Poaceae, Brachypodium distachyon L. is a new model plant for wheat and barley as well as several potential biofuel grasses such as switchgrass. Vegetative growth is vital for biomass accumulation of plants, but knowledge regarding the role of protein phosphorylation modification during vegetative growth, especially in biofuel plants, is far from comprehensive.Protein phosphorylation is one of the most important post-translational modifications involved in the regulation of plant growth and development as well as diverse stress response. As a member of the Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total of 1470 phosphorylation sites in 950 phosphoproteins were identified, and these phosphoproteins were implicated in various molecular functions and basic cellular processes by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Among the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. Conservation analysis showed that 93.4% of the 950 phosphoproteins had phosphorylation orthologs in other plant species. Motif-X analysis of the phosphorylation sites identified 13 significantly enriched phosphorylation motifs, of which 3 were novel phosphorylation motifs. Meanwhile, there were 91 phosphoproteins with both multiple phosphorylation sites and multiple phosphorylation motifs. In addition, we identified 58 phosphorylated transcription factors across 21 families and found out 6 significantly over-represented transcription factor families . Eighty-four protein kinases (PKs), 8 protein phosphatases (PPs) and 6 CESAs were recognized as phosphoproteins.In this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in B. distachyon. We revealed a MAPK cascade network that might play the crucial roles during the phosphorylation signal transduction in leaf growth and development. The phosphoproteins and phosphosites identified from our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocots.Through a large-scale bioinformatics analysis of the phosphorylation data in seedling leaves, a complicated PKs- and PPs- centered network related to rapid vegetative growth was deciphered in The online version of this article (doi:10.1186/1471-2164-15-375) contains supplementary material, which is available to authorized users. In thisB. distachyon, O. sativa, A. thaliana and M. truncatula  is phosphorylated at a Tyr residue during the activation process [MAPK cascades are universal signal transduction modules in plants and are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes . Activat process . A previ process . Interes process –59.FigurArabidopsis response regulator  was found in the cytokinin regulated pathway. B-ARR is a DNA-binding transcriptional regulator, whose activity is regulated by phosphorylation of its phospho-accepting receiver domain through the His-kinase-mediated His to Asp phosphorelay and lack of this domain disturbs the development of young seedlings [In plants, many signal pathways are involved in vegetative growth. In the present study, KEGG pathway analysis showed that the plant hormone signal transduction pathway  studies of temperate grasses [B. distachyon, O. sativa and A. thaliana, was phosphorylated at Ser149 , as a kind of RLK, has the potential to serve as both linkers of the cell wall to the plasma membrane and as a signaling molecule. WAKs also function in pathogen response and cell expansion and elongation [The ll walls , 63. B.  grasses , 64. Cel grasses . The RLK49 Table . Hématy e et al. found a ongation , 66, 67.ongation , 68. IntA. thaliana, CESA1, CESA3 and CESA6 interact with each other and are largely responsible for cellulose production during formation of the primary cell wall [B. distachyon, namely BdCESA1, BdCESA2, BdCESA4, BdCESA7, BdCESA8 and BdCESA9 -β-D-glucan, small quantities of structural proteins, and low levels of pectin and xyloglucan , 69–71. ell wall –75, wherell wall , 76, 77.ell wall . This st, 3; 1, 4e plants . All cele plants . The amie plants . The RINe plants . Kurek e [et al. confirme[et al. demonstrn et al. found thPoales[HvCslF6 are maximal in 4- to 5-d coleoptiles, at the time when -β-D-glucan content of coleoptile cell walls also reaches its maximum level [Arabidopsis[B. distachyon, the CSLH gene showed a much higher transcript level and played a prominent role in -β-D-glucan synthesis [-β-D-glucan is mainly detected in some families of the Poales. RecentlPoales, 85. Theum level . HvCSLH1ynthesis .2-enriched phosphopeptides in Bd21 seedling leaves detected by LC-MS/MS, and MaxQuant software were used to identify the phosphoproteins and phosphorylation sites. To our knowledge, this is the first large-scale phosphoproteome analysis in B. distachyon and provides an overview of in vivo phosphorylation events in seedling leaves. A total of 950 phosphoproteins containing 1470 unambiguous phosphorylation sites were identified. In-depth GO, conservation, and KEGG pathway analyses of these phosphoproteins revealed the phosphorylation profiling during rapid seedling leaf growth. GO analysis showed that the 950 phosphoproteins mainly functioned in protein binding, nucleotide binding and kinase activity. Conservation analysis revealed that 887 (93.4%) of the phosphoproteins had phosphorylation orthologs in other species. Many fundamental biological pathways and two signal transduction pathways were identified through KEGG pathway analysis.In this work, TiOB. distachyon. A total of 127 phosphoproteins contained multiple phosphorylation sites and 91 of them contained two or more phosphorylation motifs. Several signal transduction and cell wall expansion associated phosphoproteins that may have vital functions in the rapid growth of B. distachyon seedling leaves were found such as B-ARR, PTEN, CESA and CSL. Our overall results provide an important resource for further research and new insights into the regulation of protein post-translational phosphorylation during rapid vegetative growth of Poaceae species.Fifty eight TFs, 94 PKs and 8 PPs were identified from the large dataset and analyzed in depth. Thirteen enriched motifs specific for various Ser/Thr PKs were identified from Brachypodium distachyon Bd21, kindly provided by Dr. John Vogel, USDA-ARS, Albany, CA, were surface sterilized in 5% sodium hypochlorite for 5 min, and rinsed 4 times in sterile distilled water. Seeds were submerged in water for 12 h at room temperature, and then transferred to wet filter paper for 24 h to germinate at room temperature (22–25°C). Uniformly germinated seeds were selected and grown in three plastic pots containing Hoagland’s solution [Seeds of solution ; the Hoaet al.[2, 20 mM DTT) and 4 mL phenol  in a 10 mL tube, and 1 mM phenylmethanesulfonyl fluoride (PMSF) and PhosSTOP Phosphatase Inhibitor Cocktail , were added to inhibit the activities of proteases and phosphatases. The mixtures were thoroughly vortexed thoroughly for 30 s and the phenol phase was separated by centrifugation at 14,000 × g and at 4°C for 15 min. The upper phenol phase was pipetted to fresh 10 mL tubes and four volumes of cold methanol plus 100 mM ammonium acetate were added, and the mixture was stored at –20°C for at least 30 min. After centrifugation at 14,000 × g and at 4°C for 15 min, the supernatant was carefully discarded and the precipitated proteins were washed twice with cold methanolic ammonium acetate (100 mM) and ice-cold 80% acetone, respectively. Finally the pellet was vacuum-dried and then dissolved in lysis buffer  over 3 h at 4°C. The protein mixtures were harvested by centrifugation at 14,000 × g and 4°C for 15 min to remove insoluble materials. The concentrations of the extracted protein mixtures were determined with a 2-D Quant Kit  using BSA (2 mg/mL) as standard, and the final protein solution was stored at –80°C for later use.Total proteins were extracted from seedling leafs according to the method of Wang et al. with minet al.[2 beads  were incubated in 400 μL loading buffer containing 65% Acetonitrile (ACN)/2% trifluoroaceticacid (TFA)/saturated by glutamic acid. A total of 2 mg of tryptic peptides were dissolved in 600 μL loading buffer, and incubated with an appropriate amount of TiO2 beads. After washing with 600 μL wash buffer (65% ACN/0.1% TFA), phosphopeptides were eluted twice with 300 μL elution buffer (500 mM NH4OH/60% ACN) and the eluates were dried down and reconstituted in 0.1% formic acid (FA)/H2O for MS analysis.Extracted protein mixtures were directly reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and subsequently digested with endoproteinase Lys-C and trypsin as previously described . The enret al. with modμm I.D., 150 mm length) , which directly connected the nano electrospray ion source to a LTQ-Orbitrap XL mass spectrometer . Pump flow was split to achieve a flow rate at 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% FA (A), and 0.1% FA and 80% ACN (B). A five-step linear gradient of 5% to 30% B in 105 min, 35% to 90% B in 16 min, 90% B in 4 min, 90% to 2% B for 0.5 min and 2% B for 14.5 min was performed. The spray voltage was set to 2.0 kV and the temperature of the heated capillary was 240°C.Enriched phosphopeptides were separated on a self-packed C18 reverse phase column  with lock mass option enabled and was followed by a data-dependent top 10 MS/MS scans using collision induced dissociation (CID). The threshold for precursor ion selection was 500 and mass window for precursor ion selection was 2.0 Da. The dynamic exclusion duration was 120 s, repeat count was 1 and repeat duration was 30 s. The analyzer for the MS scans was Orbitrap and for the MS/MS scans LTQ (37% relative collision energy). Three biological replicates were performed independently from sample collection to phosphopeptide identification using LC-MS/MS.B. distachyon protein database  in Phytozome  concatenated with a decoy of reversed sequences. The following parameters were used for database searches: cysteine carbamidomethylation was selected as a fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/MS spectra. The false discovery rate (FDR) was set to < 1.0% for both peptide and protein identifications, the minimum peptide length was set to 6.The raw files were processed with MaxQuant (version 1.2.2.5)  and searPhosphorylation site localization was based on PTM scores that assign probabilities for each of the possible sites according to their site-determining ions. In this study, MaxQuant (version 1.2.2.5) was used to calculate PTM scores and PTM localization probabilities. Potential phosphorylation sites were then grouped into three categories depending on their PTM localization probabilities , 37, 88,http://pfam.sanger.ac.uk/search) [B. distachyon protein database  in Phytozome . For pathway analysis, the proteins were searched in the KEGG B. distachyon database and mapped to B. distachyon-specific pathways with KEGG Mapper. The enrichment of TF family significantly regulated by phosphorylation was performed using a two-sided test of the hypergeometric distribution. The significantly enriched phosphorylation motifs were extracted from phosphopeptides with unambiguous phosphorylation sites (class I) using the Motif-X algorithm [B. distachyon protein database in Phytozome (version 9.1) was used as the background database to normalize scores against random distributions of amino acids. The occurrence threshold was set to 5% of the input data set at a minimum of 20 peptides, and the probability threshold was set to P < 10-6. The sequences of all the phosphoproteins were used for BLAST analysis with the National Center for Biotechnology Information (NCBI) clusters of the KOG database to obtain the KOG numbers of those proteins. Then a dataset containing all the KOG numbers was used for protein-protein interaction (PPI) analysis by using the STRING database  [http://www.enzim.hu/hmmtop/) was used to predict the transmembrane helices in proteins [The GO biological processes, molecular functions and cellular components of the identified phosphoproteins were examined by using Blast2GO software . GO enri/search) . Proteinlgorithm . The pho-db.org) . Only in-db.org) . HMMTOP proteins .B. distachyon. (A) Total phosphorylation sites and phosphoproteins. (B) Annotation of the phosphoproteins. (XLSX 506 KB)Additional file 1: Table S1: Phosphorylation sites and phosphoproteins identified from seedling leaves of Additional file 2: Figure S1: Representative tandem mass spectrometry spectra of the seven phosphopeptides containing the eight phosphosites in Bradi1g66870.1. (A) KHLPSQDITDAS(ph)GDEEEGSR. (B) HWS(ph)PSAGRR. (C) LKDFS(ph)ADPELNK. (D) MNYLGT(ph)PPS(ph)DLEK. (E) NS(ph)PISIK. (F) QFDS(ph)PDDSLVK. (G) RIS(ph)PPSGR. (PDF 275 KB)B. distachyon. (XLSX 122 KB)Additional file 3: Table S2: Conservation analysis of phosphoproteins identified from Additional file 4: Figure S2: GO biological process enrichment of 582 highly conserved phosphoproteins. The statistical significance of the enrichment analysis is represented by a scale of red tones whose intensity is proportional to the degree of significance starting from FDR < 0.05. (PDF 382 KB)Additional file 5: Table S3: Pathway analysis of the identified phosphoproteins by KEGG. (A) Pathway search results of all identified phosphoproteins and highly conserved phosphoproteins. (B) KEGG identifiers of all the identified phosphoproteins. (XLSX 23 KB)B. distachyon pathway database. The phosphoproteins were used to search the KEGG B. distachyon database and mapped to B. distachyon specific pathways with KEGG Mapper. In each pathway map, objects with red foreground color and blue background color represent the highly conserved phosphoproteins; objects with pink foreground and blue background represent the other conserved phosphoproteins; objects with yellow foreground and blue background represent proteins identified as novel phosphoproteins; objects with green background represent the total proteins in the B. distachyon KEGG database. Details of the pathways can be found in Additional file Additional file 6: Figure S3: Pathway mapping of identified phosphoproteins by KEGG. Phosphoproteins were analyzed in the KEGG Additional file 7: Table S4: Enriched phosphorylation motifs, and the phosphopeptides and the corresponding phosphoproteins in each phosphorylation motif. (A) Phosphorylation motifs enriched by Motif-X and the putative protein kinases. (B) Phosphopeptides and the corresponding phosphoproteins in each phosphorylation motif. (C) Motifs of phosphoproteins with multiple phosphorylation sites. (XLSX 60 KB)B. distachyon. GO enrichment graphs of phosphoproteins contained each of 11 motifs were displayed by Blast2GO software. No GO terms were significantly enriched from phosphoproteins containing motif 7 or motif 11. The statistical significance of the enrichment analysis is represented by a scale of red tones whose intensity is proportional to the degree of significance starting from p < 0.05. (PDF 2 MB)Additional file 8: Figure S4: GO enrichment of phosphoproteins represented by the phosphorylation motifs in Additional file 9: Figure S5: Protein-protein interaction network of the phosphoproteins. The confidence (score) was set as the highest (0.900) and only the phosphoproteins identified in this study were used to construct the interaction profiles. Protein kinases and protein phosphatases are highlighted in red and green respectively and other phosphoproteins identified in this study are shown with sky-blue nodes. (PDF 630 KB)Additional file 10: Figure S6: Phosphorylation sites of the six BdCESAs. (A) and (B) The tandem mass spectrometry spectra of the phosphopeptides EFSGS(ph)LGNVAWK from BdCESA2 (Bradi1g04597.1) and VTIASQLS(ph)DR from BdCESA4 (Bradi2g49912.1). (C) Sequence alignment among the six BdCESAs. Phosphorylation sites identified in this study are highlighted in red. The RING domain and transmembrane regions are highlighted in yellow and green, respectively. (PDF 1 MB)


Studies were conducted to determine the role of nitric oxide (NO), if any, on the release of arachidonic acid in platelets. The cytosolic Ca2+ was visualized and quantitated by fluorescent spectroscopy by using QUIN-2. NO was measured by methemoglobin method. Arachidonic acid was determined by HPLC. TXA2 was measured as ThromboxaneB2 (TXB2) by ELISA. Treatment of platelets in platelet-rich plasma (PRP) with different aggregating agents resulted in the inhibition of nitric oxide synthase (NOS) which inhibited the production of NO synthesis and increased TXA2 synthesis. Furthermore, the treatment of washed PRP with different platelet aggregating agents resulted in the increase of [Ca2+] in nM ranges. In contrast, the pre-treatment of washed PRP with aspirin increased platelet NO level and inhibited the Ca2+ mobilization and TXA2 synthesis. These results indicated that the aggregation of platelets by different aggregating agonists was caused by the cytosolic Ca2+ mobilization due to the inhibition of NOS.Platelet aggregation by different aggregating agonists is essential in the normal blood coagulation process, the excess of which caused acute coronary syndrome (ACS). In all cases, the activation of arachidonic acid by cycloxygenase was needed for the synthesis of thromboxane A The cell suspension was then kept at room temperature in a stoppered plastic tube. Before measurements were made, aliquots of the suspension were equilibrated at 37°C for several minutes. The measurement of [Cad before -15. The 2. The disrupted mass was centrifuged at 30,000 g for 30 min at 0°C and the pellet which contained platelet membranes was resuspended in Kreb’s buffer. The membrane suspension was incubated with 1 nM CaCl2 at 37°C for 10 min. The lipids were extracted by centrifuging the sample at 3000-4000 rpm to obtain cell free plasma (CFP). To the CFP, 1(N) HCl and Chloroform/Ethanol mixture was added and was mixed. The mixture was then centrifuged and the supernatant was extracted, evaporated under vacuum and stored at -70°C for High performance liquid chromatography (HPLC) analysis (The platelets from washed PRP (30 mL) were disrupted by repeated freezing and thawing in liquid Nanalysis .18 (ID: HiQ Sil C18 HS) column with size 4.0 mm × 250-mm. The solvent system consisted of a mixture of water and acetic acid and acetonitrile. A sample volume of 10 μL was injected and the flow rate was maintained at 2 ml/min. All injections for quantitative measurements were made with Hamilton syringe.The fatty acids were separated at room temperature (26°C) using a Waters Associates (Milford MA) Model with Model 515 solvent delivery pumps, a Rheodyne injector and a model 486 tunable absorbance detector operated at 280 nm. Separation was achieved on Waters C2 in its TXB2 form by ELISA by using TXB2 antibody and TXB2 as antigen.The prostaglandins synthesis was determined as TXA2+ release and TXA2 formation as described above.Typically 5 mM EGTA was added to washed PRP and incubated for 30 min at 37°C. The cell suspension was further used for measurement of Cap<0.05 were considered significant. Where appropriate, the Pearson’s correlation coefficients (r) of the results were also determined. Pearson score “r”, is such that -1 ≤ r ≤ +1 is acceptable where the (+) and (-) signs are designated as positive linear correlations and negative linear correlations, respectively. GraphPad Prism software  and Micro Cal origin 6.0 software were used for the statistical analyses.The obtained results are presented as mean ± SD, while the significance of the results was determined by using Student’s t-test. Values of 2 synthesis leading to the aggregation of platelets . The negative Coefficient of correlation indicates that they (NO and TXA2) are highly but negatively correlated.When different aggregating agents including P Figure  and the l-NAME, a well known inhibitor of NOS that is reported to decrease the basal NO level in platelets  in vitro  could be seen in fluorescence spectrophotometer in case of all aggregating agents  which resulted in the inhibition of aggregation of platelets by nearly 60% with simultaneous reduction of TXA2 from 0.35 ± 0.02 pmol in the presence of 4.0 μM ADP to 0.158 ± 0.007 pmol/108 platelets .When the platelets were pre-incubated with 200 μU/ml insulin instead of aspirin and subsequently 4 μM ADP added, the NO level increased from the basal 0.08 ± 0.001nmol to 0.367 ± 0.02 nmol/10When the washed PRP was incubated with either 80 µM aspirin or 200 µU/ml insulin or 0.8 nM NO followed by the addition of 4 µM ADP, no release of arachidonic acid from the platelet membrane was observed in HPLC and as seen without any change in transmission in fluorimeter. The treatment of Quin-2 loaded washed PRP with 80 µM aspirin demonstrated the presence of lesser amount of fluorescence similar to control (washed PRP only) as shown in Figure 2 synthesis is reported to be due to the ADP related activation of platelets  independent of ADP related interaction.It could be argued that the effect of ADP on TXAlatelets . HoweverThese results suggest that the basal NO level in platelets could be important both in the aggregation and in the inhibition of aggregation of platelets induced by all currently known platelet aggregating agents.2 for the aggregation of platelets  did not release arachidonic acid suggesting that these agents by itself was not the cause for the release of arachidonic acid from platelet membrane phospholipids. Indeed all known aggregating agents including ADP were following similar metabolic pathway for the release of arachidonic acid irrespective of their receptor activity, indicating that the release of the platelet membrane arachidonic acid is a “post receptor interaction” phenomenon leading to the increase of the cytosolic Ca2+ through the inhibition of platelet cytosolic NOS.Furthermore, the availability of Cad Figure . That arC Figure . In thisl-arginine, the substrate for NOS  with corresponding changes in the optimum substrate concentration (Km) for OS Table . These r+ Figure .2 (Figure-2 and to the aggregation of platelets (Figure 2 synthesis with no appearance of Ca2+ in the platelet cytosol (Table Although it has been repeatedly claimed that ADP was capable of activating platelet membrane COX, the availability of arachidonic acid in platelets was adequate to produce TXA2 Figure- which les Figure . The incs Figure  or by ins Figure  or by tr2+ has been reported to be a biologic messenger in various cellular events (2+ and for the activation of COX for the aggregation of platelets to follow. Conversely increase of NO level in platelets associated with the inhibition of platelet aggregation which resulted in the inhibition of both TXA2 synthesis and in the appearance of Ca2+ in the platelet cytosol.Finally it should be mentioned here that NO has been reported to act as the biologic “messenger molecule” before  and the r events . Our res2+. The Ca2+ thus liberated from the dense granule of the platelet cytoplasm in turn activated Phospholipase A2 releasing arachidonic acid from the platelet membrane phospholipids and thus affected the aggregation of platelets through the COX induced prostaglandin synthesis.In conclusion, it could be summarized that the inhibition of NO synthesis by different aggregating agents resulted in the increased intracellular mobilization of Ca


Tshr was silenced in mice, the phosphorylation of HNF-4α decreased, and cytoplasmic HNF-4α decreased while nuclear HNF-4α increased. In conclusion, our study revealed a novel mechanism by which TSH regulated the hepatic HNF-4α subcellular localization, suggesting the possibility that one of the effects of TSH is to reduce the expression of HNF-4α target genes.Hepatocyte nuclear factor-4 alpha (HNF-4α) is an orphan nuclear receptor with important roles in hepatic metabolism. Protein phosphorylation plays a functional role in its nuclear localization, DNA binding, and transactivation. Thyroid-stimulating hormone (TSH) is a hormone produced by the anterior pituitary gland, whose direct effect on the metabolic pathway has been observed. Our previous study demonstrated that TSH significantly decreases hepatic nuclear HNF-4α expression. However, whether TSH can influence HNF-4α phosphorylation is unclear. Here, we discovered that TSH can increase HNF-4α phosphorylation and modulate its subcellularlocalization. When HepG2 cells were treated with TSH, the phosphorylation of HNF-4α increased and its nuclear localization was interrupted. Cytoplasmic HNF-4α increased, while nuclear HNF-4α decreased. When the cAMP/PKA pathway was inhibited by the PKA inhibitor H89 and the adenylate cyclase (AC) inhibitor SQ22536, the TSH-mediated phosphorylation of HNF-4α was disrupted. When  Hepatocyte nuclear factor-4α (HNF-4α) is an orphan nuclear receptor (NR) that plays a critical role in hepatocyte differentiation1245681011PCK1), L-pyruvate kinase , glucose-6-phosphatase ] and xenobiotic and drug metabolism . HNF-4α is also known for its regulation of genes involved in lipid transport, especially those encoding apolipoproteins  that are linked to circulating levels of cholesterol and triglycerides and, hence, to atherosclerosis.Hundreds of HNF-4α target genes have been identified in the liver, pancreas, and colon24et al. found that glucagon and cAMP can increase the phosphorylation of HNF-4α, which loses its ability to bind to the CYP7A1 gene and results in the inhibition of human CYP7A1 gene transcription.Protein phosphorylation is a common post-translational modification among transcription factors and has been shown to play a functional role in nuclear localization, DNA binding, and transactivation1315in vivo and in vitroThyroid-stimulating hormone (TSH) is produced by the anterior pituitary gland, whose main function has been widely considered to be to regulate thyroid hormone (TH) synthesis and release in the thyroid aloneIn this paper, we demonstrated that TSH can increase the phosphorylation of HNF-4α via the cAMP/PKA pathway, which reduces the nuclear translocation of HNF-4α and attenuates its transcriptional activity in the liver. Our results will contribute to understanding the pathophysiological mechanism for the disruption of glucose and lipid homeostasis under thyroid and pituitary diseases accompanied by an elevated TSH level.Cyp7a1 also decreased, which is consistent with our previous studyThe HNF-4α transcription factor regulates many liver-specific genes. In the previous study, we demonstrated that the nuclear HNF-4α expression was reduced in HepG2 cells treated with TSH, and its target genes were also regulatedCYP7A1 via the PKA-mediated phosphorylation of HNF-4αWe also demonstrated the possible mechanism that TSH modulates the subcellular localization of HNF-4α. A recent report suggested that phosphorylation of a highly conserved serine (Ser78) in HNF-4α resulted in its impaired nuclear localizationTshr(−/−) mice are described in Tshr-KO littermate mice .Compared with the littermate wild-type mice, the phosphorylation of HNF-4α decreased in the Tshr-KO mice  is a type of thyroid function abnormality that is defined as increased serum TSH concentrations and normal serum TH levelsOur study also has some limitations. For example, HNF-4α has been reported to be phosphorylated by multiple kinases at multiple sites. PKA phosphorylates HNF-4α at S133 and 134 in the DBD and impairs its DNA binding activityIn summary, our study demonstrated a novel mechanism in which TSH regulated the hepatic HNF-4α subcellular localization. By regulating HNF-4α localization and thereby its transcription factor activity, TSH is likely to modulate the expression of several hepatocyte genes, which could contribute positively to a pathophysiological mechanism for the disruption of glucose and lipid homeostasis.Tshr-knockout (KO)  heterozygous mice [Tshr(+/−)] were obtained from the Jackson Laboratory, USA. Heterozygous mice were intercrossed to produce the homozygous mice[Tshr(−/−)] used in this study. The genotyping was determined from tail DNA. Because of the absence of Tshr gene expression, serum T4 was undetectable in Tshr(−/−)mice. To exclude the effect of thyroid hormones, the mice were placed on a hormone-replacement diet supplemented with 100 ppm desiccated thyroid powder (Sigma).Wild-type [Tshr(+/+)]C57BL/6 mice were used as a control. The mice were housed at 23 °C in a 12-hour light-dark cycle  and humidity-controlled (60%) environment.At 6–8 weeks of age, littermate male mice were sacrificed between 10:00 P.M. and 2:00 A.M. after fasting for approximately 12 h. Blood samples from the animals were obtained for analyses of serum levels of T4, TSH and liver function. In addition, liver tissues from all animals were collected. A partial tissue sample was fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline for histological analysis. The remaining tissue was immediately stored in liquid nitrogen until further analysis.The Shandong Provincial Hospital Animal Care and Use Committee approved the procedures for all animal experiments, and the methods were carried out in accordance with the approved guidelines. The human HepG2 hepatocellular carcinoma cell line and the human L02 liver cell line were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The HepG2 cells and the L02 cells were cultured in EMEM medium and RPMI 1640 medium with 10% FBS, respectively. As appropriate for the experiments to be performed, the cells were washed twice with PBS and then starved in serum-free medium overnight before they were treated with bovine TSH  for 24 h or 48 h. For some experiments, the PKA inhibitor H89 (20 μM) was added 1 hour before TSH treatment in serum-free medium.Liver tissue samples were ground into powder in liquid nitrogen. The tissue powder and cells were lysed in RIPA buffer to obtain total protein according to the manufacturer’s protocol. The nuclear and cytoplasmic proteins were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce). The protein concentration was measured using the BCA method.Equal amounts of protein from different samples were subjected to 10% SDS-PAGE followed by electrotransfer from the gel to polyvinylidene difluoride membranes (Millipore). The membranes were incubated overnight at 4 °C with anti-CYP7A1  and anti-HNF-4α  antibodies. β-actin protein was evaluated as a loading control  for cytoplasmic protein and Lamin B1 protein as a loading control  for nuclear protein. Immune complexes were detected using the Enhanced Chemiluminescence Plus Detection System (Amersham).Cells were incubated in serum-free medium without antibiotics containing 1mM IBMX  for 30 minutes, and then treated with 50 mIU/ml bTSH, 200 mIU/ml bTSH or 50 μM forskolin  for 1 hour. Intracellular cAMP levels were determined using a commercially available cAMP Direct Immunoassay Kit (BioVision) according to the manufacturer’s instructions and were normalized against total cellular protein concentration.PKA activity was assessed using a kit  according to the manufacturer’s instructions. In brief, 5 μg protein was added to a buffer containing substrate and then measured at an absorbance of 340 nm at 0 min and 5 min. The difference between the absorbance at the fifth minute and the immediate absorbance represented the activity.HNF-4α phosphorylation was assayed according to the method previously describedCells attached to cover slips were washed with PBS twice and fixed in 4% paraformaldehyde. The cells were blocked with serum that was homologous to the secondary antibody, followed by overnight incubation with the primary antibody (mouse anti-HNF-4α). The sections were subsequently incubated with a FITC-conjugated secondary antibody and mounted with DAPI. The analysis and photo-documentation were performed using a fluorescence microscope .t-test for two-group comparisons or a one-way analysis of variance (ANOVA) (Dunnett’s t or LSD test) for multiple comparisons. All of the calculated P values are two-sided. Differences were considered significant at P < 0.05.The data were analyzed using SPSS 17.0 and are expressed as the mean ± standard deviationor number. Differences among the groups were compared using an unpaired Student’s How to cite this article: Song, Y. et al. Thyroid-Stimulating Hormone Increases HNF-4α Phosphorylation via cAMP/PKA Pathway in the Liver. Sci. Rep. 5, 13409; doi: 10.1038/srep13409 (2015).


Cyclic nucleotide-dependent protein kinases  in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ- A critical point is whether the concentration of cCMP in vivo is sufficient to activate the identified signaling proteins. Moreover, a selective signaling pathway for cCMP has not yet been demonstrated. Although cCMP could act via pathways that are usually activated by other cyclic nucleotides such as cAMP/cGMP, further downstream effectors may exist. Moreover, the stimuli inducing cCMP synthesis could differ from those of the other cyclic nucleotide systems, and therefore a specific role for cCMP could be elucidated. Hence, the discovery of further cCMP targets is important for the identification of probable cCMP functions. To elucidate possible targets of cCMP in tissues, we performed cCMP kinase activity assays and cCMP-affinity chromatography of lung or intestinal murine tissues and analyzed the relevant proteins using mass spectrometry and immunoblotting analysis. These tissues were selected because cGKI and/or cGKII are present in high concentrations. cGKI was originally purified from lung (which contains high amounts of cGKI)  ATP (100 cpm/pmol) and ± cNMP at increasing concentrations). The Ka values for the different cyclic nucleotides were determined with GraphPad Prism based on the results of three to four experiments, using increasing concentrations of cNMP (1 nM to 50 mM). Ka values in experiments with tissue lysates were determined in the same way as in the experiments with purified enzyme, but only two representative concentrations were shown. To eliminate cAK-phosphorylation, a cAK-inhibitor peptide (AS5-24) was applied in all assays with tissues. This substance is a potent, synthetic peptide inhibitor of cAMP-dependent protein kinase with a Ki value of 2.3 nM . Therefore, both VASPtide and IRAGtide are suitable peptide substrates for the kinase assays. Interestingly, the measured kinase activation differed slightly between the substrate peptides. This result suggests that substrate specificity for these kinases is mediated by peptide sequences not integrated into the phosphorylated consensus sequence.In previous studies, we showed that cCMP is able to stimulate purified cAMP- and cGMP-dependent kinases cAK and cGKI ,8. Based of IRAG . Fig 1 d5-24) was added to eliminate cAK phosphorylation. Treatment of WT tissue lysates with cCMP (100 μM) resulted in a 2- to 2.5-fold stimulation compared with water alone (ctrl) (32P) ATP kinase assays using increasing cAMP or cGMP concentrations (1 nM-50 mM). 5-24) was added to eliminate cAK phosphorylation, a crossreaction with other kinases is possible. In these experiments the kinases in jejunum WT tissue possessed a ~70fold lower Ka value (2±0.5 μM) for cGMP (in vivo to activate endogenous kinases. The affinity of cCMP for cGKs is much lower than that of cGMP.The next step was to examine whether cCMP could stimulate endogenous cGKs in jejunum and lung tissue lysates. Both tissue types are known to contain cGKs in high concentrations –12. Wilde (ctrl) . Comparafor cGMP  than forfor cGMP , using ITo illuminate the exact role of cCMP, we sought to identify proteins that interact with this cyclic nucleotide. Therefore, lung and jejunum WT tissue lysates were analyzed by cCMP-affinity chromatography and gel electrophoresis of the bound proteins, followed by silver staining  or WesteWhile searching for other cCMP-interacting proteins, we found several publications that postulated a possible role for cyclic nucleotides and protein kinases interacting with the MAPK (mitogen-activated protein kinase) signaling pathway –25. TherNext, we performed a co-immunoprecipitation to detect a possible protein-protein interaction between cGK and MAPK . MAPK wa5-24, suggesting a stimulatory function for PKA in cCMP-mediated MAPK phosphorylation. There is a need for further studies analyzing MAPK activation by other cyclic nucleotides. To elucidate the role of cGK in lung and jejunum tissues in this process, we used cGKII knockout (cGKII KO) and cGKI knockout (cGKI KO) mice. The experiment was conducted as described above (the same experiment using WT tissue). We detected phosphorylation both in the jejunum and lung tissues from cGKI KO and cGKII KO mice  were treated identically to the DB-cCMP stimulated samples except that water was added instead of DB-cCMP water. Treatment of lung WT tissue lysate  with DB- KO mice , S7 Fig.inter alia regulated by cCMP. The role of cCMP/cGKII in these tissues would most likely be involved in intestinal secretion or lung alveolar fluid clearance [in vivo might be less important than that recently described in an investigation into CREB-phosphorylation in S49 lymphoma cells [In this study, we used cCMP kinase activity assays and affinity chromatography to identify cGKII and MAPK as possible cCMP effector molecules, and confirmed that cGKI and cAK function as cCMP interactors. The activation of the kinase activity of the cGKI isoforms in addition to cGKII and cAK by cCMP using IRAGtide as a substrate is in agreement with previously published data . Intereslearance ,30. As plearance , cCMP/cAma cells . MAPK stma cells . Therefoma cells . The newma cells ,35. Howema cells , and anoma cells . There ama cells ,37,38. Oma cells . Here, wS1 Fig(A/B) Stimulation of endogenous cGKs in lung or jejunum tissue lysates after activation with water alone (ctrl) or cGMP (50 nM or 5 μM). (C/D) Same panel as (A/B) using cAMP (500 nM or 100 μM). Data were expressed as x-fold stimulation relative to control samples . Error bars indicate mean±SEM of three independent experiments. Asterisks indicate statistically significant differences, ns: not statistically significant.(TIF)Click here for additional data file.S2 FigFor the competition experiments, cCMP (2 mM) was added (+) or omitted (-) as described in the Material and Methods. Untreated tissue lysate  and purified enzyme (1.5 ng/μL) were used as the controls. Purified enzymes are designated with * (B) Same panel as (A) using jejunum (WT) lysate. cCMP-binding proteins were analyzed by electrophoresis and immunoblotting with antibodies directed against cGKIc, cGKII or cAKRIα/β (C) Jejunum WT tissue lysate was incubated with 4-AH-cCMP agarose beads or EtOH-NH-agarose beads (ctrl). For the competition experiments, cAMP (2 mM) was added (+) or omitted (-). Untreated tissue lysate  was used as control; bound proteins were analyzed by electrophoresis and immunoblotting (cGKIc antibody).(TIF)Click here for additional data file.S3 FigFor the competition experiments, cCMP (2 mM) was added (+) or omitted (-) as described in the Material and Methods. Untreated tissue lysate  was used as an additional control. cCMP-binding proteins were analyzed by electrophoresis and immunoblotting with an antibody directed against p44/42 MAPK (B) Same panel as (A) using jejunum tissue lysate .(TIF)Click here for additional data file.S4 FigLung and jejunum WT tissue lysates were incubated with 8-AET-cGMP agarose beads or EtOH-NH-agarose beads (ctrl). For the competition experiments, cGMP (200 μM) was added (+) or omitted (-) as described in the Material and Methods. Untreated tissue lysate  was used as an additional control. cGMP-binding proteins were analyzed by electrophoresis and immunoblotting with antibodies directed against cGKIc, cGKII or p44/42 MAPK.(TIF)Click here for additional data file.S5 Fig5-24 cAK inhibitor, 10 μM) was added or omitted . The phosphorylation of MAPK was detected by immunoblotting using a phospho-p44/42 MAPK antibody (pMAPK). Total MAPK was measured by stripping the membrane and retreating with the respective antibodies. Densitometry analysis of 4–6 independent experiments (numbers in columns) was performed to quantitate the p44/42 MAPK levels. Data were expressed as x-fold MAPK phosphorylation relative to untreated control samples. Error bars show mean ± SEM. Asterisks indicate statistically significant differences, ns: not statistically significant.Lung tissue lysate (WT) was treated with 100 μM DB-cCMP for the indicated times (15/30/60 min). A protein kinase A inhibitor (AS (TIF)Click here for additional data file.S6 FigWT tissue lysates (lung or jejunum) were treated with 100 μM DB-cCMP for the indicated times (15/30/60 min). The phosphorylation of MAPK was detected by immunoblotting using a phospho-p44/42 MAPK antibody (pMAPK). Total MAPK was measured by stripping the membrane and retreating with the respective antibodies. Densitometry analysis of 5–6 independent experiments (numbers in columns) was performed to quantitate pMAPK levels. Data were expressed as x-fold MAPK phosphorylation relative to untreated control samples. Error bars show mean ± SEM. Asterisks indicate statistically significant differences, ns: not statistically significant.(TIF)Click here for additional data file.S7 FigTissue lysate was stimulated with 100 μM DB-cCMP for the indicated times (15/30/60 min). As control, untreated  and unstimulated tissue lysate  was used. Control samples (‘us’) were treated like DB-cCMP stimulated samples but, instead of DB-cCMP, water was added. The phosphorylation of MAPK was detected by immunoblotting using pMAPK antibody. Total MAPK was measured by stripping the membrane and retreating with the respective antibody. Densitometry analysis of 3–6 independent experiments (numbers in columns) was performed to quantitate p44/42 MAPK levels. Data were expressed as x-fold MAPK phosphorylation relative to untreated control samples. cGKII KO and cGKI KO data were compared with WT data. Error bars show mean ± SEM. Asterisks indicate statistically significant differences, ns: not statistically significant.(TIF)Click here for additional data file.S1 TableBands of interest were excised from the silver stained gel and handled as described in Material and Methods. The analysis was carried out using a nano-LC-MS/MS system and the tandem MS-spectra were aligned with the Uniprot-database.(DOCX)Click here for additional data file.


Background: Genetic loss of SAMHD1, a catabolic enzyme preventing dNTP accumulation outside S phase, leads to disease in humans but not in mice.Results: Allosteric regulation by dNTPs works similarly in pure recombinant human and mouse SAMHD1.Conclusion: Human SAMHD1 is more efficiently regulated at low dNTP concentrations.Significance: Inside cells dATP and dTTP are the main regulators of both human and mouse SAMHD1. 1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3–4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent mK for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower mK constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G SAMHD1The name SAMHD1 was coined from its protein structure that contains two previously known modules: an N-terminal SAM domain preceded by a nuclear localization signal and followed by a catalytic HD domain plus a disordered ≈40 amino acid-long C terminus. The complete protein as well as the isolated HD domain show triphosphohydrolase activity , 6, 7 anThermus thermophilus, Pseudomonas, and Enterococcus faecalis demonstrated that dNTPs not only are substrates for the enzymes but also work as allosteric effectors. Their crystal structures revealed a hexameric homo-oligomer as the biologically active unit of the T. thermophilus triphosphohydrolase  of SAMHD1 is enzymatically active and has a dimeric structure , 14. In 1 phase. After removal of the siRNA the pools progressively normalized and growth restarted, suggesting that in proliferating cells SAMHD1 limits dNTP pool size during G1 before entry into S phase. A similar arrest in G1 had been reported earlier in yeast cells with constitutively high concentrations of dNTPs . The crude bacterial extract was adsorbed to a 1-ml column of nickel-nitrilotriacetic acid resin and purified by stepwise elution with imidazole buffers of increasing imidazole concentrations. Enzyme purity was checked at each step. The material eluting at 100 or 250 mm imidazole was divided in small aliquots, frozen at liquid nitrogen temperature, and used for the experiments. TM Any kDaTM precast gels (Bio-Rad).We are grateful to Dr. F. Perrino for the gift of the plasmid for mouse SAMHD1 , coding 3H]dNTP , various effector combinations, and 20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 2 mm DTT, 50 mm NaCl, 2.5 mm mercaptoethanol, 1 mg/ml BSA, and 12.5% glycerol. After addition of SAMHD1 (50 ng of mouse enzyme or 150 ng of human enzyme) the tubes were incubated at 37 °C for 20 min, the reaction was stopped with 1 ml of 5 mm EDTA, and the solution was passed through a 1-ml column of AG1-X2 ion exchange resin to retain unreacted nucleotides. The produced isotopic deoxyribonucleoside was eluted with 4.5 ml of 50 mm acetic acid. From the total radioactivity in the combined run-through fraction + eluant and the specific radioactivity of the dNTP used for the reaction we calculated the dephosphorylation rate (nmol of deoxyribonucleoside/min/mg of enzyme) shown on the ordinate in most figures. In all cases <15% of the labeled dNTP was dephosphorylated during the incubation. We determined the mK constant for a substrate from incubations at 20 μm effector (GTP or dATP) concentration . Typically we conducted with each dNTP at least three independent experiments and in mK values ± S.E. For constants derived from single experiments we report in the tables the S.E. of the curve fitting provided by the software. We calculated kcat constants (turnover number) by dividing the Vmax values obtained from the Michaelis-Menten equation by 60 and by the nanomoles of SAMHD1 subunits used in the individual experiments. Graphs show results from representative experiments.A typical experiment consisted of separate parallel tubes containing in a final volume of 0.05 ml, the different concentrations of one [ntration . To dete3H]dATP  and 100 ng of recombinant mouse or 300 ng of human SAMHD1 was incubated for 0 or 20 min under assay conditions and chromatographed isocratically by HPLC together with nonlabeled carrier deoxyadenosine and dATP on a nucleosil C18 column. Flow rate was 1 ml/min for 30 min with 0.2 m NH4H2PO4, pH 3.5, followed by a 20-min gradient to 30% methanol. The reaction products were identified from their UV absorption, and their radioactivity was measured by counting 0.5-ml fractions of the eluate.To test the specific hydrolysis of dATP to deoxyadenosine and triphosphate by SAMHD1 , [3H]dATE. coli. On denaturing gels each enzyme gave a single band at the position expected for the monomer . The human enzyme was not stimulated by GTP at any concentration of dGTP (E). The activation of dATP hydrolysis was also simple, with either dGTP or GTP stimulating dephosphorylation by both the mouse and human enzymes (B and F). A more detailed comparison of GTP and dGTP as effectors is presented in the next section. Turning to the dephosphorylation of pyrimidine dNTPs, we found that GTP together with dATP gave optimal stimulation. GTP alone strongly stimulated the dephosphorylation of dTTP by both enzymes (C and G) and that of dCTP by human SAMHD1 (H). With the mouse enzyme, however, hydrolysis of dCTP at concentrations ≤100 μm was slightly but consistently inhibited when GTP was the only effector (D). When we tested at tighter intervals more dCTP concentrations we observed that above 120 μm substrate the hydrolysis started to be stimulated by GTP not reaching a plateau even at 1500 μm (data not shown). At all concentrations of dCTP as substrate, dATP  in combination with GTP further stimulated the reaction (D).The hydrolysis of purine dNTPs appeared more straightforward than that of pyrimidine dNTPs where attainment of the highest activity required more than one effector. dGTP presented the simplest case as the nucleotide efficiently stimulated its own dephosphorylation. Mouse SAMHD1 at concentrations of dGTP below 100 μubstrate A. The hu of dGTP E. The ac enzymes , B and F enzymes , C and Gn SAMHD1 H. With teffector D. When wreaction D.mK constants for all four substrates varying between 120 and 340 μm, but with kcat constants between 2 and 4 nmol · s−1/nmol of mouse SAMHD1 and 1 nmol · s−1/nmol of human enzyme . With both enzymes low concentrations of either effector stimulated the reaction maximally. The effector mK constants of the mouse enzyme were 0.5 μm for GTP and 0.8 μm for dGTP, the corresponding values of the human enzyme were 0.15 μm (A and B). At the low effector concentrations used for the calculations of the effector mK values, a competition between substrate and effector sites for dGTP binding can be excluded. At higher concentrations competition became apparent for dGTP, but not for GTP, showing that GTP does not bind to the substrate site.We begin with the effectors GTP and dGTP in their stimulation of dATP hydrolysis by mouse and human SAMHD1 , A and B 0.15 μm . GTP not 0.15 μm , A and BG and 3C). At 100 μm dTTP, a concentration at which dTTP bound both the catalytic site and allosteric site 2, the effector mK constant for GTP was 0.15 μm, similar to the mK of GTP during the hydrolysis of dATP . In the presence of 20 μm GTP, the effector mK of dATP was 11 μm for mouse SAMHD1 and 1.2 μm for the human enzyme. Increasing the GTP concentration to 500 μm had little effect. The mouse enzyme now had an effector mK for dATP of 8 μm, the human enzyme 0.9 μm. By testing the two concentrations of GTP we show that GTP does not compete with dATP at allosteric site 2.dATP worked as an effector for the hydrolysis of dCTP by both the mouse and human enzymes , A and BC, see also Fig. 2C), with a mK constant of 2.5 μm at 20 μm GTP and 1.5 at 500 μm GTP .dATP also stimulated the hydrolysis of dTTP by mouse SAMHD1 when GTP was present  working as effector in the presence of GTP with a mK constant of 2.1 μm. The corresponding activation by the mouse enzyme (D) occurred with a 20-fold higher mK of 50 μm  or dTTP (B) (30 and 100 μm) served as substrates. In all instances the combination of GTP and dATP gave the highest activation, agreeing with the results in Fig. 5B) dGTP alone produced a small stimulation only at 100 μm dTTP, and dCTP alone was inactive as effector and it did not potentiate the stimulation by GTP. Finally, the combination of GTP with dGTP resulted in a stimulation of dTTP hydrolysis intermediate between those observed with each nucleotide by itself suggesting a competition between the two effectors.We analyzed with the mouse enzyme different combinations of effectors, each at the fixed concentration of 10 μ of dCTP A or dTTP or dTTP B  and Fig. 5D (30 and 100 μm [3H]dTTP). Again, the combination of GTP and dATP gave the best activation with both substrates, but now GTP and dGTP alone were similarly active and strongly stimulated the hydrolysis of dCTP , in contrast to what was observed in Fig. 5A with mouse SAMHD1. The difference between the two enzymes may be explained by dCTP not only being a substrate for SAMHD1 but also an effector that stimulates its own hydrolysis as is the case for dTTP (see C). If, in analogy to dTTP, dCTP as effector has a much lower mK constant for human than for mouse SAMHD1, the contrast between A and C, is resolved.Similar experiments with human SAMHD1 but with all effectors at a concentration of 3 μdTTP see C. If, inm, and a high concentration of dATP, 100 μm, that saturated site 2. Thus, we limited the competition between GTP and dGTP to site 1. Raising the concentration of dGTP from 0 to 10 μm in the absence of GTP or with <0.5 μm GTP stimulated the hydrolysis of dATP. However, at concentrations of GTP ≥0.5 μm the addition of dGTP inhibited the reaction, and the degree of inhibition depended on the relative proportions of the two effectors (Fig. 3A, GTP is a better activator for the hydrolysis of dATP compared with dGTP. The data in In the next experiment we investigated in more detail the competition between the two guanine nucleotides at allosteric site 1 of the mouse enzyme by using different combinations of GTP and dGTP to activate the hydrolysis of dATP . To avoiffectors . As showmK constant of 50 μm (m dATP became the only effector responsible for the activation. These results show that both dATP and dTTP bind to the same effector site and that dATP, in agreement with its smaller mK constant (At allosteric site 2 we could demonstrate a competition between dATP and dTTP as effectors for the dephosphorylation of dCTP by mouse SAMHD1 . In the of 50 μm . On addiconstant , effectimK constants for each substrate (kcat values. Both enzymes dephosphorylated single dNTPs also in the absence of effectors and were activated up to 4-fold by GTP in combination with various dNTPs. Thus, dNTPs are both substrates and effectors of SAMHD1. For each given dNTP the mK constant as effector was considerably smaller than its mK as a substrate (mK values of the mouse enzyme for dCTP possibly being an exception. A second general observation was that the mouse enzyme had much larger effector mK values than the human enzyme. From competition experiments we conclude that allosteric site 1 has a very high affinity for guanine nucleotides and that site 2 is specific for deoxyribose and binds dNTPs with lower but still high affinity.The allosteric regulation of mouse and human SAMHD1 was incompletely understood when we started this investigation. Since then two publications about the crystal structure of the tetrameric protein in complex with effectors and substrate provided a precious key for the interpretation of the present results , 14. Eacubstrate , but theubstrate  and 2, wmK constant at site 2. Small constants favor the dephosphorylation of low concentrations of intracellular dNTPs. The smaller mK constants of the human compared with the mouse enzyme provide a superior mechanism to maintain low intracellular dNTP concentrations.We propose the following general scenario for the allosteric regulation of both enzymes. Site 1 accommodates GTP, the most abundant guanine nucleotide in the cell, stabilizing the tetrameric structure of the protein and resulting in the formation of site 2 , 22. NowmK constants influence the in vivo function of SAMHD1? The mK of GTP for site 1 is low, and at the physiological concentrations of the nucleotide site 1 will be saturated by the effector and SAMHD1 will normally be a tetramer, ready to be activated by dATP, dGTP, or dTTP at site 2. SAMHD1 is expressed mainly in quiescent cells which contain very small dGTP pools, thus a major regulatory function of this nucleotide is unlikely. The intracellular concentrations of dATP and, in the case of the human enzyme, also dTTP lie well in the range covered by the mK constants of the two enzymes. We therefore believe that these two deoxynucleotides, and in particular dATP, are the most important regulators of SAMHD1 activity.How do the effector m, to support nuclear DNA replication, but rapidly decline as replication is completed, and the R2 small subunit of ribonucleotide reductase disappears (m for dGTP (mK values reported here we can predict that in quiescent or differentiated cells SAMHD1 is not working at its maximum potential but still contributes significantly to the regulation of dNTP pools. The enzyme works outside S phase as a guardian of pool sizes by sensing dNTP fluctuations, e.g. due to variations of extracellular deoxyribonucleoside levels or due to invasion by viruses provided with their own dNTP synthesizing enzymes such as Herpes viruses. It was shown that SAMHD1 restricts not only HIV-I proliferation but also that of other human viruses, including Herpes (The concentrations of dNTPs are highly dependent on the proliferation state of cells , 25. Thesappears . Outsidefor dGTP , but adefor dGTP . SAMHD1 for dGTP . Loss offor dGTP , 21, silfor dGTP , or regufor dGTP , leads tg Herpes , 28.Returning to the different phenotypes of SAMHD1 deficiency in humans and mice, our present data demonstrate quantitative but not qualitative differences in the regulation of the two enzymes. Alone they are probably not sufficient to fully explain the different phenotypes of SAMHD1 deficiency in the two species. Other as yet unknown factors are likely involved. A solution for this problem requires further work with intact cells, but it will have to incorporate the results of our present investigation.


Homo erectus while indicating that their activities at the site were aimed at hunting, rather than scavenging.Animal movements in the Kenya Rift Valley today are influenced by a combination of topography and trace nutrient distribution. These patterns would have been the same in the past when hominins inhabited the area. We use this approach to create a landscape reconstruction of Olorgesailie, a key site in the East African Rift with abundant evidence of large-mammal butchery between ~1.2 and ~0.5 Ma BP. The site location in relation to limited animal routes through the area show that hominins were aware of animal movements and used the location for ambush hunting during the Lower to Middle Pleistocene. These features explain the importance of Olorgesailie as a preferred location of repeated hominin activity through multiple changes in climate and local environmental conditions, and provide insights into the cognitive and hunting abilities of  Their herding experience corroborates to our edaphic analysis of favoured and disfavoured grazing areas .Theropithecus oswaldi), elephant (Elephas recki) and hippopotamus (Hippopotamus gorgops)127Crocuta crocuta), and the number of individuals found is low. A relative lack of more dangerous predators, such as lions, would have added to the attraction for Homo. However, low predator numbers means that hominins would not have been able to reliably scavenge kills of other predators and must have been competent in close-encounter kills of larger-bodied and dangerous animals.We consider that the distribution of palaeofauna will have been subject to the topographic and nutritional constraints outlined. The palaeofauna of Olorgesailie  include We conclude that the Olorgesailie site was attractive to hominins for six independent reasons: (a) it was located close to a lake providing reliable drinking water; (b) workable stoneHomo exploited this part of the Kenya rift not because it was generally “good” for herbivores, but because it was generally “bad”, and constrained their movements to predictable pathways which allowed them to be exploited by early hunters. This, in conjunction with raw material availability, low predator numbers and suitable ambush positions explains the repeated attraction of the site within the wider region.The landscape complexity and the patchy nature of healthy grazing and browsing provide a way to understand how Pleistocene animals moved through the region and explain the hominin hunting and butchery activities at the Olorgesailie site over periods of significant climate fluctuationsThis study combines two new approaches to explain the hominin presence in the region and provides new insights into the significance of the location of the Olorgesailie site. Our preliminary results around Kariandusi and Baringo indicate that they share similar features, and that the methods we describe can be applied more widely in the East African Rift and that hominins at these other sites were practising similar techniques of ambush hunting during the Palaeolithic.How to cite this article: Kübler, S. et al. Animal movements in the Kenya Rift and evidence for the earliest ambush hunting by hominins. Sci. Rep.5, 14011; doi: 10.1038/srep14011 (2015).


Insulin/IGF-1 signaling involves phosphorylation/dephosphorylation of serine/threonine or tyrosine residues of the insulin receptor substrate (IRS) proteins and is associated with hormonal control of longevity determination of certain long-lived mice. The stimulation of serine phosphorylations by IGF-1 suggests there is insulin/IGF-1 crosstalk that involves the phosphorylation of the same serine residues. By this mechanism, insulin and IGF-1 mediated phosphorylation of specific IRS-1 serines could play a role in longevity determination.Ames dwarf mice to examine whether: (a) IGF-1 stimulates phosphorylation of IRS-1 serines targeted by insulin; (b) the levels of serine phosphorylation differ in WT vs. Ames fibroblasts; and (c) aging affects the levels of these serine phosphorylations which are altered in the Ames dwarf mutant. We have shown that IRS-1 is a substrate for IGF-1 induced phosphorylation of Ser307, Ser612, Ser636/639, and Ser1101; that the levels of phosphorylation of these serines are significantly lower in Ames vs. WT cells; that IGF-1 mediated phosphorylation of these serines increases with age in WT cells. We propose that insulin/IGF-1 cross talk and level of phosphorylation of specific IRS-1 serines may promote the Ames dwarf longevity phenotype.We used fibroblasts from WT and  Ames and Snell dwarf mice is attributed to the attenuation of the insulin/IGF-1 signaling pathways [The extended lifespan of the pathways . In thespathways . Reducedpathways .(+/−)] provided direct evidence that IGF-1 plays a role in controlling mouse longevity  mice is attributed to decreased levels of tissue and circulating insulin and IGF-1[Ames fibroblasts and their age-matched WT controls to IGF-1 treatment indicate that Ser307 phosphorylation is stimulated in WT fibroblasts by ~6-fold at 15 minutes and ~10-fold at 30 minutes and remains elevated up to 60 minutes of treatment  is a Ser/Thr kinase that upon endothelin-1 (ET-1) stimulation associates with IRS-1 thereby promoting ET-1 mediated Serradation . GRK2 thsistance . Furtherc injury , 25; typc injury . obesityc injury , 27.612 in WT fibroblasts whereas the Ames fibroblasts do not respond to this treatment  young Ames fibroblasts are resistant to IGF-1 stimulated phosphorylation and (b) the inducible level of Ser612 phosphorylation in the aged WT is similar to that of the young cells and c) the level of induction of the WT cells remains elevated for 60 minutes, whereas in the Ames cells it peaks in 5 minutes and declines rapidly  and degradation [636/639 phosphorylation in young WT mice whereas it is virtually non-responsive in dwarf fibroblasts  mice maintained on a high protein diet. Nutrient dependent activation of S6K1 is thus associated with insulin resistance in mice and humans in part via Ser1101 phosphorylation [Infusion of amino acids into humans leads to the activation of S6K1 phosphorylation of IRS-1 Serl muscle . Mutatiol muscle . Phosphorylation .1101 and treatment with IGF-1 results in further increased phosphorylation at this site Prop1 mutation are stabilized and maintained in fibroblasts derived from these mice [Using genetically identical young and aged WT and pathways . Importaese mice , 30, 31307, Ser612, Ser636/639, and Ser1101 is attenuated in Ames cells. Since these phosphorylations regulate multiple signaling pathways and metabolic processes the overall reduced levels of phosphorylation will, for example, affect levels of protein synthesis, fatty acid metabolism, glucose metabolism and stress response activity characteristic of the long-lived Ames mice [612 which is virtually absent in the fibroblasts from young Ames mice. Since phosphorylation of IRS-1 Ser612 is a physiological characteristic of cardiovascular pathology [612 in young WT mice may promote cardiovascular pathology and also serve as a marker of this disease as well as of aging. Alternatively, the virtual absence of Ser612 phosphorylation in young Ames fibroblasts and rapid loss of P-Ser612-IRS-1 by aged Ames fibroblasts suggests a decreased risk of cardiovascular disease associated with longevity. The fact that phosphorylation levels are significantly elevated in fibroblasts from aged WT and dwarfs suggest that this increased endogenous activity and strong stimulation of phosphorylation may represent major age-associated changes in the IRS-1 targeted metabolic functions.The IGF-1 mediated phosphorylation of IRS-1 Sermes mice . This isathology , 32, typathology , obesityathology , 27, our307, Ser612, and Ser636/639 phosphorylation in aged WT vs. aged Ames fibroblasts. For example, the level of phosphorylation in aged WT fibroblasts remains elevated up to 60 minutes whereas the phosphorylation declines after 15-30 minutes in the age-matched Ames fibroblasts. Thus, the WT-specific, sustained and elevated level of phosphorylated IRS-1 Ser residues may be part of the mechanism of inhibition of IRS-1 activity associated with resistance to insulin/IRS-1. On the other hand, although the Ames fibroblasts respond strongly to IGF-1, the decrease of Ser phosphorylation suggests this rapid recovery may be the mechanism that protects against the consequences of IRS-1 hyperphosphorylation.There is a significant difference between the induction of Ser307 and Ser636/639 that occur in animal models of insulin resistance [Ames mutants [The insulin/IGF-1 and mammalian mTOR pathways are highly conserved physiological processes that protect against the development of the aging phenotype. The multiple physiological consequences of IRS-1 phosphorylations are seen in increased activation of the mTOR pathway along with enhanced phosphorylation of IRS-1 at Sersistance , 34. Redsistance , 36. Thu mutants . That de(+/−) mouse down regulates the principle pathways stimulated by IGF-1 [(+/−)Igf1r mice showed a 50% reduction in IGF-1R levels and a marked reduction in IGF-1 induced tyrosine phosphorylation of its substrate, IRS-1. This suggests that the down-regulation of IGF-1R in haploinsufficient mice down-regulates the principal pathways stimulated by IGF-1 and that the general decrease of IGF-1 via a decrease in IGF-1R can increase mammalian lifespan [It has been shown that the haploinsufficient IGF-1Rby IGF-1 . Embryonlifespan .307 exemplifies a common site that integrates heterologous inhibition of insulin signaling by multiple factors  and cultured for 3 days in DMEM medium containing 15% FBS. The day before treatment, the medium was replaced with a medium containing 0.5% FBS. The recombinant human IGF-1 (Sigma) was prepared in distilled H2O and filter sterilized. The IGF-1 stock solution (25 ng/ml) was diluted in DMEM medium containing 0.5% FBS and the fibroblasts were treated with IGF-1 at a final concentration of 2 ng/ml. The cells were harvested and cytoplasmic and nuclear extracts were prepared [The isolation of fibroblasts from young (3-6 mos) and aged (21-24 mos) wild-type and escribed . In thisprepared . The proprepared . The pro307 IRS-1, anti-phospho Ser612 IRS-1, anti-phospho Ser636/639 IRS-1 and anti-phospho Ser1101 IRS-1. The antibody was from Sigma.Western blot analyses and immunoprecipitation assays were performed as described . AntibodAmes dwarf mutants. The normalized values of protein and phosphorylated protein bands were analyzed using the 2-tailed t-test to test the mean difference between age-matched groups at a significance level of 0.05. The symbols (*) indicate statistical significance for the values represented by the bar or time-point.Statistical analyses were performed for age-matched comparisons, the single dependent variable being the


Ag/AgCl), thus allowing the detection of the mustard agents with no electrochemical interferences. The suitability of this novel bioassay was tested with the nitrogen mustard simulant bis(2-chloroethyl)amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. The bioassay proposed in this work allowed the detection of mustard agent simulants with good sensitivity and fast response, which are excellent premises for the development of a miniaturised sensor well suited for an alarm system in case of terrorist attacks.In this work a novel bioassay for mustard agent detection was proposed. The bioassay is based on the capability of these compounds to inhibit the enzyme choline oxidase. The enzymatic activity, which is correlated to the mustard agents, was electrochemically monitored measuring the enzymatic product, hydrogen peroxide, by means of a screen-printed electrode modified with Prussian Blue nanoparticles. Prussian Blue nanoparticles are able to electrocatalyse the hydrogen peroxide concentration reduction at low applied potential (−50 mV  CWA are in all probability the cruellest produced among the Weapons of Mass Destruction (WMD). The Chemical Weapons Convention (CWC) has classified them based on their volatility, chemical structure or the physiological effects produced on humans. Regarding the latter feature, CWAs comprise nerve agents, vesicants (blistering agents), bloods agents (cyanogenic agents), choking agents (pulmonary agents), riot-control agents (tear gases), psychomimetic agents, and toxins . Among tSince 1993, CWC has promulgated several regulations, with new implementations since 1997, to prohibit the production and the use of chemical weapons, including mustard agents. Nevertheless, MAs and their degradation products, represent nowadays one of the most toxic environmental pollutants, being persistent in the environment for long term and causing high toxicity on biota and humans. MA exposure may occur through the skin, respiratory system, genital tract, ocular surface and gastrointestinal system, with serious acute and long term complications . Several50: dose required to kill half the members of a tested population).For these reasons, MAs remain some of the agents of highest internationally concern and are receiving increasing attention regarding their decontamination and degradation, but in particular for their detection in water and food or in human biological fluids. Gas chromatography (GC) and liquid chromatography (LC), combined with mass spectrometry (MS) are the main techniques employed for the screening of MAs in environmental samples  or in biIn this context, biosensor technology has a great potential to address these challenges, through the development of tailor-made, fast and cost effective, small and portable instruments –12, withet al. in 1948, focused on the effects of mustards on the structural properties and chemical activity of different enzymes involved in metabolic processes. Among these enzymes, choline oxidase has been indicated as the most sensitive to the action of nitrogen mustards [In the present work, we propose a new analytical device able to deal with the drawbacks related to sensitivity, time response, and portability. In particular, we developed an amperometric bioassay to reveal sulfur and nitrogen mustards based on their capability to inhibit the enzyme choline oxidase. This inhibitory effect was demonstrated in the work of Barron mustards .Alcaligenes sp. was chosen as biological recognition element for the development of the bioassay, and the simulants nitrogen mustard bis(2-chloroethyl)amine and sulfur mustards 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide (Scheme 1) were selected as target analytes and amperometrically detected by following the enzymatic product hydrogen peroxide.Thus, choline oxidase from 2.2.1.Alcaligenes sp. and its substrate choline, potassium hydrogen phosphate, hydrogen peroxide, the nitrogen mustard simulant bis(2-chloroethyl) amine and the sulfur mustard simulants 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide were purchased from Sigma Chemical Co. . Potassium chloride, potassium ferricyanide and iron chloride were obtained from Carlo Erba . A PalmSens3  Potentiostat was used for amperometric/voltammetric measurements.All chemicals from commercial sources were of analytical grade. Choline oxidase (EC 1.1.3.17) from 2.2.2 [Screen-printed electrodes (SPEs) were produced in our laboratory by thick film technology (TFT) with a 245 DEK  screen-printing machine. SPEs consisted of a working electrode in graphite modified with Prussian Blue nanoparticles (PBNPs), a reference electrode in silver/silver chloride, and a counter electrode in graphite . Graphit2 . The SPE2 . This so2.3.vs. Ag/AgCl was applied and a signal current was registered. The electrode surface was washed with distilled water. Then, 100 μL of a solution containing 0.05 M phosphate buffer + 0.1 M KCl pH 7.4, 180 mU/mL choline oxidase and 0.5 mM choline was drop-casted on the electrode surface and a signal current was registered for a period of 3 min to follow the current signal increasing, corresponding to the enzymatic reaction progress. The final current signal corresponds to A0. The electrode surface was thus washed with distilled water once more. To quantify the inhibitor, 70 μL of a solution containing 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4 and 180 mU/mL choline oxidase was incubated for 20 s with 10 μL of the inhibitor to a final concentrations ranging from 0 to 2.5 mM and drop-casted to the electrode surface. After incubation 20 μL of choline for a final concentration of 0.5 mM was added and a signal current increasing was registered for 3 min, corresponding to the enzymatic reaction progress. The final current signal corresponds to A1. Then, the inhibition percent was calculated according to the equation:0 is enzyme activity in the absence of inhibitor, A1 is enzyme activity in presence of inhibitor.The amperometric measurements were performed exploiting PBNPs-SPEs as electrochemical sensor connected to a potentiostat  able to apply the adequate potential and send to a computer the registered response signals. 100 μL of a solution containing 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4 was drop-casted on the sensor surface being attention to cover all three electrodes . A potential of −50 mV 2.4.Mustard simulant stock solutions were prepared under appropriate safety conditions. Operators were dressed with lab dresses, gloves, mask, and glasses to avoid contact or inhalation with powder and/or vapour. In addition, a dedicated hood has been used during sample preparation and analysis.3.Alcaligenes sp. as biological recognition element for the detection of nitrogen and sulfur mustard agent simulants: bis(2-chloroethyl)amine, 2-chloroethyl ethyl sulfide and 2-chloroethyl phenyl sulfide. Several parameters were optimised to highlight the operational conditions of the analysis, including enzyme concentration, substrate concentration, and incubation time between the enzyme and simulant. The type of inhibition was further investigated to highlight if the mechanism was reversible or irreversible. Reversible inhibition is characterised by non-covalent interactions between inhibitor and enzyme with the consequent restoration of the initial activity after the inhibition measurements. On the contrary, in the case of irreversible inhibition, characterised by covalent bonding between the enzyme and the inhibitor, the restoration of the initial activity requires a reactivation of the enzyme using specific compounds. From an analytical point of view, these mechanisms should be clarified since the reversible inhibition does not require incubation time and the measurement is then characterised by a short time of analysis. On the other hand, extended incubation times are necessary in the case of irreversible inhibition to enhance the sensitivity.The amperometric bioassay was set up by exploiting choline oxidase (CO) from 3.1.2O2) as a by-product, an electroactive molecule successively reduced on PBNPs-SPEs, since PBNPs are able to electrocatalyse the reduction of H2O2 at an applied potential of −50 mV vs. Ag/AgCl [2O2 concentration produced by the enzyme. Through the kinetic study of the production of H2O2, the enzymatic activity can be monitored in the presence of possible inhibitors . These inhibitors were able to decrease the enzyme activity, with a consequently reduction of H2O2 concentration enzymatically produced. The reduction of H2O2 leaded to a decrease of the amperometric signal proportional to the inhibitor concentration.Choline oxidase belongs to the family of oxidoreductase enzymes and catalyses the hydrolysis of acetylcholine. The enzymatic reaction produces hydrogen peroxide  current values of about 60 nA were obtained with a RSD of 4.3%; instead, using a higher enzyme concentration , the higher current values of about 500 nA were reached with a lower RSD of 2.9%. As expected, a lower repeatability was obtained using 3 min as reaction time with 20 mU/mL enzyme; indeed an extended reaction time was required to reach current intensities with higher accuracy. On the other hand, an enzyme concentration of 500 mU/mL allowed a lower RSD of 2.3%; however, the analysis was more expensive due to the cost of the additional enzyme.In order to optimize the concentration of choline oxidase, a study on the enzyme response as a function of enzymatic units was carried out. According to the experimental data presented in 2O2 concentration and consequently to the reaction rate as a function of a substrate concentration increase. The Michaelis-Menten constant, KM, for choline was determined using the Michaelis-Menten equation:Using an enzyme concentration of 180 mU/mL, a calibration curve for choline measurement was recorded . An incrM of 0.47 ± 0.4 mM, which is, for instance, higher than the one found using the immobilised enzyme since usually in solution the enzyme has a better affinity for the substrate compared to the immobilised enzyme [Thus resulting in a Kd enzyme .A final concentration of 0.5 mM choline was chosen for the measurements, since a higher concentration could compromise the identification of a competitive inhibition of reversible nature.3.3.2O2. A decrease of the amperometric signals occurred upon increasing of the inhibitor concentration. In particular, the grey line represented an analysis of a solution of 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4 in the absence of choline oxidase, where the signal was constant and close to zero; green line represented an analysis of a solution of 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4 in the presence of choline oxidase and choline, where the signal indicated the production of H2O2 due to the enzymatic reaction; whereas blue and pink lines were signals of a solution of 0.05 M phosphate buffer + 0.1 M KCl, pH 7.4 in the presence of choline oxidase, choline and bis(2-chloroethyl)amine at a concentration of 0.25 mM and 25 mM, respectively. At high concentration of simulant (25 mM) the inhibition percentage was close to 100%.The sulfur mustard agent simulants  and the nitrogen mustard agent simulant bis(2-chloroethyl)amine were used as target analytes. Since MAs are highly toxic and their use in no-security laboratories is restricted, the bioassay was developed using these simulants employed also in literature . Figure The inhibition mechanism and its percentage was further investigated in function of the incubation time and enzymatic units (U/mL), with the aim of highlighting which kind of inhibition (reversible or irreversible) takes place between the enzyme and the sulfur or nitrogen mustard simulants. Since the reversible inhibition is not dependent by the abovementioned parameters, while the irreversible one is directly proportional to the incubation time, but inversely proportional to the enzyme units, the enzyme inhibition mechanism can be unequivocally demonstrated. Thus, studies on incubation time and enzyme units were provided for each simulant.3.3.1.Analyses of the percentage of inhibition due to the simulant bis(2-chloroethyl)amine were investigated as a function of incubation time and enzyme units. As reported in y = (10.8 ± 1.4)x + (19.5 ± 4.5) with a regression coefficient (R2) of 0.940, where y is the degree of inhibition and x is the concentration of inhibitor. A limit of detection of 0.45 mM corresponding to an inhibition of 20% was obtained. As expected, the IC50 found (2.8 mM) was much higher than the one (0.001 mM) reported using the mustard agent tris(β-chloroethyl)amine (HN3) [This reversible inhibition, described by means of Lineweaver-Burk diagram so called double reciprocal plot, a graphical representation of the equation of enzyme kinetics , was repne (HN3) .Inhibitory studies on the enzyme choline oxidase have been conducted with nitrogen mustards, as reported in . In thisAs previously described for bis(2-chloroethyl)amine, incubation time and enzyme units were investigated for the inhibition due to 2-chloroethyl ethyl sulfide, showing that the inhibition percent did not vary significantly thus indicating a reversible inhibition . Since ay = (844 ± 66)x + (14 ± 3) with a regression coefficient (R2) of 0.967, with a limit of detection of 7 μM corresponding to an inhibition of 20%. In this case, a lower IC50 was found (0.04 mM), if compared with the previous one (2.8 mM) obtained using nitrogen mustard simulant. The limits of detection obtained using the proposed method were higher than standard laboratory set-up analyses , by which very sensitive limit of detection were obtained (237 ng/g) [Additionally, a calibration curve for 2-chloroethyl ethyl sulfide was constructed with a non-saturating substrate concentration. The obtained curve showed a linear range between 7 μM and 0.053 mM described by the equation 37 ng/g) . However37 ng/g) .50 equal to 0.7 mM. In this case the IC50 was higher than the one previously obtained using 2-chloroethyl phenyl sulfide as the sulfur mustard, which can probably be ascribed to the steric hindrance of the phenyl group. Furthermore, the best reproducibility was observed in the case of the nitrogen mustard simulant tested.Incubation time and enzyme units were also investigated due to the inhibition by 2-chloroethyl phenyl sulfide . ResultsTaking into account the obtained results, a reversible mechanism was suggested for the analysed simulants, having advantages in terms of short analysis time and higher number of analyses using immobilised enzymes ,27. The 4.In this work we have carried out preliminary studies on the inhibition of the enzyme choline oxidase due to mustard agent simulants, in order to develop a bioassay for the determination of the modeled mustard agent compounds in contaminated environmental sites, considering crucial features for tailor-made investigations such as short time response, simple equipment, reduced costs, and in-field analyses.Based on the few biochemical studies reported in the literature on the inhibitory effect of mustards on choline oxidase ,27,28, w


In natural signals, such as the luminance value across of a visual scene, abrupt changes in intensity value are often more relevant to an organism than intensity values at other positions and times. Thus to reduce redundancy, sensory systems are specialized to detect the times and amplitudes of informative abrupt changes in the input stream rather than coding the intensity values at all times. In theory, a system that responds transiently to fast changes is called a differentiator. In principle, several different neural circuit mechanisms exist that are capable of responding transiently to abrupt input changes. However, it is unclear which circuit would be best suited for early sensory systems, where the dynamic range of the natural input signals can be very wide. We here compare the properties of different simple neural circuit motifs for implementing signal differentiation. We found that a circuit motif based on presynaptic inhibition (PI) is unique in a sense that the vesicle resources in the presynaptic site can be stably maintained over a wide range of stimulus intensities, making PI a biophysically plausible mechanism to implement a differentiator with a very wide dynamical range. Moreover, by additionally considering short-term plasticity (STP), differentiation becomes contrast adaptive in the PI-circuit but not in other potential neural circuit motifs. Numerical simulations show that the behavior of the adaptive PI-circuit is consistent with experimental observations suggesting that adaptive presynaptic inhibition might be a good candidate neural mechanism to achieve differentiation in early sensory systems. Typically, sensory input signals vary slowly for most of the time and abrupt changes in value occur relatively rarely , 2. ThusA number of different neural mechanisms exist and have been proposed that have the ability to respond strongly to the informative changes in the sensory input, while staying relatively silent to slowly varying, redundant inputs, and thus act as differentiators. In principle, to amplify a step increase in input intensity over the baseline steady state level, an excitatory feedforward drive has to be coupled with a (delayed) negative component that retracts the transient rise to a lower plateau. Roughly two kinds of neural mechanisms can achieve such a differentiator response: 1) inhibitory activity (circuit-based) and 2) purely synaptic modifications . In circuit-based differentiators, the negative components are generated by inhibitory neurons, either by feed-forward inhibition , 7 or feWhile thus a differentiator can have different designs in neural circuitry, not all possible mechanisms might fit to the requirements of early sensory systems. In particular, a suitable differentiator for early sensory systems has to cope with the high dynamic range of natural signals, often spanning multiple orders of magnitude , much hipresynaptically mediated feedforward inhibition functions in the widest dynamical range of all tested differentiator circuits. Crucially, despite the wide dynamical range, vesicle resources are maintained regardless of the input intensity, a property that is in stark contrast to the other circuit mechanisms investigated. This advantageous property might be the reason why presynaptic inhibition, an axo-axonic form of inhibition, is specifically observed in early sensory systems such as the retina . To ensure a large dynamic input range this modulation signal was then given to our model in exponentiated form according toTo fit such a LN model to the response of our circuit motifs, we followed  and firsF and nonlinearity N of the LN model, N(F * n(t)), was then fitted to the circuit motif response nmin, nmax] of the modulation signal  and the circuit response n(t) and response n(t), which can be done in the Fourier domain:The estimation of the filter see e.g. ). The mon(t), and t. The estimated filter F was then normalized that the variance of the LN model response was equal to the variance of the input [h = F * n(t) is the linearly filtered response. The nonlinearity N(h) was then calculated by examining the relation of h.Here he input , i.e.,VPreE, receives external sensory inputs, feedforwardly connects to a post-synaptic excitatory population, PostE  as a differentiator system  and thus divisively modulates its strength  input firing rate analysis , 30, we ρ, the PostE neurons display different kinds of transient activities and steady state values , becr1 to r2, the firing rate of PostE neurons exhibits a transient dynamics. In case of the perfect differentiator setting, the response amplitude of the transient dynamics r1 and find a linear relationship. If instead Δr is fixed and the baseline r1 is changed, we also find a linear relationship when r1 /r1. In PostE firing rate decays to its stationary state within a shorter time period if the contrast is high, whereas the decay becomes markedly slower for low contrast stimuli. Similarly, if we estimated the temporal filter of the neural circuit , the smaller is τu at the initial stage after the stimulus change  or short-term facilitation (STF), we compared the adaptive range of neural responses when one of STD or STF (or both) are blocked see . Using ttion see .We have found that the adaptive PI-circuit response is contrast dependent, with slower transient activity decay for low contrast inputs than for high contrast inputs. This adaptivity arises from the input dependent transient activity of the vesicle resource and the release probability. Experiments have measured the linear filtering and nonlinearity property of different cell types in the retina . It was In summary, the STP-PI circuit seems to well reproduce the experimental observations suggesting that the observed adaptability in the retinal circuit might well arise from short-term plasticity of synaptic transmission and not necessarily from other intra-cellular mechanisms.We compared a number of simple circuit motifs in their capabilities to respond adaptively to sudden changes in the input. We found that unlike other feedforward inhibition motifs that have been described previously , 7, our Presynaptic inhibition is a form of synaptic inhibition that is mediated via an axo-axonic synapse complex . Inhibitdivisively balanced by inhibitory inputs, while for postsynaptic feedforward inhibitory circuit, excitation and inhibition are subtractively balanced. Thus, in contrast to the common notion of balanced excitation and inhibition in neural circuits [In our analysis, we found that whether feedforward inhibition is mediated pre- or post-synaptically is a crucial difference. Although both mechanisms can show perfect differentiation, the way of establishing the balance of excitation and inhibition is qualitatively different. In the presynaptic feedforward inhibition circuit, excitatory current is circuits , 50, in Divisive or subtractive balance differs in several aspects in regard to their signal processing properties. First, presynaptic inhibition maintains the vesicle resource and release probability at a proper value regardless of the input intensity, thus enabling efficient utilization of vesicle resource without potential depletion of resources for high input intensities. In contrast, in case of postsynaptic inhibition, vesicle resources will decrease in proportion to the presynaptic firing rate and thus cannot be maintained. Second, when the excitatory synapse exhibits short-term depression, the feedforward inhibitory pathway would also need an identical form of short-term depression, i.e. the very same depression time constant, to guarantee a detailed balance of excitation and inhibition. This seems highly unlikely to be realizable in practice. Finally, because of the divisive nature of the presynaptic inhibition, the PI circuit can reduce higher-order statistics of sensory inputs (as shown in the In the PI circuit, perfect differentiation is largely due to presynaptic modulation of the excitatory synaptic transmission efficacy. To achieve perfect differentiation in the circuit, the strength of the presynaptic inhibition has to be reversely proportional to the inhibitory activity. In this case, excitation and inhibition is perfectly balanced with each other. It has been reported that the balance of excitation and inhibition could be learned in a feed-forward network with inhibitory synaptic plasticity , 52. ThuResponses according to the law of Weber-Fechner ensure that the dynamic range of input signals is compressed into the dynamic range of single neurons. The discussed presynaptic inhibition mechanism ensures a dynamic range compression by relying on a specific connectivity between different neuron types, namely a specific type of feed-forward inhibition. It was reported that the dynamic range of input signals might also be compressed through single neuron properties, for instance by active dendrites or electrical synapses , 54. SinExperimentally, it was found that sensory neurons act as differentiators in response to strong enough sensory inputs but can also act as integrators for weak inputs , 15. ThiPostE neuron spiking mechanism, rather than to an inapplicability of the PI circuit to the experimental data. When our circuit model would be extended to additionally include postsynaptic shunting inhibition [Our results suggest a novel mechanism for contrast adaptation in early sensory system. In particular, we found that STP mediated the contrast adaptivity of the PI-circuit. STP has been only recently more thoroughly investigated theoretically and STP was suggested to play various important computational roles, e.g. in the generation of memory traces , persisthibition  and neurIn summary, our results indicate that the experimental findings of contrast adaptivity might indeed be a result of the plasticity of synaptic contacts rather than caused by other intra-cellular mechanism. Whether this suggestion is correct could be tested in future experiments.