downstream effectors, IκB kinase (IKK) and the mitogen-activated protein kinases (MAPKs), which control the production of inflammatory cytokines. However, the association between phosphorylation and ubiquitination of TAK1 is not yet clear. Here, we examined the crosstalk between phosphorylation and polyubiquitination of TAK1 and further investigated the mechanism of distinct activation of MAPKs and IKK. Inhibition of TAK1 phosphorylation enhanced Lys63-linked polyubiquitination of TAK1. Conversely, ubiquitin modification was counteracted by phospho-mimic TAK1 mutant, TD. Moreover, using LC-MS analysis, Lys562 of TAK1 was identified as a novel Lys63-linked ubiquitination site and as the key residue in the feedback regulation. Mutation of Lys562 of TAK1 leads to a decrease in TAK1 phosphorylation and specific inhibition of the MAPK pathway, but has no effect on formation of the TAK1-containing complex. Our findings demonstrate a feedback loop for phosphorylation and ubiquitination of TAK1, indicating a dynamic regulation between TAK1 polyubiquitiantion and phosphorylated activation, and the molecular mechanism by which IKK and MAPKs are differentially activated in the TLR4 pathway. Transforming growth factor β-activated kinase 1 (TAK1 or MAP3K7), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, was first identified in the TGF-beta signaling pathwayMap3k7 deletion in neutrophils enhances activation of NF-κB and MAPKs after LPS stimulationThe IkappaB kinase (IKK)-nuclear factor-κB (NF-κB) pathway and MAPK cascades, such as p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) are two major effectors of TAK1. TAK1 functions as an essential and positive regulator of NF-κB and MAPK signaling in macrophages, fibroblasts and B cells, but serves as a negative regulator in neutrophils, indicating that TAK1 regulates signals in a cell-specific and receptor-specific mannerTLR4 is the most-studied of the TLRs. In general, TLR4 signaling is mediated by recruitment of adaptor proteins, such as MyD88 and toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-beta (TRIF), to the cytoplasmic portions of the receptors and classified as either MyD88-dependent or TRIF-dependent69Helicobacter pylori, and regulates the kinase activity of TAK126272830In addition to translocation to the cytosol, activation of TAK1 is tightly regulated through its binding partners and protein modifications. TAK1 activation requires the formation of complexes with its binding proteins TAK1-binding protein (TAB) 1, TAB2 or TAB3131416201123Biochemical and genetic evidence accumulated so far leaves no doubt that TAK1 plays a major role in the activation of NF-kappaB and the MAPKs in the TLR4 pathway, which is critical for innate immunity. However, the molecular mechanisms underlying TAK1-mediated signal activation, in particular the crosstalk between phosphorylation and ubiquitination, still await elucidation. In this study, we examined the role of TAK1 phosphorylation in activating the IKK and MAPK pathways, and in its interplay with polyubiquitination. Using mass spectrometry, we identified the Lys562 residue of TAK1 as a novel Lys63-linked ubiquitination site that forms a regulatory feedback loop with phosphorylation, and demonstrated that the polyubiqutination of TAK1 at Lys562 is critical for the activation of the MAPKs, but not IKKs, in the TLR4 pathway.In agreement with previous reports4in vivo ubiquitination assay was enhanced when TA- or D156A-TAK1 was expressed R mutant R mutants were overexpressed in HEK293T cells, respectively. Polyubiquitination was significantly decreased in K562R and KR, but not in K563R mutant R mutant showed decreased non-Lys48-linked polyubiquitination comparing with WT, and TA mutant enhanced non-Lys48-linked polyubiquitination. When TAK1 mutant TA, KR was co-expressed with ubiquitin WT or K48R mutant in HEK293T cells, the enhancement in polyubiquitination was blocked . HoweverSince non-Lys48-linked polyubiquitination was still able to be detected in K562R or KR mutated TAK1 as shown in The results above suggested that Lys562 of TAK1 is a novel non-Lys48-linked ubiquitination site. Next, to dissect the role of Lys562 in TAK1-mediated signaling TAK1-silenced RAW264.7 cells were reconstituted with TAK1 WT or KR mutant, and TAK1-mediated signal activation, including IKK, p38 and JNK, in TLR4 pathway was analyzed. Unlike the Lys158 site of TAK1 that has been reported to be required for TAK1-mediated IKK and MAPK activation in response to TNF stimulationWe previously demonstrated that MyD88-dependent TAK1 signaling in TLR4 proceeds via a two stage mechanism that involves receptor-induced assembly of a multiprotein complex, containing MyD88, TRAF6, IKKγ/NEMO, TAK1 and TRAF3, and degradative TRAF3-mediated translocation of the MyD88-associated signaling complex to the cytosolTAK1 mediates IKK and MAPK activation which in turn induce inflammatory cytokines, such as TNF and IL-6. Next, the role of Lys562 in TAK1-mediated gene expression was investigated. TAK1-silenced RAW264.7 cells were reconstituted with either TAK1 WT, TA, K562R, or KR mutants, and TAK1-induced mRNA expression, including that of TNF and IL6, in response to LPS stimulation was analyzed with RT-qPCR . It was 3738TLR4 signaling is a double-edged sword that protects the host from infection but also causes tissue damage, and therefore requires careful regulation and fine tuningIn this report, we examined the crosstalk between phosphorylation and polyubiquitination of TAK1 and investigated the mechanism through which the distinct activation of MAPKs and IKK is regulated. Although TAK1 inhibitor, 5Z-7-oxozeaenol, that inhibits kinase activity leads to inhibition of both IKK and MAPKs pathways, the results of the reconsititution of TAK1-silenced cells with TAK1 WT or mutant that blocks phosphorylation at Thr184 and Thr187 of the activation loop, suggest that phosphorylated TAK1 is important for MAPKs signaling, but less critical for the IKK pathway in response to LPS. We assume that the basal kinase activity of TAK1 is enough for IKK activation, which needs for further investigation. Moreover, TAK1 phosphorylation status is able to modulate the level of polyubiquitination. By inhibiting TAK1 activation with TA mutant or specific inhibitor, 5Z-7-oxozeanenol, TAK1 polyubiquitination is enhanced. Conversely, ubiquitin modification can be counteracted by the TAK1 phospho-mimic mutant, TD. These results reveal a feedback loop for phosphorylation and ubiquitination of TAK1, and indicate dynamic regulation of TAK1 polyubiquitiantion and phosphorylation activation. Moreover, Lys562 of TAK1 was identified as a Lys63-linked ubiquitination site and the key residue in the feedback regulation. Mutation in Lys562 of TAK1 leads to inhibition of TAK1 and subsequent MAPK pathway activation.in vivo ubiquitination assay to be modified with a Lys63-linked polyubiquitin chain. However, the role of TAK1 Lys562 does not conflict with the other ubiquitination sites. Although mutation in the Lys158 residue is unable to reverse the enhancement of polyubiquitination caused by blocking of TAK1 phosphorylation, TAK1 K158R mutant leads to inhibition of TAK1 and the downstream effector, NF-κB pathwayIt has been reported by different groups that TAK1 is modified with Lys63-linked polyubiquitination at specific residues, including Lys34, Lys158 and Lys209, in response to different stimuli. Those reports inferred that regulation of TAK1 occurs in a cell-type specific and ligand specific manner, and indicated the existence of multiple ubiquitination sites. Unlike the Lys63-linked ubiquitination sites2528In summary, our study demonstrates that the Lys562 residue of TAK1 is a novel Lys63-linked ubiquitinated site that is involved in feedback regulation of TAK1 phosphorylation. As shown in the working model in The following commercial antibodies were used: HRP-conjugated anti-mouse I gG (NA931), and anti-rabbit IgG (NA941) from GE Healthcare , HRP-conjugated anti-rat IgG (AP136P), and anti-ubiquitin Lys63 specific (05–1313) from EMD Millipore , anti-TAK1 (sc-7162), anti-IKKα/β.(sc-7607), anti-IκBα.(sc-371), anti-JNK (sc-7345), anti-p38 (sc-728), anti-Tab1 (sc-6052), anti-β-actin (sc-69879), and anti-ubiquitin (sc-8017) from Santa Cruz , anti-p-TAK1 (4531), anti-p-IKKα/β.(2078), anti-p-IκBα.(9246),.anti-p-JNK (9251), and anti-p-p38 (9211) from Cell Signaling , anti-HA (11867423001) from Roche , and anti-Flag  from Sigma . LPS, TNF, IL-1β,.5Z-7-oxozeaenol, dithiothreitol (DTT), iodoacetamide (IAA), and formic acid were obtained from Sigma. Acetonitrile (ACN) and ammonium bicarbonate were obtained from Aldrich . Sequencing-grade trypsin was purchased from Promega .Human HEK293T, human THP-1 and mouse RAW264.7 cells were obtained from American Type Culture Collection . The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 1% (v/v) penicillin-streptomycin solution, which were from Biological Industries . For ligand stimulation, cells were washed once with PBS, and incubated with 100 ng/mL LPS, 100 ng/mL Pam3CSK4, 0.1 μg/mL TNF or 0.1 μg/mL IL-1β, respectively, for the indicated times. When TAK1 inhibitor was used, the cells were pre-treated with 100 nM 5Z-7-oxozeaenol for 30 minutes.in vivo ubiquitination assay. pDUO-mMD2/TLR4 was purchased from Invivogen , and CMV-Flag-mouse TLR2 was kindly provided by Li-Chung Hsu . AP-1 Luc reporter vector from Michael Karin , and pGL4.32[luc2P/NF-κB-RE/Hygro] vector and pRL-TK Renilla luciferase control reporter vector purchased from Promega were for luciferase reporter assay. The lentivirus-based specific Map3K7 gene knockdown construct in the pLKO_TRC2 vector, and the packaging plasmids, pCMVΔ8.91 and pMD.G, were from the National RNAi Core Facility . The target sequence for 3′UTR region of mouse Map3K7 is as follow: 5′- AGAGAAGACAAACCATTATAA-3′. The target sequence for 3′ UTR region of human MAP3K7 is as follow: 5′- CGGAACCTTTAGGGATAGTTC-3′. Expression vectors encoding specific proteins were constructed by standard recombinant DNA technology. Site-directed mutagenesis was conducted using QuikChange mutagenesis kit from Agilent . All mutations were confirmed by DNA sequencing.The lentiviral vector, pLU-PL3, for protein expression was a gift from Andrei V. Budanov . pEF-HA-Ub was kindly provided by Ze’ev Ronai (Sanford-Burnham) for Lentiviral packaging was as previously describedhttp://rsb.info.nih.gov/nih-image/). The relative amount was calculated and the band with the highest intensity was set as 1. The data was presented as mean values from duplicated experiments.Cells were lysed in ice-cold RIPA lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethyl sulfonyl fluoride and 20 μg/ml of aprotinin. After centrifugation, cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting. The membranes were probed with the indicated antibodies. Blots were visualized by chemiluminescence reagent  exposed on X-ray film (Fujifilm). For densitometric quantification, the Western blot images were analyzed with ImageJ 1.48  was added into the lysis buffer. All samples were incubated with protein G–Sepharose (Roche) for 2 hours at 4 °C. The beads were washed three times with ice-cold lysis buffer or immunoprecipitation buffer, separated by SDS-PAGE and analyzed by immunoblotting with indicated antibodies. Blots were developed with chemiluminescence reagent.Flag-tagged protein and HA-tagged ubiquitin were coexpressed in HEK293T cells. 24 hours post transfection, cells were lysed with RIPA lysis buffer containing protease inhibitors and 20 mM NEM (Sigma). The lysate was immunoprecipitated with Flag antibody overnight at 4 °C, and pulled-down with protein G beads. The samples were washed three times with RIPA lysis buffer, gel-separated and analyzed by immunoblotting.Flag-tagged TAK1 WT, K562R, or KR mutants and Myc-tagged TRAF6 were overexpressed in HEK293T cells, respectively. The tagged proteins were immunoprecipitated with anti-Flag or anti-Myc antibodies. The Flag-tagged proteins were eluted from beads by incubating with 3 × Flag peptide at 4 degrees for 30 mins. The supernatant containing TAK1 proteins was collected and incubated with or without Myc-TRAF6 immunoprecipitated beads in the present of ATP, UBE1, E2 UbcH enzyme set, and either ubiquitin K63 only or ubiquitin K48R mutant (Boston Biochem) for 1 hour. The supernatant and beads were isolated and the reactions were stopped by adding sample buffer and heating at 95 degrees for 2 mins. The supernatant containing TAK1 protein and the beads containing TRAF6 were subjected for immunoblotting analysis.5 cells was isolated with TRIzol (Invitrogen), and used to synthesize first-strand cDNA with iScript cDNA synthesis kit (Bio-Rad). mRNA amounts were quantified by real-time quantitative polymerase chain reaction (RT-qPCR)cyclophilin A (PPLA) mRNA expression.Total cellular RNA from 1 × 105) were seeded in a 12-well culture plate overnight. After stimulating with the indicated ligands for 24 hours, the cytokine-containing medium was collected, and the protein level was measured with Mouse TNF-alpha DuoSet ELISA Development kit  or Mouse IL-6 ELISA set .Cells  following the manufacturer’s instructions.in vivo ubiquitination assay. Cell lysate with 25 mg total protein was incubated with 250 μl Flag-agarose (Sigma) at 4 oC overnight. Samples were gel-separated, and stained with Coomassie blue. Protein samples containing ubiquinated-TAK1 were recovered from the gel, and subjected to enzyme digestion according to the following procedure. After thermal denaturation at 95 °C for 5 min, 10 μL of protein samples (0.1 mg/mL) were reduced through the addition of DTT to a final concentration of 10 mM and incubated at 50 oC for 30 min. Alkylation was performed by adding IAA to a final concentration of 20 mM. After 30 min of incubation at room temperature in the dark, a second aliquot of DTT was added to quench the reaction. Trypsin solution (20 ng/μL) was added to protein samples  and the reaction was incubated at 25 oC for 12 h. The enzymatic digestions were quenched through the addition of a 10 μL formic acid (10%). Digested peptides were dried by vacuum concentrator  prior to mass analysis.Flag-tagged TAK1 and HA-tagged ubiquitin were co-expressed in HEK293T cells. After 24 hours, cells were collected and underwent m/z 350 to 2,000 for the survey full-scan MS mode and data dependent MS/MS acquisition. The ten most abundant ions detected in a full-scan were subjected to a MS/MS experiment performed in a LTQ mass spectrometer. Ion accumulation (automatic gain control target number) and maximal ion accumulation time for a full-scan and MS/MS were set at 1 × 106 ions, 1000 ms, and 5 × 104 ions, 200 ms. Ions were fragmented by use of collision induced dissociation (CID) with the normalized collision energy set to 35%, activation Q at 0.3 and activation time at 30 ms. For data analysis, all MS/MS spectra were converted to mzXML and mgf format from the experimental RAW file using MM File Conversion Tools (http://www.massmatrix.net)Mass spectral data were acquired using a Thermo LTQ-FT mass spectrometer  equipped with a nanoelectrospray ion source , an Agilent 1100 Series binary high-performance liquid chromatography pump , and a Famos autosampler . The vacuum-dried digested samples were reconstituted with mobile phase A buffer and 5 μL of samples were injected on to a self-packed pre-column  at a flow rate of 10 μL/min. Chromatographic separation was performed on a self-packed reversed phase C18 nano-column  using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in 80% acetonitrile as mobile phase B operated at the flow rate of 300 nL/min. Separation was attained using a solvent gradient ramping from 10% to 40% of mobile phase B in 45 min. The mass scanning range was set from Data are expressed as the mean ± SD. All statistical analyses were conducted using Prism (version 6.01). The significance differences between groups were determined by Dunnett t-test with unequal variances between groups. How to cite this article: Chen, I.-T. et al. Polyubiquitination of Transforming Growth Factor β-activated Kinase 1 (TAK1) at Lysine 562 Residue Regulates TLR4-mediated JNK and p38 MAPK Activation. Sci. Rep.5, 12300; doi: 10.1038/srep12300 (2015).


In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the “classical” protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed. Steroid hormones are synthesized in steroidogenic cells of the adrenal gland, ovary, testis, placenta, and brain and are required for normal reproductive function and body homeostasis. Unlike cells producing polypeptide hormones, which store large amounts of hormone in secretory vesicles ready for rapid release, steroidogenic cells store low amounts of steroids. Thus, a rapid steroidogenic response requires a rapid synthesis of new steroid molecules.The transport of cholesterol from the outer to the inner mitochondrial membrane (IMM) is the rate-limiting step of steroidogenesis , 2, and +, in addition to ACTH. These stimuli promote phosphorylation events, which are not dependent on cAMP/PKA. Indeed, K+ activates voltage-operated Ca2+ channels, while Ang II, bound to Ang II type 1 receptors, acts through the inositol 1,4,5-trisphosphate IP3–Ca2+/calmodulin system  transcription but also the transcriptional regulation of several steroidogenic-related genes  StAR or mutant StAR S194A to rescue StAR knockout mice – have demonstrated that Ser194, a conserved site among species, is an essential residue for normal StAR function in mice adrenal cortex and testis . These dig cells . Strikinig cells . This coig cells .In the same line, work by Stocco’s group has explored the regulation mechanism of StAR expression and steroidogenesis in conjunction with PKA and PKC pathways in MA-10 Leydig cells . This stIn addition to PKA activation importance for trophic hormone-stimulated steroid biosynthesis, it is also known that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and upstream activator mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) participate in the regulation of steroidogenesis. Indeed, several reports describe the role of members of the MAPK family in the regulation of steroid synthesis acting at both genomic and non-genomic levels.One of the first published works in the field indicates that cAMP-induced steroid synthesis depends on ERKs phosphorylation and activation . These aAlthough already demonstrated, PKA involvement in ERK activation continues to generate controversy; Le and Shimmer have shown that ACTH increases MEK and ERK phosphorylation in Y1 adrenocortical murine cells. This effect has also been detected in Kin-8 cells, a PKA-deficient mutant Y1-derived cell line , which sSTAR gene in Y1 cells  and increased steroid production through increased transcription of the Y1 cells . The actY1 cells .Luteinizing hormone receptor cascade activation in Leydig cells also promotes ERK1/2 phosphorylation, which is mediated by PKA through Ras activation . More reIn agreement with findings in adrenocortical cells , MartineIn turn, 3-day treatment with Ang II in cultured rat adrenal glomerulosa cells increases aldosterone secretion through a mechanism involving both ERK1/2 and p38 MAPK pathways . In addiSTAR gene expression  enhances cAMP-dependent pathways , 44. AKAKTKLTWLLSI) lies between amino acids 235 and 244 and is conserved among MEK1/2, MAPK phosphatase, and ERK substrates (232PS) has a 90% probability of phopshorylation and is adjacent to the docking D domain, while the probability of Ser277 is only 5%. Besides, Ser277 is relatively less conserved among species. In agreement of a predicted StAR–ERK binding, the treatment of subcellular fractions with pERK–GST has shown that StAR protein interacts with pERK1 just in the mitochondrial fraction, but not in the cytosol. Together, MEK phosphorylation PKA-dependent, mitochondrial StAR and pERK1/2 activity increase cholesterol transport and mitochondrial synthesis of progesterone in cell-free assays , we demonstrated that this residue is indeed the target of ERK  does not produce such effect, which suggests that the negatively charged amino acid partially mimics the negative charge of the phosphate group present in the phospho-Ser .Taking together, PKA phosphorylates StAR protein and also activates mitochondrial MEK1/2. Then, phosphorylated MEK1/2 activates a non-phosphorylated mitochondrial pool of ERK1/2 when the three kinases interact at the OMM, a crucial site for cholesterol transport forming a mitochondrial multi-complex with StAR.The degree of tyrosine phosphorylation of a given protein is the result of the action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Protein kinases have been the focus of the research for a long time. Proportionately much less research has focused on protein phosphatases.Whereas PTPs were initially regarded as household enzymes with constitutive activity and capable of all-substrate dephosphorylation, evidence in favor of tight regulation of PTP activity by various mechanisms is now accumulating. Like protein phosphorylation, dephosphorylation by PTPs is required in a cell compartment-specific manner. Protein–protein interaction domains and compartment-specific targeting domains in PTPs serve to localize PTPs all over the cell compartments .Based on the amino acid sequence of their catalytic domain, PTPs are classified into four groups, Class I, II, III , and IV (Asp-based PTPs), each with a specific range of substrates. The largest family is the Class I (Cys-based PTPs), comprising the 38 “classical” PTPs, and the 61 “dual-specificity” PTPs (DSPs), which is the most diverse group in terms of substrate specificity. The group of the classical PTPs, with strict tyrosine specificity, consists of the receptor-like (transmembrane) and non-receptor (intracellular) classes. In the human genome, these PTPs comprise 21 and 17 genes, respectively , 53.The cross talk between pathways that involve Ser/Thr phosphorylation and Tyr dephosphorylation has been described in the regulation of steroid synthesis. Our group has reported that ACTH treatment causes an increase in the activity of PTPs located in the cytosol of adrenal zona fasciculata (ZF). The stimulation is detected very soon after ACTH stimulation (5 min), reaches a maximum (twofold) after 15 min, and returns to basal levels after 30 min . Incubatin vivo  2-containing PTP (SHP2) is classified among the non-receptor, classical PTPs. It is widely expressed and plays an essential role in many organisms from lower eukaryotes to mammals . In contThe rapid increase in PTP activity induced by ACTH may prove that this activity is necessary in the stimulation of steroidogenesis. Studies on PTPs role in the acute steroidogenic response to hormones have been performed using incubation of rat adrenal ZF cells with two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), and evaluation of the steroid production upon stimulation by ACTH and 8Br-cAMP. It has been proven that those PTP inhibitors block ACTH- and cAMP-stimulated corticosterone production, but exert no effects on basal steroidogenesis . SimilarPhenylarsine oxide and PV also reduce LH/hCG- and cAMP-stimulated steroid production in testicular interstitial cells  and in M+-induced steroid synthesis in a dose-dependent fashion in H295R cells, a cell line derived from human ZG tumor  has a structure very similar to the PTP substrate, exerting its inhibitory action on PTPs by competitive inhibition of the enzyme. However, both inhibitors, PAO and BPA, inhibit Ang II- or KZG tumor , and in ZG tumor .+), acting by different signal transduction pathways, conveys on PTPs as common intermediaries  release , 69. AA The activity of Acsl4 and Acot2 are needed for AA release, StAR induction, and steroidogenesis. This statement is supported by the fact that the reduction of the expression of both, Acsl4 and Acot2, causes an inhibition of steroid production in two steroidogenic systems , 80. MorOur group has also linked the sequential action of PTPs, Acsl4, and StAR to the hormone-stimulated steroid production , 81. In These results brought about a challenge to determine the identity of the PTP involved in the stimulation of steroid synthesis through AA release.By means of overexpression and suppression approaches, SHP2 has been proven to be at least one of the PTPs playing an obligatory role in steroidogenesis. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, has been shown to reduce Acsl4 protein levels in Acsl4-rich breast cancer cells and steroidogenic cells. In addition, overexpression of an active form of SHP2 has increased Acsl4 protein levels in MA-10 Leydig cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on Acsl4, which could be specifically attributed to SHP2, as phosphatase knockdown reduces Acsl4 mRNA and protein levels. Through the action on Acsl4 protein levels, SHP2 affects AA–CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 leads to increased (or decreased) steroid production .The downregulation of SHP2 also modifies StAR expression. StAR expression increases in MA-10 Leydig cells treated with cAMP, an effect impaired by a short hairpin RNA (shRNA) against SHP2. Also, cAMP treatment causes a significant increase in StAR levels in mock-transfected cells, whereas SHP2 shRNA treatment prevents this effect. The involvement of AA in this process receives strong support from the fact that AA addition to SHP2 shRNA-treated cells bypasses the inhibitory effect produced by SHP2 knockdown . In thisin vitro phosphorylation of SHP2 by PKA dramatically increases its phosphatase activity (The Src homology domain (SH) 2-containing PTP is phosphorylated upon ACTH treatment . Moreoveactivity . Althougactivity –86.The Src homology domain (SH) 2-containing PTP has been identified in mitochondria, being the first evidence of the presence of a tyrosine phosphatase in such organelle . SeveralThe translocation of ERK to mitochondria is abolished by SHP2 knockdown in MA-10 cells. Moreover, the pronounced rearrangement of mitochondria that occurs after hCG stimulation is reduced by the downregulation of SHP2 expression . Then, tGiven that MAPK activation depends on Thr/Tyr protein phosphorylation, the magnitude and duration of their activity are related to protein phosphatases. MAPK phosphatases (MKPs) are a family of dual activity (Thr/Tyr) protein phosphatases, which dephosphorylate specifically members of MAPKs , 91. SevOn the basis of evidence showing that ACTH can regulate the activity of MAPKs , 92, theThe hormone-dependent expression of MKP-1 has also been analyzed in MA-10 Leydig cells, where hCG/cAMP rapidly increases MKP-1 gene induction in a transient manner . BesidesThe functional role of MKP-1 in the regulation of steroidogenesis has also been analyzed in MA-10 Leydig cells. Work by our group demonstrates that MKP-1 overexpression downregulates the effects of cAMP on phospho-ERK1/2 levels, StAR expression, and steroidogenesis, while MKP-1 downregulation produces opposite effects. In summary, these data demonstrate that in Leydig cells, MKP-1 expression is regulated at multiple levels as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis .Casal et al. have demonstrated the expression and regulation of MPK-1 also in primary cultures of bovine adrenal glomerulosa cells . These aIn MA-10 Leydig cells, LH receptor stimulation also induces MKP-2  and MKP-In conclusion, stimuli promoting MAPK activity also regulate MKPs expression at multiple stages as a negative feedback regulatory mechanism to modulate hormonal actions on ERK1/2 activity and steroidogenesis , and adenine nucleotidetranslocase (ANT) , 98.The precise mechanism of StAR action has been widely explored, but still remains elusive. StAR is synthesized as a 37-kDa protein with a typical mitochondrial leader sequence that directs the protein to the mitochondria for the import and cleavage to an intramitochondrial form of 30-kDa –102. AftThis mechanistic model of StAR action suggests that the active form of StAR is partially unfolded, with the N-terminal domain entering the mitochondria and the partially unfolded C-terminus interacting with the OMM. Direct evidence has been presented showing that StAR exists as a molten globule. While certain native structure is retained at the N-terminal domain, the C-terminal domain folding appears to be less tight at the low pH that StAR may undergo on the mitochondrial membrane. Then, the tightly folded N-terminal domain could make StAR halt as it enters the mitochondria, extending the time window for the C-terminus to act.Steroidogenic acute regulatory protein exhibits constitutive activity on the OMM, but no activity when localized to the intermembrane space (IMS) or to the matrix . MitochoEven when N-62 StAR form  does not access to the mitochondria, a few molecules of this protein are associated with the OMM as it is shown by immuno-electron microscopy , 109. Thvia mitophagy, of depolarized mitochondria , with a vast importance in regulation of Catabolism –113.Mfn2 in the ER bridges mitochondria and ER by forming homotypic and heterotypic complexes, with Mfn2 or Mfn1 on the mitochondrial surface. Therefore, Mfn2 is critical for MAM formation by tethering ER to the mitochondria. A mitochondrial ubiquitin ligase, MITOL, has been described as the regulator of the ER–mitochondria interaction by controlling Mfn2 activity . InteresDynamin-related protein 1 (Drp1) is required for mitochondrial fission. It is a cytosolic protein, which is recruited to the OMM by a poorly characterized multiprotein complex. In neurons, Drp1 phosphorylation by PKA in the mitochondria results in its inactivation and concomitant mitochondrial elongation . On the 2+ signal into the mitochondrial matrix (Although it is well recognized the relevance of mitochondrial dynamics in several cellular processes, its role in steroid synthesis is poorly described. Nevertheless, a work published 30 years ago described hormone-induced changes in intracellular location of the mitochondria and in the morphology of this organelle . Later, l matrix , which rl matrix . The autl matrix , 120. Mol matrix , suggestSteroid synthesis requires mitochondrial fusion induced by in a hormone-dependent fashion . The facIn silico molecular modeling has demonstrated that cholesterol binding to StAR could elicit a conformational change in StAR C-terminal domain, which in turn might favor the exposure of StAR Ser 232 and the docking domain for ERK. Therefore, StAR could be a substrate for ERK binding and phosphorylation, only when this protein is bound to cholesterol (lesterol . This molesterol . The OMMlesterol , 53. In Cholesterol acts as an allosteric modulator of its own binding to StAR  and is sSteroidogenic acute regulatory protein molecular structure has been partially studied –126, andOur group has shown that StAR S232A expression significantly diminishes the localization of StAR in the mitochondria induced by hCG or cAMP. ERK phosphorylation affects mitochondrial StAR levels posttranscriptionally, as the expression of transfected StAR S232A is independent of cellular endogenous regulation . The mitSteroidogenic acute regulatory protein activity is determined by its localization on the OMM, and not its cleavage from the 37- to 30-kDa form . Hence, Taken together, these results offer new insights into StAR regulation by kinases and phosphatases and their impact on StAR site of action. The phosphorylation–dephosphorylation of StAR would contribute to modulate its affinity for cholesterol and to increase pregnenolone synthesis with a few molecules of StAR. In this work, we have reviewed StAR mechanism of action on cholesterol transport to the P450scc to achieve maximal steroid production. We have also described the role of phosphorylation–dephosphorylation events and mitochondrial fusion as novel regulators of the localization of StAR protein in order to carry out its action in steroidogenic cells.Serine/threonine phosphatases have an important role in the regulation of adrenocortical cell functions, mainly steroid synthesis. In this context, the participation of PKA and PKC appears relevant, as phosphorylation events mediated by these kinases are involved in the expression and activation of StAR. Although StAR activation mechanism has not been fully described, it is known to require hormonal action on mitochondrial dynamic. Studies from other and our laboratory show that MAPKs, particularly ERK1/2, play an important role in StAR induction as well as in its posttranslational regulation in the mitochondria. In addition, ACTH-activated ERK1/2 regulates adrenal cell proliferation.A field scarcely described is the role of PTPs in steroidogenic cells. We presented data on PTP activation triggered by ACTH through a PKA-dependent mechanism. In this context, PTP SHP2 has a role in the stimulation of steroidogenesis involving Acsl4 protein induction. In turn, Acsl4 promotes AA release, StAR induction, and steroidogenesis. Moreover, SHP2 along with ERK could also have a role in steroidogenesis promoting mitochondrial fusion. MKPs, a group of dual activity phosphatases that inactivate MAPK, are also regulated by steroidogenic hormones at multiple levels. While MAPK activation is linked to steroid production activation and cell proliferation, MKP induction is associated with the turn-off of hormonal signal through MAPK inactivation and, consequently, the downregulation of ERK-dependent events.Ernesto J. Podesta, Cristina Paz, Cecilia Poderoso and Ana F. Castillo wrote the paper. All authors provided critical revisions of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Despite the view that only βs-selective - and the Gs- and Gi-activating -fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.We used the G50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective -fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.PTX amplified both the - and -fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The ECi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.Together, these data indicate that G In 1995, Xiao et al. made the intriguing observation that pertussis toxin (PTX), known to cause ADP-ribosylation of the Gαi subunit, uncoupling the GPCRs from Gi, enhanced β2AR- but not β1AR-mediated positive inotropic responses in isolated rat cardiomyocytes s, the β2ARs also couple to Gi in native systems while the β1ARs do not.The major stimulatory input in the myocardium comes from the β1- and β2AR-mediated inotropic response (βAR-IR) and cAMP accumulation in isolated ventricular cardiomyocytes were increased by PTX pre-treatment 2AR 1AR-evoked contractility in the heart may also be regulated by Gi activity. Due to conflicting data on the role of Gi in β1AR signalling, we wanted to further investigate this issue.However, we recently reported that the isoproterenol-mediated inotropic response in left ventricular muscle strips was potentiated by PTX s-coupled receptors can be increased by prior PTX treatment in various cell types i2 as well as by Hussain et al. i may have receptor-independent effects, whereby it directly inhibits AC activity. We examined the possibility that Gi may have receptor-independent effects in the absence of constitutively active receptors by using the unique properties exhibited by stereoisomers of the β2AR agonist fenoterol. -fenoterol was first characterized by Woo et al. (2009) to selectively activate only the Gs pathway of the β2AR, whereas the other stereoisomers, including  used in this study, activate both the Gs and Gi pathways s- and Gi-coupling of the β2AR i by using the direct AC activator forskolin, as well as the effects of PDE3 and 4 inhibition in the absence and presence of known antagonists or inverse agonists.It has previously been reported that AC activity evoked by forskolin and agonists at Gi in receptor signalling, this G protein exerts a constant intrinsic inhibition upon AC independent of receptor activation.Our data indicate that in addition to the traditional role of Gad libitum.Experiments and animal care were conducted in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes  and approved by the Norwegian Animal Research Authority. The animals were housed with a 12/12 h cycle at 21°C, food and water available 2+, equilibrated and field-stimulated at 1 Hz max was measured and inotropic responses were expressed as increase in (dF/dt)max. Concentration-response curves were constructed by estimating centiles (EC10 to EC100) and calculating the corresponding means, and the horizontal positioning is expressed as –logEC50 values Left ventricular strips (diameter ∼1.0 mm) were prepared from male Wistar rats  weighing 300–350 g, mounted in 31°C organ baths containing physiological salt solution with 1.8 mM Cain vivo  and in cardiomyocytes (see below), we measured the level of PTX-catalysed incorporation of [32P]ADP-ribose from [32P]NAD into available Gi as previously described In a subset of rats, PTX was administered at a dose of 60 µg/kg i.p. as a single injection three days prior to isolation of the left ventricular muscle strips. Control rats were given a saline injection of equal volume. Data from animals treated with PTX were only included if carbachol inhibition of the βAR-IR was completely abolished . To conf2+-free JOKLIK-MEM solution and enzymatic digestion using collagenase (90 U/mL) as previously described 1AR blocker CGP20712 (300 nM), the non-selective PDE inhibitor IBMX (0.5 mM) or PDE3 and 4 inhibitors cilostamide (1 µM) and rolipram (10 µM), as indicated. cAMP accumulation was measured by radioimmunoassay as previously described Adult left ventricular cardiomyocytes were isolated from excised male Wistar rat hearts by retrograde aortic perfusion with a nominally Ca2AR-mediated inotropic response, Gs-selective -fenoterol and Gs- and Gi-activating -fenoterol were assessed by conducting a concentration-response experiment in the presence of a selective β1AR antagonist (300 nM CGP20712). Likewise, - and -mediated activation of AC was assessed by incubating cardiomyocytes for 10 min with increasing concentrations of either stereoisomer (0–100 µM) in the presence of 300 nM CGP20712. All cAMP accumulation experiments in this subset were conducted in the presence of the non-selective PDE inhibitor IBMX (0.5 mM).To evaluate the βi activity was assessed through forskolin concentration-response curves (50 nM-10 µM) in left ventricular strips and isolated cardiomyocytes in the presence of the non-selective βAR antagonist timolol (1 µM) and α1-adrenoceptor antagonist prazosin  with and without prior pre-treatment with PTX (as described above). To determine if spontaneous intrinsic activity of Gi regulated basal AC activity, the inotropic response in ventricular strips or cAMP accumulation in cardiomyocytes pre-treated with or without PTX was measured in the presence of both the PDE3 inhibitor cilostamide (1 µM) and PDE4 inhibitor rolipram (10 µM) in the presence of the non-selective βAR antagonist timolol (1 µM) and α1-adrenoceptor antagonist prazosin .Receptor-independent GData are expressed as mean ± SEM from n animals. p<0.05 was considered statistically significant (One-way ANOVA and student's t-test). When appropriate, Bonferroni corrections were made to control for multiple comparisons.3H]cAMP was from PerkinElmer .Prazosin hydrochloride, (-)isoprenaline hydrochloride, timolol maleate, atropine sulphate, lidocaine  hydrochloride, L-ascorbic acid and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich . CGP20712 dihydrochloride, CGS 15943, cilostamide and rolipram were purchased from Tocris Bioscience . Forskolin was from LC Laboratories . - and -fenoterol were a kind gift from J. Kozocas . Isoflurane  was from Abbot Scandinavia . Pertussis toxin was from Merck chemicals . [in vivo PTX treatment to inhibit Gi was assessed by measuring PTX-catalysed incorporation of [32P]ADP-ribose from [32P]NAD into available Gi in ventricular tissue. An ∼80-90% reduction was seen in the ability of subsequent PTX to ADP-ribosylate Giin vitro in animals pre-treated with PTX compared to animals given saline injection . Importantly, in the PTX-treated group, data were included only if carbachol-induced inhibition of the βAR-IR was abolished -fenoterol . Maximal cAMP accumulation evoked by 3 µM -fenoterol was enhanced by 60% from 55 pmol/mg protein in control to 92 pmol/mg protein in the presence of PTX. The EC50 was not significantly different after PTX treatment (140±48 nM in control and 245±22 nM after PTX treatment) -fenoterol at the β2AR - and -fenoterol-induced AC activation was assessed by measuring cAMP accumulation in isolated left ventricular cardiomyocytes in the presence of the non-selective PDE inhibitor IBMX. Surprisingly, PTX inactivation of Genoterol . -featment) . These E2AR (100 µM) was enhanced by 62% from 38 pmol/mg protein in control to 61 pmol/mg protein in the presence of PTX. The EC50 value was not significantly shifted from 3.5±0.5 µM in control to 5.0±0.9 µM after PTX treatment -fenoterol of 3.7 ± 0.3 µM i exerts inhibitory activity upon AC downstream of receptor coupling to G protein.As expected, -fenoterol concentration-response curves were similarly modified by PTX treatment. Maximal cAMP accumulation evoked by the highest selective concentration of -fenoterol for the βreatment . Again, 1AR antagonist CGP20712 present; 2AR stimulation by adrenaline in the presence of CGP20712 .In untreated (control) left ventricular muscle strips, the highest concentration given of - and -fenoterol elicited very small inotropic responses of 8.6±2.7% and 4.4±2.9% above basal, respectively - and -fenoterol were significantly amplified by either PTX pre-treatment  or PDE3 and 4 inhibition  . These d50 values for either fenoterol isoform, due to the very small inotropic response. However, in the presence of PTX, the EC50 of -fenoterol was 139±18 nM and of -fenoterol 4.1±0.4 µM - and 0.44±0.01 µM for -fenoterol), indicating that both Gi and PDE3 and 4 regulate translation of the cAMP signal to a functional inotropic response.In the absence of PTX treatment or PDE inhibition it was not possible to determine the EC1±0.4 µM , very si1±0.4 µM  and corri on AC activity. To eliminate the influence of constitutive βAR activation of Gs (known to enhance forskolin responses), all experiments were conducted in the presence of the βAR inverse agonist timolol. cAMP accumulation in response to the maximum concentration of forskolin tested  was enhanced by 85% by PTX treatment from 269±41 pmol/mg protein in control to 498±69 pmol/mg protein in the presence of PTX  at maximal tested concentrations  evoked an inotropic response 120±15% above basal with an ECs through β1- or β2ARs. As previously reported s-coupled receptors. To test whether the effect of PTX occurred as a result of removing constitutive receptor activation of Gi, we evaluated the effect of two established Gi-coupled receptor systems in the heart. Neither the muscarinic inverse agonist atropine (1 µM) nor the non-selective adenosine inverse agonist CGS 15943 (1 µM), shown to be an inverse agonist at the Gi-coupled A1 adenosine receptor The following experiments were conducted in the presence of βAR blockade , to eliminate a possible effect of residual endogenous noradrenaline or constitutive activation of Gi-coupled receptors, since neither atropine (1 µM) nor CGS 15943 (1 µM) alone or in combination in the presence of simultaneous inhibition of PDE3 and 4 mimicked the effect of PTX treatment  PTX treatment increased responses evoked by both the Gs-selective - and the dually coupled -fenoterol isoforms -fenoterol have been shown to only activate Gsi; and (3) PTX treatment revealed intrinsic AC activity upon basal responses only after inhibition of PDE3 and 4, whereas inverse agonists at muscarinic receptors and adenosine receptors were without effect after PDE3 and 4 inhibition  nor the non-selective adenosine receptor antagonist CGS 15943, alone or in combination, elicits effects similar to PTX , further highlighting the inhibitory potential of presumably inactivated Gi. There is a good basis indicating that Gs∶Gi ratio ranging from ∼1∶10 to 1∶40 i would be the predominant G protein activated under these conditions. Since AC may already be maximally inhibited by spontaneously active Gi, it is expected that no additional effect of Gpp(NH)p is seen. However, when the stable GDP analogue GDPβS was used, basal AC activity increased, presumably through relief of tonic Gi inhibition. Under this condition, subsequent addition of Gpp(NH)p caused inhibition of AC, presumably through competition with GDPβS i intrinsic activity.Further, Piacentini et al. i2 has been studied in cardiomyocytes derived from embryonic stem cells i2i. Interestingly, basal inotropic and lusitropic responses were reduced by ∼10% in these mice compared to wildtype s-dependent stimulation of beating rate by the β2-AR agonist procaterol was nearly abolished in foetal RGS-insensitive Gαi2-cardiomyoctes via a PTX-sensitive mechanism, further indicating that Gi exerts tonic inhibition on AC Regulators of G protein signalling (RGS) are GTPase-accelerating proteins that promote rapid GTP hydrolysis and consequently inhibit signal transduction by inactivating G proteins and returning them to the GDP-bound heterotrimeric state. The effect of mutated, RGS-insensitive Gαi. AC6 basal activity was low, but could be inhibited by Gαi if previously activated with GTPγS-Gαsi activity.Chen-Goodspeed et al. i activity. Consistent with this hypothesis, Graham et al. i2 and Gαi3, inhibited cAMP accumulation evoked by constitutively active Gs or forskolin in 293T cells In addition, data suggest that AGS proteins  may promote G protein activation by either directly acting as a guanine nucleotide exchange factor or by interfering with subunit association/dissociation/trafficking (both dependent and independent of nucleotide exchange) preventing heterotrimeric Gαβγ formation i exerts intrinsic receptor independent inhibition upon spontaneous AC activity, the mechanism is still unknown. Further, we cannot completely rule out the possibility that PTX treatment removes an unknown constitutively active Gi-coupled receptor. Despite these limitations, it is reasonable to hypothesize that the ratio of Gs∶Gi has important implications for the regulation of basal AC activity. As reported here, a minimum of 80-90% ADP-ribosylation of Gi was necessary to reveal functional effects of PTX treatment in our models. Under this scenario, the normal reported ratio of Gs∶Gi of ∼1∶10 to ∼1∶40 would be altered to ∼1∶1 to ∼1∶4, giving Gs more favourable terms to compete for binding to AC. This is consistent with Lutz et al. i activity), as both basal AC activity and Gs-coupled receptor activation would be enhanced by PTX inactivation of Gi. The mechanism that mediates these effects of PTX treatment remains to be determined.Although the current data support the hypothesis that Gs and Gi compete for an apparently limited pool of βγ i becomes ADP-ribosylated by PTX, Gi sequesters βγ from the total shared pool. Under this scenario, over time, there will be less βγ available for Gs, consequently increasing the probability of Gs to be in its active state (GTP-bound) in addition to the corresponding removal of intrinsically active Gi. A plausible hypothesis would be that Gi acts as a βγ sink. In support, overexpression of βARK-ct, a βγ scavenger, resulted in increased AC stimulation It has recently been reported that Gi and Gs proteins. NDPK B is a G protein histidine kinase that regulates cAMP synthesis and cardiomyocyte contractility independent of GPCRs through the formation of an NDPK B-Gβγ complex that activates G protein through a phosphotransfer from NDPK B to His-266 in Gβi over Gs, NDPK B would predominantly activate Gi and the stimulatory effects of Gs upon basal cAMP generation may be neutralized i is inactivated by permanent ADP-ribosylation. Thus, NDPK B would predominantly activate (potentiate) Gs activity leading to both increased basal contractile force and cAMP accumulation that becomes revealed after PDE3 and 4 inhibition , non-selective muscarinic inverse agonist atropine (1 µM) or non-selective adenosine receptor inverse agonist CGS-15943 (1 µM) upon PDE3  and PDE4 -evoked inotropic response. Data are mean ± SEM. * P<0.05 vs. PTX, One-way ANOVA with Bonferroni post test adjustment for multiple comparisons.(TIF)Click here for additional data file.


Monitoring guidance for clinical trials recommends the use of central and on-site monitoring practices. Comparing the efficiency of on-site monitoring visits targeted as a result of central statistical monitoring (CSM) procedures, versus those that were not, could help to improve trial conduct.The monitoring plan in a large long-term multicentre international trial required that sites received routine monitoring visits every nine months. Oversight of this trial was augmented by central statistical monitoring that identified high scoring sites as priority for further investigation. To validate this approach high scoring sites, and some low scoring sites (in the same countries) identified by the country teams as potentially problematic were visited.Over a twelve month period 21 sites  received a comprehensive monitoring visit from the senior monitor. Only 1 site identified by CSM had no findings, versus 7 of the comparator sites. Minor findings indicative of ‘sloppy practice’ were identified at 12 sites (10 CSM versus 2 comparator). At 1 site identified by CSM there were serious findings indicative of an under-performing site.On-site visits triggered by CSM are more efficient than undirected routine visits. Furthermore, information from CSM can help focus the nature of on-site visits and any interventions required to improve site quality, e.g. additional training on data collection or event reporting. Study costs will be reduced if resources are directed to improving the performance of sites of concern, leaving lower risk sites to receive less frequent on-site monitoring.


Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change.Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic  They are widely distributed in marine and freshwater sediments, anaerobic sewage digestors, and animal gastrointestinal tracts7Methanohalophilus portucalensis FDF1T, belongs to the Methanosarcinales order and is cultivated from a naturally hypersaline environmentT utilizes only methanol, monomethylamine (MMA), dimethylamine (DMA), and trimethylamine (TMA) as carbon and energy sources for growth through the methylotrophic methanogenesis pathway. Intriguingly, a non-canonical amino acid, pyrrolysine (Pyl), can be incorporated by an in-frame amber codon (UAG) of three distinct methylamine methyltransferases, TMA methyltransferase (mttB), DMA methyltransferase (mtbB), and MMA methyltransferase (mtmB), to initiate methanogenesis from methylamines10amber suppressor tRNAPyl (pylT) gene in Methanosarcina acetivorans disabled methane production from precursor MMA, DMA, or TMAMethanogens can utilize three types of methanogenic pathways for energy conservation: COM. portucalensis FDF1T has been reported to possess the capability for de novo synthesis of betaine through three-step methylation from precursor glycine via glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT)2021T, and the amount of monovalent ions modulates the activity of rate-limited enzyme GSMTT T has an immediate on-off switch in the betaine synthesis process to respond to environmental changes.Importantly, methane produced by halophilic methanogens contributes significantly to the carbon mineralization in marine and hypersaline environments where large amounts of the greenhouse gas methane are stored14161718Methanosarcina barkeri, which converts methanol to methane via the methylotrophic methanogenesis pathwayMethanosaeta harundinacea2627M. portucalensis FDF1T.Reversible protein phosphorylation is the most common cellular mechanism to regulate many physiological and adaptational processes. The first identified methanogen phosphoprotein with a known function was the methyltransferase activation protein from E. coli system had uncovered the ability to overcome hyper-salinity stress via expression of the osmolyte betaine synthesizing enzymes, GSMT and SDMT, from strain FDF1T In this study, we provided a genome-wide, shotgun and phosphorylation site-specific investigation in a halophilic methanogen through MS-based systematic phosphoproteomic analysis. Both Ser/Thr/Tyr and His/Asp phosphorylation-based signaling systems were involved in diverse biological processes, especially in the single-carbon energy metabolism for methane production and osmolyte biosynthetic pathways. Although a previous study in the M. portucalensis FDF1T was acquired through high accuracy LC-MS/MS analysis to derive a global and site-specific phosphoproteomic data set. In order to analyze a comprehensive phosphoproteome, protein extracts prepared from mid-exponential phase cultures were subjected to a combination of gel-free and gel-based approaches. In addition, TiO2-based HAMMOC was applied for efficient phosphopeptide enrichment29TGTGFNSVR from GSMT carrying one phosphorylated site on Thr-68. Detailed information about all identified phosphopeptides and matched MS data are listed in The phosphorylation-mediated cellular signaling network in methylotrophic halophilic M. portucalensis FDF1T were categorized by cellular localization and biological function to gain insight into their functional roles   codon, which encodes pyrrolysine (Pyl) during protein synthesis . The pepPF_0343) . Intrigutructure , which iM. portucalensis FDF1T synthesizes glycine betaine de novo as a preferred osmolyte for turgor adjustmentN-methyltransferase (GNMT)M. portucalensis GSMT (MpGSMT)  , suggestSequence alignment and predicted MpGSMT structure indicate that the first four phosphorylated sites  are located in the SAM binding region, and the remaining phosphosites  are located in the lid structure for substrate glycine or sarcosine binding . NotablyM. portucalensis in response to betaine biosynthesis through phosphorylation/de-phosphorylation. Additionally, circular dichroism (CD) analysis showed that the secondary structures of T68A and T68D mutant proteins had no conformational change  and phospho-mimetic isoforms  . These rl change , indicatE. coli MKH13 with heterologous expression of Mpgsmt-sdmt (WT) and mutant Mpgsmt-sdmt (T68A- or T68D-GSMT) genes. Media containing 0 mM, 500 mM or 700 mM NaCl were prepared in solid form for salt shock and liquid form for salt adaptation growth tests. The growth patterns of E. coli MKH13 with WT-, T68A- or T68D-GSMT were similar under the non-saline condition , compared with 2.5% of the halophilic archaeon Halobacterium salinarumM. portucalensis  for Pyl-tRNAorylated . BuildinE. coli MKH13 RIL , respectively. The archaeal strain Methanohalophilus portucalensis strain FDF1T (=DSM 7471)The bacterial strains used for cloning and heterologous expression were M. portucalensis FDF1T were cultured in medium containing 120 g L−1 NaCl and 20 mM trimethylamine as a sole carbon and energy source, as described in the literature 540 at 0.5) cell pellets were resuspended in fresh lysis buffer , 6 M urea, and 2 M thiourea) and disrupted by sonication on ice. The protein concentration was quantified by the Bradford protein assay (Bio-Rad).2 beads  packed into 10-μL C8-StageTips, as described previously29In order to compile a comprehensive phosphoproteome data set, the total protein was treated with trypsin in both gel-based and gel-free processes following procedures described in the literatureThe peptide mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on a nanoAcquity system  coupled to an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Scientific) equipped with a PicoView nanospray interface (New Objective). Detailed detection conditions are described in http://www.maxquant.org/)M. portucalensis strain FDF1T with all 2, 131 protein sequences and a well-annotated genome of M. mahii DSM 5219M. portucalensis strain FDF1T and contains 1, 987 sequences . The protein-encoding genes from the M. portucalensis strain FDF1T genome sequence were previously predicted by Glimmer 2.13−5, identity >40%, and matched length >30%). The detailed search criteria are described in M. portucalensis strain FDF1T were reported and listed in All MS and MS/MS raw data were analyzed using MaxQuant software (version 1.4.1.2) (Mpgsmt (GenBank: AEG64703) (EC 2.1.1.156) was cloned into the pET28a expression vector (Novagen)Mpgsmt construct and its mutant derivatives were transformed into E. coli BL21(DE3)RIL for heterologous expression as described in literatureThe MpGSMT exhibiting GMT and SMT activities were determined in the forward direction, measuring sarcosine and dimethylglycine formation from glycine (Sigma) and sarcosine (Sigma) substrates. A modified acid-washed charcoal method was used to detect methyltransferase activities following the standard protocol described in literature21Mpgsmt-sdmt co-expressing MpGSMT/SDMT was initially introduced into the osmolyte uptake mutant E. coli MKH13 as described in literatureMpgsmt-sdmt were defined as wild type (WT), while cells carrying the plasmid with a mutated version of Mpgsmt at Thr-68 were indicated as mutant T68A or T68D. For growth tests, three E. coli strains  were grown in M9 minimal medium without NaCl for at least three generations, and then subcultured in different NaCl concentrations in solid and liquid medium to investigate the influence of salt shock and salt adaptation, respectively. To assess the effects of salt shock, the overnight cultures of the three strains were diluted to OD600 of 1.0 using M9 medium without NaCl, and then used for further serial dilution to be dotted in equal volumes of each dilution, ranging from 100 to 10−7, on M9 agar plates containing 0, 500, or 700 mM NaCl. All the conditions were carried out in triplicate and incubated at 37 °C. To assess the effects of salt adaptation, the three strains were acclimated in M9 liquid medium containing 0, 500, or 700 mM NaCl for three generations. The cells in 0 mM NaCl were inoculated at an initial OD600 of 0.01 for growth curves. In order to shorten the lag period prior to initiating growth in high concentrations of 500 or 700 mM NaCl, the initial OD600 value was fixed at 0.03. The growth rates of each strain in different salt conditions were determined in duplicate experiments.The plasmid pUHE21-How to cite this article: Wu, W.-L. et al. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation. Sci. Rep.6, 29013; doi: 10.1038/srep29013 (2016).


Pedicle screw fixation in the thoracic spine presents certain challenges due to the critical regional neurovascular anatomy as well as the narrow pedicular corridor that typically exists. With increased awareness of the dangers of intraoperative radiation, the ability to place pedicle screws with anatomic landmarks alone is paramount. In this study, we reviewed the literature from 1990 to 2015 for studies that included freehand pedicle screw placement in the thoracic spine with special emphasis on entry points and the trajectories of the screws. We excluded studies that used fluoroscopy guidance, navigation techniques, cadaveric and biomechanical articles, case reports, and experimental studies on animals. The search retrieved 40 articles, and after careful selection, seven articles were analyzed. Over 8,000 screws were placed in the different studies. The mean accuracy for placement of the thoracic screws was 93.3%. However, there is little consensus between studies in entry points, sagittal, and axial trajectories of the screws.We complete this review by presenting our step-by-step technique for the placement of freehand pedicle screws in the thoracic spine. Pedicle screw and rod constructs have become widely accepted as the ideal option to provide stable spinal fixation -8.Traditionally, freehand pedicle screw placement in the lumbar spine has had wide acceptance; however, pedicle screw placement in the thoracic spine has posed more challenges due to more challenging anatomy -10.A variety of different techniques is currently used to aid the surgeon for accurate placement of pedicle screws in the thoracic spine. These techniques include intraoperative C-arm fluoroscopy, intraoperative computer tomography (CT), and computer-assisted navigation -16. AlthFreehand pedicle screw placement in the thoracic spine is commonly performed by many surgeons and is both safe and effective , 21-23. The goal of this study was to comprehensively review the existing published literature on freehand thoracic pedicle screw placement techniques, focusing on similarities and differences. Additionally, we present our step-by-step technique, which relies on a uniform entry point and sagittal trajectory for all levels.A review of the literature using the National Library of Medicine via the PubMed database was performed from 1990 to August 2015. Search words included: “freehand”, “thoracic pedicle screw”, and “technique”. Only articles written in English were included. The title and abstract were screened and a first selection was done to include only the papers that discussed the specific techniques in the analysis. Specifically, we collected data on the number of patients, the number of inserted pedicle screws, and parameters of the surgical technique . We excluded studies that involved fluoroscopy guidance, navigation techniques, cadaveric and biomechanical articles, case reports, and experimental studies on animals.In order to encompass as many relevant studies as possible, we also performed a manual search of the references of selected articles.The search yielded a total of 40 published articles. Following the first selection by title and abstract, we identified seven , 26-27 eIn Table Spine PathologiesThere were several pathologies involved in the placement of freehand thoracic screws: from moderate scoliosis (idiopathic and neuromuscular scoliosis) to severe scoliosis (Cobb angle > 90), to infection, Mal de Pott, trauma, tumors, metastasis, and congenital problems Table  1-4, 2323, 26-27Entry PointsVarious entry points have been proposed that are mainly based on the level of the thoracic spine.Six of the seven studies focused on several thoracic levels. Only one study  focused TrajectoriesSix studies evaluated axial and sagittal trajectories , 23, 28.Accuracy of Freehand Thoracic Pedicle ScrewsThe accuracy of thoracic pedicle screws placement was reported in all the studies. In general, pedicle screw accuracy was defined as “having the entire screw contained within the cortices of each respective pedicle” .Mean accuracy rate for placement of pedicle screws in the studies was 93.34% (standard deviation of 3.54). The lowest reported accuracy was 87.4% .In studies with multiple surgeons, the accuracy for five surgeons was 93.8%  and for Breach of the Vertebral BodiesThere was a consensus in all the studies of a “safe zone” from 2 to 4 mm for pedicle screw breach of the vertebral bodies. This safe zone allows the medial or lateral wall breach by the screw without clinical consequences for the patient , 28. FurOverall, a lateral breach was more common than a medial breach of the vertebral bodies. There was a range of 2.5% to 21.6% of the screws for lateral breach and 1.7% to 13.2% of the screws for medial breach, with the majority falling below 5% of the screws , 26-27.Four studies reported the most common location of the vertebral bodies’ breaches. Parker, et al.  reportedComplicationsOverall, the complication rate after freehand pedicle screw placement in the thoracic spine was low. Parker, et al. reported 4.3% incidence of durotomies in their case series, however, this includes thoracic and lumbar freehand screw placement .Thoracic pedicle screw fixation can be challenging due to the complex morphology of the thoracic vertebrae, which can lead to screw malposition with potential injury to adjacent structures , 14, 29.The accuracy of thoracic pedicle screw placement is high. In this review, Parker, et al. reported the highest rate of accurate placement 98.3%) [% [3]. ThIn this review, we found a high range of reported lateral breaches of the vertebral bodies. The highest reported was by Modi, et al. of 21.6% and may be due to the fact that these screws were placed in patients with severe scoliosis where the anatomical landmarks may be more difficult to delineate . NonetheKim, et al. suggested a “safe zone” for vertebral bodies’ breach  as follows: breach of < 2 mm as a “definite safe zone”, a breach of 2 to 4 mm as a “probable safe zone”, and a breach of 4 to 8 mm as a “questionable safe zone” . HoweverThe complication rate in the reviewed studies was low. The majority of the studies reported no neurological or vascular complications following freehand placement of thoracic screws. The study by Kim, et al. provides the strongest evidence of the safety of this technique; they have the longest follow-up (10 years) without any neurological or vascular complication reported . MoreoveThe results of this review highlight the fact that, while freehand thoracic screw placement techniques may be widely employed and are common, only a handful of published studies delineate the nuances of these techniques. Furthermore, the published studies highlight various starting points and/or trajectories for each level of the thoracic spine, making the adoption of the technique cumbersome to the spinal surgeon or trainee.We have recently proposed a technique that relies on less variable parameters and may be easier to teach . We haveStep 1After the incision, dissection of the subcutaneous tissue and muscles the thoracic spine is exposed. The surgical exposure should include the superior articulating processes and transverse processes of the vertebrae. The inferior articulating process can be removed, if needed, for osteotomy but is not mandatory for screw placement alone Figure .Step 2Then, we proceed with a decortication of the bone in the location of the entry point using high-speed electrical or pneumatic drill. For all levels in the thoracic spine, we use a uniform entry point, which is approximately 3 mm caudal to the junction of the lateral margin of the superior articulating process and the transverse process Figure .Step 3The pedicle is cannulated from the entry point and the gearshift probe is further advanced to the desired level based on a preoperative CT analysis, which also determines the length and diameter of the screw to be inserted. A straight gearshift is adequate, and the medial trajectory is approximately 30 degrees for T1 and T2, and 20 degrees for T3 through T12. The pedicle is cannulated in an orthogonal trajectory in relation to the dorsal spine Figures .Step 4The gearshift probe is removed, and a ball-tipped instrument is inserted to assess for any penetration (breach) of the bony wall in five directions: medially, laterally, superiorly, inferiorly, and anteriorly.Step 5With the use of a tap , we undertap the pedicle tract. Then, the ball-tipped instrument is used to revise any possible vertebral body breach. After this, the pedicle screw can be inserted. Screw insertion can be performed using manual or powered drivers, our preference being the latter for ergonomic ease and better precision.Step 6The sagittal trajectory of the pedicle screw insertion should be maintained orthogonal to the curvature of the dorsal spine at each level.Step 7C-arm fluoroscopy with anterior-posterior (AP) and lateral views is used for initial localization and subsequent confirmation of appropriate screw insertion. Additional fluoroscopic images with pedicle markers in place are not necessary.                      The uniform entry point that we identified is reproducible at each thoracic level as long as the cranial-caudal orthogonal cannulation trajectory is used as opposed to the earlier studies , 8, 21 iFreehand thoracic pedicle screw placement is safe and effective and may decrease operative times and radiation exposure. While many of the existing techniques are effective and generally accepted, we have proposed more uniform parameters that could make freehand thoracic pedicle screw placement easier to teach and learn.


Recent retreat of outlet glaciers in Wilkes Land, East Antarctica, is driven by changes in sea ice. The dynamics of ocean-terminating outlet glaciers are an important component of ice-sheet mass balance. Using satellite imagery for the past 40 years, we compile an approximately decadal record of outlet-glacier terminus position change around the entire East Antarctic Ice Sheet (EAIS) marine margin. We find that most outlet glaciers retreated during the period 1974–1990, before switching to advance in every drainage basin during the two most recent periods, 1990–2000 and 2000–2012. The only exception to this trend was in Wilkes Land, where the majority of glaciers (74%) retreated between 2000 and 2012. We hypothesize that this anomalous retreat is linked to a reduction in sea ice and associated impacts on ocean stratification, which increases the incursion of warm deep water toward glacier termini. Because Wilkes Land overlies a large marine basin, it raises the possibility of a future sea level contribution from this sector of East Antarctica. This trend continued into the most recent epoch (2000–2012), when 65% of glaciers advanced at a median rate of 17.9 m year−1.Our results show clear decadal-scale patterns of terminus position around the entire EAIS marine margin (table S1). During the 1974–1990 epoch, 65% of glaciers retreated at a median rate of 17.8 m year−1). Between Queen Mary Land and Kemp Land (DB8 to DB12), there was a less obvious change, with 53% of glaciers retreating . During the periods 1990–2000 and 2000–2012, however, outlet glaciers within every drainage basin showed a dominant signal of advance  switch from the 1990s, when 75% of glaciers in Wilkes Land advanced. This is the first demonstration that glacier terminus changes in the region are linked to recently observed trends in mass loss  between each epoch reveal clear spatial trends across Antarctica . In EastMore recently, there has been only a small change in the overall average number of sea-ice days per year between 2000 and 2012, compared to the 1990s . HoweverCoastal polynyas are important in driving sea-ice variability in Antarctica. They produce around 10% of total sea ice, despite representing only 1% of the maximum sea-ice area . We suggChanges in the amount of sea-ice production can alter the mixing of shelf waters, which can drive variability in the basal melt rate of glacier termini , Thematic Mapper (TM), and Enhanced Thematic Mapper Plus (ETM+) satellites to map the terminus position of 176 outlet glaciers along the coast of East Antarctica across four approximate time steps: 1974, 1990, 2000, and 2012, stretching from Queen Mary Land to the Ronne Ice Shelf. This extends the record of a previous study (www.metoffice.gov.uk/hadobs/en4/download-en4-0-2.html). The objective analysis data set covers the entire study period on a monthly basis and is available at a 1° × 1° spatial resolution, with data obtained from the WOD09, GTSPP, Argo, and ASBO collections (www.metoffice.gov.uk/hadobs/en4/download-en4-0-2.html).We use the EN4.0.2 subsurface ocean temperature objective analysis data set . The data set offers near-complete coverage of the Antarctic sea-ice zone on a daily basis since July 1987 and every 2 days before this stretching back to October 1978 at a spatial resolution of 25 km × 25 km. There are brief gaps in the data set in August 1982  and 1984 (13 to 23 August). These have been interpolated where missing pixels were present (see http://nsidc.org/data/nsidc-0079).We used the Bootstrap sea-ice concentrations derived from the Nimbus-7 satellite and Defense Meteorological Satellite Program (DMSP) satellites (In line with other studies .https://legacy.bas.ac.uk/met/READER/). In addition, mean monthly 0.25° ERA-Interim (http://apps.ecmwf.int/datasets/data/interim-full-moda/levtype=sfc/) from 1979 to 2012 were extracted from the Wilkes Land coastline. For the purpose of this study, we defined the Wilkes Land coastline as a shapefile consisting of the coastline of DB13 with a 25-km buffer.Monthly mean surface air temperature records from Casey station were extracted from the Scientific Committee on Antarctic Research (SCAR) Met-READER project (


Leuconostoc mesenteroides URE 13 strain was carried out. All the tested terpenoids showed an inhibitory effect on glycosyltransferases from strain URE 13 at concentration 0.34 mmol. Out of all studied diterpenoids splendidin showed the strongest inhibitory effect decreasing the activity of both glycosyltransferase complex and dextransucrase with 70% and 90%, respectively. The triterpenoid ursolic showed the second strongest inhibitory effect as the enzyme complex and dextransucrase from strain URE 13 retain 27% and 13% of their initial enzyme activity. Despite the higher degree of inhibition of purified dextransucrase, compared to the enzyme complex, a complete inhibition of the enzyme was not observed at the highest used terpenoid concentration (3.42 mmol). When acarbose was used as an inhibitor, a complete inhibition of dextransucrase was observed at concentration of 6.9 mmol, while the enzyme complex retained 8% of its enzyme activity. Ki values of 0.28 mmol for splendidin, 0.37 mmol for ursolic acid and 0.29 mmol for acarbose were determined from the kinetic studies of purified dextransucrase.A study of the influence of different plant terpenoids and amino sugar derivate acarbose on the activity of glycosyltransferase complex and purified dextransucrase from  Streptococcus.[Actinomycetales sp. is acarbose: pseudotetrasaccharide consisting of two glucose units, 4-amino-4,6-dideoxy glucose unit and unsaturated cyclitol unit. The inhibitory effect of acarbose is ascribed to cyclohexan ring and glycosidic nitrogen linkage that mimics the transition state for cleavage of glycosidic linkages in normal glycosidase substrates.[Terpenoids are a large class of natural hydrocarbons produced mainly by plants. Generally, their concentrations are comparatively high in plant reproductive organs, leafs and resins during and immediately after flowering. Up to notococcus. Such an tococcus. One of tbstrates.Leuconostoc, Streptococcus, Lactobacillus and Weissella.[L. mesenteroides strains has been achieved.[While acarbose is well soluble in aqueous solutions, the terpenoids as lipophilic compounds are soluble only in solutions containing organic solvents and this hampers their study and application. One promising solution for this drawback is the modification of terpenoid molecules by attaching carbohydrate moieties to obtain their glycoside forms. The effieissella. These eneissella. Despite achieved. AccordinL. mesenteroides URE 13 strain. We also compared the effect of the studied terpenoids and acarbose on the kinetic of the enzyme reaction catalysed by purified dextransucrase.The aim of the present work is to evaluate the influence of different di- and triterpenoids on activity of glycosyltransferase complex and purified dextransucrase produced by L. mesenteroides URE 13 was obtained from the bacterial culture collection of the Department of General and Industrial Microbiology, Sofia University (Bulgaria). The strain was cultivated 6–8 h in culture media containing 4% (w/v) sucrose at 27 °C on a rotary shaker (200 rpm) for the production of glycosyltransferases.[sferases.Lavandula spica L. with methanol at room temperature for a week. The methanolic solution was concentrated by evaporation to dryness and residue was chromatographed on silica gel column  as previously described.[Scutellaria (Labiatae), and salviarin, splendidin, splenolide B were extracted from Salvia splendens. Diterpenoids were isolated by column chromatography with silica gel as previously described.[2Cr2O7/H2SO4/acetone solution leads to 3-keto-ursolic acid as described by Bozov and co-workers.[Triterpenoids ursolic acid and oleanolic acid were extracted from dried and finely powdered aerial parts of escribed. Diterpenescribed.,28 Chlorescribed. Acetate escribed. Oxidatio-workers. All terpBacterial growth was measured by a turbidimetric method at 620 nm and calibrated against cell dry-weight measurements, as previously described.The culture medium after sucrose cultivation was first centrifuged for 20 min at 7000 g and 4 °C for cell separation. The supernatant was then filtered with a Sartorius membrane (0.2 μm cut-off) to ensure the total absence of cells in the supernatant. The glycosyltransferases were separated from the supernatants and concentrated by using of polyethylene glycol (PEG) 1500 to final concentration of 20% (w/v). The glycL. mesenteroides URE 13, cultivated on sucrose media, was purified by size-exclusion chromatography with XK 16/70 column and Sepharose CL-6B medium as previously described.[An extracellular dextransucrase from escribed.2 and 100 g/L sucrose.[One unit of glycosyltransferase activity is defined as the amount of enzyme that catalyses the formation of 1 μmol of fructose per 1 min at 30 °C in 20 mmol/L sodium acetate buffer (pH 5.3), 0.05 g/L CaCl sucrose. AdditionL. mesenteroides URE 13 was studied at concentration from 20% to 70% (v/v) in the reaction mixtures. The inhibitory effect of the studied terpenoids was determined at 0.34, 1.71 and 3.42 mmol per ml end concentration in the reaction mixtures. The inhibitory effect of acarbose (Sigma) was determined at 0.3, 1.3, 2.3, 4.1 and 6.9 mmol per ml end concentration in the reaction mixtures. The reactions were performed with 1 U/ml enzyme in reaction mixtures containing 20 mmol/L sodium acetate buffer (pH 5.3), 0.05 g/L CaCl2, 100 g/L sucrose at 30 °C. D-fructose and D-glucose were determined with hexokinase, glucose-6-phosphate dehydrogenase and phosphoglucose isomerase . All the reactions were performed at least in triplicate and average values are given.The inhibitory effect of DMSO on enzyme activity of the glycosyltransferase complex and the purified dextransucrase from strain The Ki values were determined in reaction mixtures containing between 1 and 200 mmol sucrose, terpenoids from 0.34 to 3.42 mmol/ml or acarbose from 0.3 to 6.9 mmol/ml. The reactions were performed with 1 U/ml dextransucrase in 20 mmol/L sodium acetate buffer (pH 5.2) at 30 °C. D-fructose and D-glucose were determined with hexokinase, glucose-6-phosphate dehydrogenase and phosphoglucose isomerase . All the reactions were performed at least in triplicate and the average values were used for determination of Ki from Lineweaver–Burk plots.Proteins were assayed by the method of Bradford  by usingIn all the cases the software Programmable scientific calculation ‘CITIZEN’ SRP-45N and SigmaPlot 12.0  were used for data analysis.L. mesenteroides URE 13 produced a glycosyltransferase complex including 180 kDa dextransucrase, 300 kDa GTF and 120 kDa fructosyltransferase (FTF).[In our previous study, we showed that strain se (FTF). The dextse (FTF). The inflL. mesenteroides NRRL B-512F loses half of its enzyme activity in the presence of 20% DMSO.[In addition to their ability to synthesize polymers, glycosyltransferases produced by lactic acid bacteria also catalyse the transfer of glucose or fructose units from sucrose to other carbohydrate acceptors in so-called acceptor reactions. Of great20% DMSO. It appea20% DMSO.L. mesenteroides URE 13, we tested seven diterpenoids, two triterpenoids and also four their derivatives in three different concentrations  in the complex. The lack of a complete inhibition at the highest used concentration of terpenoids (3.42 mmol) gives us an opportunity for further detail studies concerning application of glycosyltransferases as a useful tool for glycosylation of natural substances with low water solubility. Several studies have reported successful glycosylation of non-carbohydrate substances as aromatic compounds, alcohols and amino acid derivatives in which the transferred sugar moieties improves not only their water solubility but also their pharmacological properties.[In this paper, we describe for the first time the influence of terpenoids on the activity of dextransucrase produced by operties. TerpenoiL. mesenteroides URE 13 was studied. At all the tested acarbose concentration the inhibitory effect on dextransucrase activity was predominant . In order to study the influence of the terpenoids splenididin and ursolic acid, and also of the amino sugar acarbose on velocity of the reaction catalysed by dextransucrase from strain URE 13, we determined and compared their inhibitory constants . In the Streptococcus spp.[L. mesenteroides B-512FMCM. The authors also defined acarbose as a non-competitive inhibitor with a Ki of 1.35 mmol.[L. mesenteroides URE 13, we obtained Ki of 0.29 mmol when acarbose was used as an inhibitor. Similar value of Ki was also obtained for the diterpenoid splendidin – 0.28 mmol. The ursolic acid was also determined as an effective inhibitor of the studied dextransucrase with Ki of 0.37 mmol. Kim and co-workers have reported also that acarbose influences the amount and type of the products obtained during the acceptor reactions catalysed by dextransucrase.[L. mesenteroides URE 13 in the presence of terpenoids and acarbose as well. On the other hand, studied terpenoid compounds show significant inhibitory capabilities, which represent a potential interest for their application as anticaries agents. Such information is already available for triterpenoids ursolic acid and oleanolic acid.[The amino sugar acarbose is known as a potent inhibitor of several carbohydrases, including α-amylase, cyclodextrin glucanotransferase, α-glucosidase and GTFs.,38 As anccus spp. Kim and .35 mmol. For dextnsucrase. In relatL. mesenteroides URE 13 showed a different degree of inhibition in the presence of terpenoids and acarbose as the purified enzyme was more sensitive. From the studied diterpenoids, the highest inhibitory effect was detected in the presence of splendidin – 90% inhibition of dextransucrase, and the triterpenoid ursolic acid showed 87% inhibition of the enzyme. The derivatives of scutalpin A and scutalpin E showed 9%–18% higher inhibitory effect than the native diterpenoids, while the ketone and the acetylated derivatives of ursolic acid caused 10%–40% higher inhibition of dextransucrase only at the lowest tested concentrations – 0.34 mmol. At the highest used concentration, none of the tested terpenoids did not show a complete inhibition of the enzyme complex and dextransucrase. When the inhibitory effect of acarbose was tested, a complete inhibition of dextransucrase was achieved at concentration 6.9 mmol, while the enzyme complex retained 8% of its enzyme activity. As for terpenoids thus for acarbose the dose/effect relation loses its linear dependence at concentrations larger than 0.3 mmol. The determination of inhibitory constant of dextransucrase from strain URE 13 when splendidin and ursolic acid were used as inhibitors showed that both terpenoids have very near Ki values – 0.28 and 0.37 mmol, to this one of acarbose – 0.29 mmol. The obtained results give us a good base for further investigations directed to study the influence of terpenoids on acceptor reactions catalysed by dextransucrase from strain URE 13 and also their glycosylation which will improve water solubility and probably biological activity of these compounds. Glycosyltransferase complex and dextransucrase produced by


Left ventricular noncompaction (LVNC) is a diagnosis often made at imaging with uncertain prognostic value. Jenni echocardiogram and Petersen cardiac MR (CMR) criteria utilize a single long-axis view to quantify the maximal ratio of noncompacted to compacted myocardial thickness, but these have not been shown to correlate with clinical status or disease progression. Quantitation of the trabecular mass is now simple to perform using advanced post-processing tools. Prior studies demonstrated that elevated LV trabeculated mass helps differentiate patients with LVNC from healthy controls and patients with other non-ischemic cardiomyopathies, but distinguishing patients with morphologic LVNC (M-LVNC) from those with clinically significant LVNC (CS-LVNC) remains problematic. The purpose of this study was to evaluate the performance of trabecular mass quantification at CMR to differentiate patients with CS-LVNC and M-LVNC.Retrospective analysis of 36 consecutive patients with a CMR meeting Petersen's criteria and an available echocardiogram. Two independent observers quantified noncompacted and compacted myocardial mass at end-diastole on QMass 7.6  using bright blood short-axis cine images. The noncompacted myocardial mass index (NCMMI) was calculated by dividing trabecular volume by body surface area and multiplying by 1.05 g/mL, the density of myocardium. CS-LVNC was defined by a clinical history of heart failure, ventricular arrhythmia, or cardioembolic event. Two-tailed t-tests were applied to compare NCMMI between groups. Subgroup analysis was performed by excluding subjects with negative echocardiograms by Jenni criteria and an ejection fraction (EF)>55%. Receiver operating characteristic (ROC) analysis was used to estimate cutoff values, sensitivities, and specificities. Interobserver agreement was assessed using the intraclass correlation coefficient (ICC).22 patients met criteria for CS-LVNC  versus 14 patients with M-LVNC . Those with CS-LVNC exhibited significantly greater NCMMI compared to those with M-LVNC . ROC analysis demonstrated fair discrimination between CS-LVNC and M-LVNC , which significantly improved after excluding 11 patients with EF≥55%  and even further after excluding an additional 10 patients with negative echocardiograms  yielding ideal cutoff values and test characteristics presented in Table Trabecular mass quantification at CMR provides an accurate and reliable means of identifying patients with clinically significant LV noncompaction, particularly among patients with a depressed EF and negative echocardiogram.None.