Objective. This study was aimed to determine the effect of Anethum graveolens extract and Anethum graveolens (dill) tablet on lipid profile, liver enzymes, and gene expression and enzymatic activity of HMG-CoA reductase in high cholesterol fed hamsters. Materials and Methods. Golden Syrian male hamsters (130 ± 10 g) were randomly divided into 6 groups (n = 6) and received daily the following: group 1 received chow + 2% cholesterol + 0.5% cholic acid (HCD), groups 2 and 3 received HCD diet plus 100 and 200 mg/kg hydroalcoholic extract of dill, respectively, and groups 4 and 5 received HCD diet plus 100 and 200 mg/kg dill tablet, respectively. Group 6 received only chow. After 1 month feeding serum biochemical factors were determined. HMG-CoA reductase mRNA level was measured  and its activity was determined spectrophotometrically. Results. Compared with hypercholesterolemic group 1, lipid profile, blood glucose, and liver enzymes significantly decreased in all dill tablet or dill extract treated groups (p < 0.05). The changes in HMG-CoA reductase gene expression level and enzyme activity significantly reduced in animals that received 200 mg/kg of extract or tablet. Conclusion. Dill extract and dill tablet showed potential hypocholesterolemic properties in hamsters by inhibition of HMG-CoA reductase activity. Anethum graveolens L., commonly known as a dill, an annual plant growing in Europe, Mediterranean region, and Asia . P. P Anethns (68%) . The hypDill was purchased from Hamadan (west of Iran) daily market and identified by our colleagues in the Department of Biology, Borujerd Azad University . Hydroalcoholic extract was prepared according to the previously described method . Dill ta2CO3 (2%), and 0.1 mL of Folin-Ciocalteu reagent (50%). The samples were then incubated for 30 min at room temperature and the absorbance of the reaction mixture was measured at 750 nm against deionized water. Total phenolic content was calculated per mg equivalents of gallic acid (GAE) per gram of each extract.The amount of total phenolic content of dill extract and/or dill tablet was determined according to previously reported method with small modification using Folin-Ciocalteu reaction . Briefly3 (10%), 0.1 mL of potassium acetate (1 M), and 2.8 mL of deionized water. After that, the mixture was incubated at room temperature for 40 min and absorbance of the mixture was measured at 415 nm against deionized water. The content of flavonoids was calculated per mg equivalents of quercetin per gram of each extract.Total flavonoids were measured using aluminum chloride colorimetric assay, according to the previously described method . Briefly3 (20 mg/mL) and 6 mL sodium acetate solution (50 mg/mL). The mixture was then incubated for 150 min at room temperature and the absorbance was determined at 440 nm. The content of total flavonols was calculated per mg equivalents of quercetin per gram of each extract.The flavonols content was measured according to the previously published method . The diln = 6) and fed as follows: group 1: chow + 2% cholesterol + 0.5% cholic acid; group 2: chow + 100 mg/kg hydroalcoholic extract of dill + 2% cholesterol + 0.5% cholic acid; group 3: chow + 200 mg/kg hydroalcoholic extract of dill + 2% cholesterol + 0.5% cholic acid; group 4: chow + 100 mg/kg dill tablet + 2% cholesterol + 0.5% cholic acid; group 5: chow + 200 mg/kg dill tablet + 2% cholesterol + 0.5% cholic acid; and group 6: chow. At the end of 30 days' experiment, animals were anesthetized and sacrificed after 12 h of fasting. Blood samples were collected from the heart and serum was separated by centrifugation of samples at 3000 rpm for 10 min.A total of 36 male golden Syrian hamsters weighing 130 ± 10 g were used in this experiment. Animals were kept for one week before experimentation and adaptation. Regular conditions with light/dark cycle (12 hour for each), relative humidity of 60 ± 5%, and temperature at 23 ± 2°C were applied during the experiments. After adaptation, hamsters were randomly divided into six experimental groups , whereas LDL-C and VLDL-C were calculated by Friedwald's formula , 9.Total RNA was isolated from liver tissue samples (100 mg) using TRIZOL reagent according to the manufacturer's protocol . The integrity of extracted RNA was determined by 1% agarose gel while its concentration and purity were assessed by Nano-Drop spectrophotometer . After that, 1 microgram of RNA was applied for reverse transcription process using QuantiTect Reverse Transcription Kit . Integrity, purity, and concentration of cDNA were also measured as described for RNA .Determination of HMG-CoA reductase mRNA level was carried out by real-time PCR using QuantiTect SYBR Green PCR Kit  in a PCR detection machine .μL cDNA template (<100 ng), 10 μL SYBR Premix (1X), and 1 μL of each primer (0.4 μM) and adjusting of volume to 20 μL by adding deionized water. HMG-CoA reductase mRNA level was measured in triplicate in 38 cycles using thermal cycler.Primers of reaction were designed by AlleleID7 software  for HMG-CoA (forward: 5′-AAGGAGCGTGCAAAGACAATC-3′ and reverse: 5′-TGAACCATGTGACTTCTAACAAG-3′) and GAPDH (forward: 5′-TGGCCTTCCTTCCTACG-3′ and reverse: 5′-TAGCCCAGGATGCCCTTCAG-3′) as internal control. The RT-PCR reaction mixture was prepared by the addition of 1 μL lysis buffer containing 1% protein inhibitor  and centrifuged for 15 minutes at 14000 rpm and 4°C. Supernatant was used for the measurement of enzyme activity using HMG-CoA reductase assay kit  based on spectrophotometrical determination of oxidized NADPH at 340 nm.Liver tissue samples were homogenized in 500 p values less than 0.05 were regarded statistically significant.Data are expressed as means ± SEM of three replicate measurements and then analyzed by SPSS package . One-way analysis of variance (ANOVA) followed by Dunnett's test were used. The Total phenol, flavonoid, and flavonol contents of dill tablet and dill extract are shown in p < 0.05) in dill tablet and dill extract treated hamsters. These reductions were higher in hamsters treated with 200 mg/kg of dill extract or dill tablet. Significant increase was observed in HDL-C level by treatment with 100 and 200 mg/kg of dill extract or dill tablet. Treatment of hamsters with 100 mg/kg of dill extract or dill tablet did not affect body weight compared with untreated high cholesterol diet fed hamsters (159 ± 6 or 155 ± 5.8 g versus 168 ± 5.1 g). However, there was a marked weight gain in high cholesterol diet fed animals compared with control (166 ± 5.5 versus 140 ± 4.0 g). Receiving 200 mg/kg of dill extract or dill tablet significantly (p < 0.05) reduced the body weight compared to the animals receiving only high cholesterol diet (146 ± 3.9 or 148.1 ± 4.4 g versus 168 ± 5.1 g). Fasting blood glucose levels significantly declined in animals treated with 200 mg/kg of dill extract or dill tablet as indicated in −ΔΔCt method. The results are summarized in The effect of dill tablet and dill extract on gene expression of HMG-CoA was determined using real-time PCR technique. To show the relative changes in the expression of this gene between the studied groups, ΔCt and ΔΔCt were calculated; subsequently fold change of expression was calculated using 2p < 0.001).Furthermore, according to statistical analysis of ΔΔCt, dill tablet and dill extract at 200 mg/kg dose reduced HMG-CoA reductase in treated group comparing to HCD group (p < 0.05), while the changes made in enzyme activity after treatment of animals with 100 mg/kg of dill extract or dill tablet did not reach statistical significance  showed significantly reduced HMG-CoA reductase activity compared with high cholesterol diet fed or control hamsters  . SuppresThis study presents preliminary results indicating that dill tablet and dill extract are able to decline total cholesterol levels by inhibiting liver HMG-CoA reductase activity. The main approach involved in hypercholesterolemia treatment is the suppression of cholesterol synthesis by declining of HMG-CoA reductase activity which is known as the rate-limiting enzyme in the cholesterol biosynthesis pathway . HMG-CoAHowever, only dill tablet and dill extract at the dose of 200 mg/kg reduced HMG-CoA reductase activity. As we expected, HMG-CoA reductase activity reduced in high cholesterol diet fed group. According to these findings, we suggest that 200 mg/kg dose of dill possibly exerts its regulatory effect at the transcriptional, translational, and posttranslational levels.But dill at the dose of 100 mg/kg only regulated at the transcriptional levels, and enzyme activity was not affected at this dose. Further experiments are required to determine the exact mechanism in HMG-CoA reductase activity inhibition.No exact mechanism has been recognized for hypocholesterolemic effects of dill so far. Yazdanparast and Bahramikia  has showSome studies have reported that rutin and quercetin (components of dill) decreased serum total cholesterol and LDL-C and reduced liver cholesterol levels . Other sEffects of the antioxidant agents  on the control of endogenous cholesterol biosynthesis have previously been investigated and antiatherosclerosis properties of these compounds have been reported in different experiments . For exaα-pinene, phenol, flavonoid, flavonol, anthocyanin, tannins, saponin, and alkaloid [Our HPLC analysis showed that AG (leaves and seeds) contains different type of flavonoids and monoterpenoid including limonene, linalool, quercetin, alkaloid . Salvamaalkaloid  reportedalkaloid  reportedPhenolic compounds also have antioxidant and scavenging activity as well as involvement in the inhibition of lipid peroxidation, mutagenesis, carcinogenesis, atherogenesis, and thrombosis . In the An increased level of serum ALT and AST was observed in hypercholesterolemic group, while these enzymes significantly reduced in dill tablet and dill extract treated animals. Serum AST and ALT have been recognized as sensitive marker of liver injury. In fact, damage of hepatocytes changes membrane permeability and cell function and causes the leakage of these enzymes into the blood stream . High chIn this study, blood glucose significantly reduced after treatment by dill tablet or dill extract (200 mg/kg). This observation is in line with previous studies by Mishra  and MadaSince we did not measure the consumed food for each animal, it can be mentioned as a limitation of this study. Furthermore, since alteration in mRNA level does not necessarily reflect changes in the related protein, determination the produced protein can indicate more precise mechanisms.Study of cellular mechanisms also can help in better interpretation of the results.This study established that dill has potential hypolipidemic activity; consequently it can be used in hypercholesterolemic patients. On the other hand, this study revealed the cholesterol lowering effect of dill via inhibition of HMG-CoA reductase activity in hamsters. Further experiments are also needed to determine the similar properties of dill in hypercholesterolemic patients.


Protein posttranslational modifications (PTMs) play key roles in a variety of protein activities and cellular processes. Different PTMs show distinct impacts on protein functions, and normal protein activities are consequences of all kinds of PTMs working together. With the development of high throughput technologies such as tandem mass spectrometry (MS/MS) and next generation sequencing, more and more nonsynonymous single-nucleotide variations (nsSNVs) that cause variation of amino acids have been identified, some of which result in the damage of PTMs. The damaged PTMs could be the reason of the development of some human diseases. In this study, we elucidated the proteome wide relationship of eight damaged PTMs to human inherited diseases and cancers. Some human inherited diseases or cancers may be the consequences of the interactions of damaged PTMs, rather than the result of single damaged PTM site. More than 200 different types of protein posttranslational modifications (PTMs) have been detected. PTMs are involved in many protein activities and cellular processes, such as protein folding, stability, conformation, and some significant regulatory mechanisms . For insWith the development of high-throughput sequencing technology, gene mutation detection has become another important resource to investigate regulatory mechanisms and cellular processes. Some databases such as dbSNP  and SNVDA PTM site that bears nsSNVs can be defined as damaged PTM. Recently, large-scale studies have shown that damaged PTMs caused by numerous inherited and somatic amino acid substitutions  have proHowever, some of these previous studies concluded the relationship between damaged PTMs and human health based on predications; some focused only on cancers and many focused on only unique type of PTM. Although data of both gene mutations and PTMs are increasing fast, the proteome-wide analysis on the relationship between damaged PTMs and human diseases is not well studied. In this work, we chose eight experimentally demonstrated damaged PTMs to elucidate their association to human diseases including inherited diseases and cancers (somatic diseases). These eight types of damaged PTMs include amino acid variations on Phosphorylation, Ubiquitylation, Acetylation, Glycosylation, Methylation, SUMOylation, Hydroxylation, and Sulfation, which have been well proved to play key roles in important cellular processes and have close relationship with human disease development; moreover, some cross talks among them have been recently revealed in the view of systematic biology , 16. In The eight human PTM data sets of Phosphorylation, Ubiquitylation, Acetylation, Glycosylation, Methylation, SUMOylation, Hydroxylation, and Sulfation were obtained from SysPTM 2.0  , which ihttps://tcga-data.nci.nih.gov/tcga/), and SNVDis [The inherited-diseases-related nsSNVs were obtained from ClinVar  , dbSNP  [P < 0.05).nsSNV affected PTM sites are defined as damaged PTMs in this work. Annotations of nsSNVs  were based on the information from the databases mentioned above and on Online Mendelian Inheritance in Man   for refeov/omim)  and the http://www.ingenuity.com/). In order to find the structure information of the damaged PTMs, we performed domain enrichment analysis for both inherited disease and cancer-related damaged PTMs based on the domain information from Pfam ; only the domains containing damaged PTMs were chosen. The enrichment results were calculated and chosen based on disease-related PTM-containing proteins using Fisher's exact test and adjusted with Benjamini-Hochberg method .To further analyze the functions and features of diseases-related damaged PTMs and their proteins, enrichment analyses were performed using DAVID 6.7  . Pathwayhttp://ptmcode.embl.de/) which compiles known and predicated PTM associations [http://string-db.org/) [As for the cross talks between some pairwise types of PTMs, positive and negative cross talks were both considered. Positive cross talk means that one PTM serves as a signal for the addition or removal of a second PTM, or for recognition by a binding protein that carries out a second modification. The negative cross talk could be direct competition for modification of one single residue on a protein, or one modification masks the recognition site of a second PTM . Some pociations . The intdb.org/) .http://dx.doi.org/10.1155/2015/124630); the percentages of the exact matched result out of all eight types of PTMs is shown in The workflow and protocol of this study are shown in We calculated the PTMs affected by inherited disease and cancer-related nsSNVs, respectively, using hypergeometric test and found that phosphorylation affected by nsSNVs was most significantly related to both inherited diseases and cancers. The next is ubiquitylation; however, based on our calculation, it is not significant in inherited diseases, albeit significant in cancers when performing the exact match. The remaining types of PTMs affected by nsSNVs were not significantly associated with inherited diseases. When we expanded to ±2 amino acids around the modified sites, the damaged PTMs significantly associated with inherited diseases included not only ubiquitylation, but also acetylation and glycosylation. Our results implied that most PTMs affected by nsSNVs were cancer-related, rather than inherited-disease-related , Serine (S), Threonine (T), and Tyrosine (Y) for phosphorylation, Lysine (K) for ubiquitylation, and made a calculation on the frequency of the appearance of nsSNVs on these modified amino acids. We found that the occurring frequency of the modified amino acids affected by nsSNVs was lower compared with their appearance on the whole proteome (data not shown). This demonstrated that the modified amino acids were less affected by mutations. Previous researches showed that PTM sites generally play a key role in normal cellular process like protein-protein interactions and signal transduction and therefore are more stable , 32, andP value = 0.01331) and 2684  out of the 7005 damaged phosphorylation sites were inherited-disease-related and cancer-related, respectively. Therefore, phosphorylation affected by nsSNVs was significantly associated with both inherited diseases and cancers   . For proP value = 0.08067) were inherited disease-associated and 651  were cancer-related sites. For acetylation and glycosylation, both were not found closely related with inherited diseases and cancers  ranked the first with 50 proteins. For example, PSN1_HUMAN, TNR1A_HUMAN, VHL_HUMA, and PSN1_HUMAN were well studied and associated with “autosomal dominant early-onset Alzheimer's disease” in human [n = 1074, corrected P value = 4.36E − 45), which ranked the top with 1074 proteins; RASK_HUMAN, P53_HUMAN, EGFR_HUMAN, and so forth were the representative ones. RASK_HUMAN is associated with adenocarcinoma in human large intestine and lung and other tissues. P53_HUMAN is well known for its associations with human colon and rectal and other cancers [n = 11, corrected P value = 3.36E − 10), and two proteins were closely related to it: TSC2_HUMAN and TSC1_HUMAN. They were reported to be associated with tuberous sclerosis syndrome in human [n = 54, corrected P value = 4.28E − 6) in Ubiquitylation. For acetylation and glycosylation, we also examined both associated inherited diseases and cancers. As to acetylation, we observed disorders of cellular development and cellular growth and proliferation besides cancers that were led by mutations on P53_HUMAN. With regard to glycosylation, the diseases were closely related to lipid metabolism and molecular transport.In order to acquire more information on related diseases, we performed enrichment analysis of diseases using IPA Figures . We perfin human . The mos cancers , 38; for cancers . As to uin human . Non-smaWe then expanded our search range to the nsSNVs that could affect the PTMs: ±2, ±7 around phosphorylation sites and ±2 for the remaining types of PTMs. First, we chose ±2 range for all the 8 types of PTMs to analyze the associated diseases. For inherited diseases, “autosomal dominant disease” and “autosomal recessive disease” ranked top three in phosphorylation, Ubiquitylation, Acetylation, Glycosylation, Methylation, Hydroxylation, and Sulfation. This was clearly different from the exact matched results. Both autosomal diseases and X-linked hereditary diseases became significant when more nsSNVs were accumulated around PTM sites. The comparison between exact-matched and ±2 range-matched results indicates that (a) mutations on PTMs are rare and, only some certain kinds of inherited diseases were indicated to be caused by them, while more kinds of diseases were indicated to be caused by nsSNVs surrounding PTM sites; (b) human inherited diseases are closely associated with disturbances on and surrounding PTM sites.Next, we analyzed the ±2 sites range-matched on cancers; the results did not introduce as many changes as exact-matched results. We also compared the data between ±2 and ±7 range around phosphorylation sites; however, their difference was not significant. The differences of human inherited diseases and cancers could be related with the damages of nsSNVs on PTM sites and phenotype: cancers are mostly caused by somatic mutations and present in the current generation; however, the damages of nsSNVs on PTM sites are not easily inherited to the next generation, so the numbers and types of inherited diseases are less compared with damaged-PTM related cancers.We performed functional enrichment analysis using DAVID. First, we performed keywords and GO association analysis (FDR < 0.01). We still divided data into two parts: exact match and ±2 amino acids (AA) match. “Disease mutation” was the most significant keyword based on the inherited-disease-related nsSNVs that appeared in all the four types of PTMs: Phosphorylation, Ubiquitylation, Acetylation, and Glycosylation. The enrichment analyses showed that the proteins we chose were more likely related to diseases when they encountered mutations. GO enrichment analysis was also performed for the four types of PTMs mentioned above. For each PTM category, the differences of functions among them are obvious (see Table S3). For example, the proteins with phosphorylation mainly involve cell activities like cell death, apoptosis, and signal transduction. Coagulation and wound healing were the GO tags for glycosylation. Through the analyses, we found that the diseases led by the damaged PTMs were closely associated with the role of these proteins played in the regulation of normal cellular processes, which indicated that the damage caused by damaged PTMs was serious.When we moved to cancer-related nsSNVs on PTMs, the keywords about them had less information about mutations, but rather directing to the function of the proteins. What interested us the most was ubiquitylation; the keywords did not show much about themselves, but other modifications on them. This indicates that ubiquitylation is more likely coexisting with other types of PTMs. Then we examined the GO terms on cancers, besides the functions of the proteins performed, also the chemical characters of them showed up. Like phosphorylation, the most significant GO term about phosphorylation was “protein amino acid phosphorylation” on both exact match and ±2 range match. For the remaining types of PTMs, GO terms more revealed protein roles on different processes; for example, “modification-dependent protein catabolic process” ranked in the top two on both range criteria of ubiquitylation.n = 13, corrected P value = 2.66E − 8) and “protein kinase domain”  ranked the first in human inherited diseases and cancers, respectively. The damaged phosphorylation on the kinases could result in damage to another phosphorylation and thus nsSNVs do not affect only one phosphorylation site. Then, in terms of ubiquitylation, “P53 DNA-binding domain”  and “Histone”  were the most significant domains. On P53_HUMAN, lots of phosphorylation and ubiquitylation sites coexisted and some of them affected the same domains, such as “P53 DNA-binding domain.” “Connexin”  and “HMG14 and HMG17”  were the domains damaged acetylation was enriched in. Glycosylation was involved in wound healing, cell-adhesion, and cellular proliferation and we found that “immunoglobulin domain”  and “class I histocompatibility antigen, domains alpha 1 and 2”  were enriched in glycosylation domains. Also for Hydroxylation, “collagen triple helix repeat (20 copies)”  was found in cancer-related dataset. For other types of PTMs, the domains were scattered compared with PTMs mentioned above. From the data of associated domains, we found that the damaged PTMs associated domains were closely related to molecular binding and protein-protein interactions, which was a major function of PTMs [Then we examined the damaged PTMs associated domains based on the data from Pfam to analyze the impact of damaged PTMs on protein structures. For damaged phosphorylation, “protein tyrosine kinase” , Ubiquitylation , and Acetylation ; “hereditary breast cancer signaling” in Phosphorylation , Ubiquitylation , Acetylation , and Methylation ; “Role of BRAC1 in DNA damage response” in Phosphorylation , Ubiquitylation , Acetylation , and Methylation . In these pathways, some are associated with their functions like “Coagulation system”  in glycosylation. As for cancers, we examined each type of PTM category and found that the pathways were more associated with their functions of the proteins, for instance, “protein kinase A signaling”  in Phosphorylation, “protein ubiquitylation pathway”  in Ubiquitylation; we found that more cancer-related damaged PTMs were associated with signaling pathways and this indicated that somatic mutations could affect normal cellular processes more often and may thus result in human cancers.In order to investigate the function of damaged PTMs in proteome-wide scale, we performed pathway analysis by IPA (details available in Table S4). In IPA analysis for inherited-disease associated damaged PTMs of the exact matched data, some pathways are significant: “ovarian cancer signaling” in Phosphorylation (corrected On the proteome-wide range, the associations among these proteins were close, and we illustrated the interactions using networks of protein-protein interactions with STRING . With a Cross talk between some paired PTMs of different types such as phosphorylation and ubiquitylation and ubiquitylation and acetylation, has become a study theme on proteomics , 16. It On the proteome-wide range, the associations were more prevalent. Then we took P53_HUMAN and TOP1_HUMAN as examples for the cross talks between different PTM sites on distinct proteins: on P53_HUMAN, we found 21 phosphorylation sites, 14 ubiquitylation sites, and 9 acetylation sites; among them, the associations were prevalent within the protein, and the damaged PTMs mostly resulted in the deficiency in the role it played in significant cellular functions ; K326R oFor the negative cross talk, where more than one kind of PTMs could happen on the same residue, could be occurred in different stage of cellular processes or on different positions. We chose three pairwise PTMs to perform the analysis: phosphorylation and ubiquitylation, phosphorylation and acetylation, and ubiquitylation and acetylation. For the first and second group, phosphorylation and ubiquitylation, and phosphorylation and acetylation, the exact match sites were not overlapped, but when we used damaged ubiquitylation and acetylation sites to match with ±7 sites around phosphorylational sites, we obtained 12 overlapping sites and 10 overlapping sites, respectively, for ubiquitylation and acetylation, and, among them, 7 and 5 sites were on P53_HUMAN, respectively. For example, K320 on TP53 could be ubiquitylated or acetylated . Then weThe damaged PTMs may cause protein functions to be out of control in canonical pathways . For resIn summary, through this work, we investigated the associations between PTMs affected by nsSNVs and human inherited diseases and cancers from diverse perspectives such as functions, pathways, and cross talks. These provided us a proteome-wide view of how the proteins, which carry modifications and nsSNVs, play roles in the development of diseases and cancers. Not only do PTMs play key roles in almost every important cellular process, but also their dysfunction could result in human diseases. We provided a practical protocol to analyze disease-related proteins that carry damaged PTMs; some valuable proteins were listed out as the candidate biomarkers for potential research and clinical use. However, still almost half of damaged PTMs did not demonstrate associations with human health based on our current analysis, and their functions need to be revealed. Moreover, what we need to do in the future is to identify the causative relationships between the damaged PTMs and human diseases, by discovering key nsSNVs on protein modifications.Supplementary Table S1: The proteins and their sites of exact matched nsSNVs on each type of PTM. The proteins were shown with their UniProt Accession number.Supplementary Table S2: The information of damaged PTM sites associated diseases annotated by SwissVar. For each type of PTM, the information of associated diseases for the exact matched and around matched PTM sites were given. The alterations between the modified amino acids of one type of PTM marked in red. Supplementary Table S3: Enrichment results of keywords and GO. Both exact matched and ±2 matched results were shown. Inherited diseases were marked in yellow and cancers were marked in green.P value and the ratio calculated based on IPA were shown.Supplementary Table S4: Pathway analysis based on IPA. The results were boxed for each type of PTM, and the analysis were performed for both inherited diseases and cancers. Supplementary Table S5: Summary of biomarker candidates. The biomarker candidates were chosen based on information from IPA and details about them were shown.


Their inhibitory mechanisms were studied comparatively. CTLA-2α contains a cysteine residue (C75), which is essential for its inhibitory potency. The CTLA-2α monomer was converted to a disulfide-bonded dimer in vitro and in vivo. The dimer was fully inhibitory, but the monomer, which possessed a free thiol residue, was not. A disulfide-bonded CTLA-2α/cathepsin L complex was isolated, and a cathepsin L subunit with a molecular weight of 24,000 was identified as the interactive enzyme protein. Crammer also contains a cysteine residue (C72). Both dimeric and monomeric forms of crammer were inhibitory. A crammer mutant with Cys72 to alanine (C72A) was fully inhibitory, while the replacement of Gly73 with alanine (G73A) caused a significant loss in inhibitory potency, which suggests a different inhibition mechanism from CTLA-2α. BCPI does not contain cysteine residue. C-terminal region (L77-R80) of BCPI was essential for its inhibitory potency. CTLA-2α was inhibitory in the acidic pH condition but stabilized cathepsin L under neutral pH conditions. The different inhibition mechanisms and functional considerations of these inhibitors are discussed.Mouse cytotoxic T-lymphocyte antigen-2 CT. CTα is t uterus . Drosophila CTLA-2-like protein, crammer, also has a cysteine residue at the homologous sequence position to CTLA-2α. Recently, Tseng et al. reported that monomeric crammer was a strong inhibitor of cathepsin L, while the disulfide-bonded dimer was not . Pr. Prα in thepsins , 33, 34.llularly . The funllularly . Cysteinllularly . Althoug


K-edge X-ray Raman spectra, showed that the molecular dissociation was enhanced up to 14 GPa and gradually suppressed on further compression to 40 GPa. The molecular dissociation was detected for a rather narrow pressure span of 2–40 GPa by the X-ray spectroscopy. The pressure variation of the dissociation yield was similar to that observed in the electric conductivity of ice VII and likely interpreted in terms of proton mobility.We investigated molecular dissociation induced by 10-keV X-ray irradiation in dense ice at pressures up to 40 GPa at 300 K. The dissociation yield estimated from the oxygen  Water ice exhibits various thermodynamically stable and quasi-stable phases at temperatures up to 2000 K and pressures up to 100 GPaDense ice has attracted broad interest in the fields of physics, chemistry, materials science, and earth and planetary sciences. There are some studies reporting the hydrogen bonding, hydrogen sublattice, or hydrogen mobility in dense ice significantly change on compression even though the body-centered arrangement of oxygen atoms remains unchanged. Careful analyses of the compressional equation of state of dense ice252O molecules by irradiation of intense 10-keV X-ray2 and O2 molecules2 molecules produced at 17.6–22.5 GPa persisted stably for two years under the confined pressure or on further compression up to 70 GPa.Another phenomenon suggesting “instability” of the hydrogen bonds in dense ice was observed at pressures up to 15 GPa: Ice VII undergoes photochemical reaction or dissociation of H2O molecules occurred only in ice VII among the dense ices, showing a maximum dissociation yield around 14 GPa. The pressure variation of the dissociation yield will be discussed in connection with that of the electric conductivity having a maximum value at the corresponding pressures.We investigated the electronic structure of oxygen, the molecular vibrations, and the atomistic structure for X-ray-irradiated dense ice by means of X-ray Raman scattering (XRS), ORS, and X-ray diffraction (XRD), respectively, and revealed that the molecular dissociation was suppressed at pressures above 40 GPa. The X-ray-induced dissociation of HR1 fluorescence lineK-edge XRS spectra were measured during irradiation. The incident X-ray energy was scanned from 10.408–10.448 keV, and scattered X-rays with energy of 9.888 keV were detected with an energy loss in the range of 520–560 eV. The incident X-ray energy was calibrated at 9.881 and 11.136 keV with the Ta L2 and L3 absorption edges, respectively, and its beams were aligned to pass diametrically through the sample chamber. The analyzer for the scattered X-rays was calibrated with the elastic line at 9.888 keV, which corresponded to the back-scattering energy of a Si 555 spherically bent crystal (Bragg angle θB = 89.32°). The scattering angle was 30°. The incident beam size of ca. 30~60 (H) × 30 (V) μm2  was small enough for or comparable to the sample thickness for the low-pressure measurements, but slightly larger than that reduced by compression above 39 GPa. Background signals from the anvils were eliminated efficiently with a clean-up horizontal slit. ORS were measured in 21-GPa and 43-GPa experiments to identify dissociation products after the 10-keV X-ray irradiation. The ORS spectra in the low- (<1700 cm−1) and high- (>2500 cm−1) frequency regions were measured using a 488.0-nm argon ion laser at 80 mW and a 632.8-nm helium-neon laser at 32 mW, respectively. Angular dispersive XRD profiles were collected with an image plate detector (RIGAKU R-AXIS IV++) at BL10XU of SPring-8Deionized water  was used for high pressure experiments with a diamond anvil cell (DAC). Water and ruby chips were placed in a sample chamber, 0.2 mm in diameter and <0.1 mm in thickness, made by drilling a beryllium metal gasket, and compressed between the opposed diamond anvils to a target pressure. Pressures inside the sample chamber were monitored via the shift of the ruby 2 molecule, a reaction product of the H2O molecular dissociation82O molecule and the peak from the σ* antibonding orbital of the O2 molecule2 molecule to the XRS main edge of H2O molecule2 molecules via 10-keV X-ray irradiation at 39 GPa.2 molecules in the dissociation product. It has been reported that the β-phase of O2 exhibits a color of pink at 7.5 GPa and 300 K8θ = 7.2° and 12.3° and shoulders at both sides of the 110 peak of ice VII basically in consistent with those reported in the earlier work22 and O2 molecules2 and ice VII here. The bcc lattice constant was 3.073(7) Å for the darken ice, slightly larger than 3.054(7) Å of the transparent ice. The observed lattice constants a, b, c, and β of ε-O2 were a = 7.86(2), b = 5.73(2), c = 3.58(1) Å, and β = 113.0(3)°, whereas those of pure ε-O2 at 21 GPa are 7.683, 5.454, 3.650 Å and 116.3°XRD patterns taken before and after 10-keV irradiation at 21 and 43 GPa are shown in 2 or H2 molecules produced slightly by irradiation even at this condition or that the size of H2O crystallites became smaller by the partial dissociation.The XRD pattern taken at 43 GPa after irradiation is almost identical to that before irradiation, and no additional peaks are observed . The X-r2 molecules in association with the molecular dissociation is confirmed from ORS spectra. −1, and one weak peak around 4250 cm−1are assigned to two O2 librons and an overtone , a O2 vibron2 vibron92 vibron was probably due to diffusion of H2 molecules into the surrounding ice; this is suggested from the peak broadening of an X-ray diffraction pattern observed for the transparent area or the outside area of the X-ray irradiation path (1g + Eg)−1 and either the O2 or H2 vibron peak is not observed in the 43-GPa spectrum; the dissociation reaction of H2O molecules was strongly suppressed.The formation of Oion path . In cont2O molecules from the XRS spectra so as to discuss the nature of the X-ray-induced dissociation. Several assumptions are made with respect to the scattering cross section of the XRS and the reaction formula. Assuming that the initial state comprises only the 1s core electrons of oxygen, the scattering intensity of the oxygen K-edge XRS spectrum between 528 and 548 eV can be expressed asWe attempt to estimate the yield of the dissociated HNoxygen is the number of oxygen atoms involved in scattering events and C0 is a constant depending on the experimental condition. Q and r are the scattering vector and the electron position operator of the oxygen 1s orbital, respectively. The initial and final states of the electronic system are expressed as where I observed at each pressure is only a sum of those of ice and the dissociation product from the following discussion. Let us define deposited energy asWe consider that value Ep is the energy of the photon irradiated on the sample (10 keV), F is the flux . The deposited energy is ~8.2 J after 2 h of X-ray exposure at 7.5 GPa. Assuming this beam is concentrated in a 30 × 30 μm2 area, the fluence would be approximately 9.1 × 105 J/cm2. Since the total deposited energy at each pressure is much more than 8.2 J /(A + B + C) should be proportional to Noxygen_product/(Noxygen_product + Noxygen_ice), where Noxygen_product and Noxygen_ice are the number of oxygen atoms belonging to the dissociation product and the dense ice, respectively.The dominant dissociation product obviously contains OB and C from the observed XRS spectra spectrum, we use the ratio R of the σ* band area to the π* peak area for pure oxygen. The pressure variation of the area ratio is approximated asTo estimate R depends on the distribution of the molecular axis of O2. Here we assume that the distribution was uniform in both of the previousA + B) to the observed area (A + B + C) can be expressed asfrom literature dataUncertainty in the area evaluation from XRS spectra was typically 10%. We consider the estimated yields from the present result are saturated values. The yields estimated from the reported XRS spectra(A + B)/(A + B + C) calculated from the present results and the previous resultsThe ratio of the dissociation product anomalies in the equation of state and the vibrational frequencies45The appearance of the maximum in the dissociation yield would be attributed to “instability” of the hydrogen bond induced in dense ice around 15 GPa, since some If the X-ray induced dissociation was related to hydrogen mobility in dense ice, the dissociation yield could show Arrhenius behavior. Although there is no data at different temperatures, we attempted to fit the X-ray induced dissociation yield to the following equation:Ao is an offset term so that the model function yields a finite value at a zero limit of the dissociation yield and Arri (i = 1 or 2) is given byD0i, βT, E0i, P, and Vi* are the prefactor, thermodynamic beta, activation energy, pressure, and activation volume, respectively. Here we assume a constant value for Vi* over the measured pressure range of 0–40 GPa and describe it in meV/GPa unit. Now, Arr1 and Arr2 can be related to the probability of the two type of hydrogen motion. The first process (Arr1) is hopping between double-well minimums along a hydrogen bond (Arr2) is hopping from a site between one O-O nearest pair to another site between another O-O pair .


DYRK1A is a pleiotropic protein kinase with diverse functions in cellular regulation, including cell cycle control, neuronal differentiation, and synaptic transmission. Enhanced activity and overexpression of DYRK1A have been linked to altered brain development and function in Down syndrome and neurodegenerative diseases such as Alzheimer’s disease. The β-carboline alkaloid harmine is a high affinity inhibitor of DYRK1A but suffers from the drawback of inhibiting monoamine oxidase A (MAO-A) with even higher potency. Here we characterized a series of novel harmine analogs with minimal or absent MAO-A inhibitory activity. We identified several inhibitors with submicromolar potencies for DYRK1A and selectivity for DYRK1A and DYRK1B over the related kinases DYRK2 and HIPK2. An optimized inhibitor, AnnH75, inhibited CLK1, CLK4, and haspin/GSG2 as the only off-targets in a panel of 300 protein kinases. In cellular assays, AnnH75 dose-dependently reduced the phosphorylation of three known DYRK1A substrates  without negative effects on cell viability. AnnH75 inhibited the cotranslational tyrosine autophosphorylation of DYRK1A and threonine phosphorylation of an exogenous substrate protein with similar potency. In conclusion, we have characterized an optimized β-carboline inhibitor as a highly selective chemical probe that complies with desirable properties of drug-like molecules and is suitable to interrogate the function of DYRK1A in biological studies. Protein kinases are pharmacologically tractable proteins that have key roles in most, if not all, cellular signalling pathways. Although protein kinases have become one of the most intensively pursued classes of drug targets, selective inhibitors suitable for functional experiments exist only for a fraction of the human kinome ,2. AlthoDYRK1A gene on chromosome 21, the over-activity of DYRK1A that results from the increased dosage of the DYRK1A gene is thought to contribute to the neurological abnormalities associated with Down syndrome . Ac. Ac44]. To evaluate the usability of the new DYRK1A inhibitors in cell culture experiments, we analyzed the effect of AnnH75 and other harmine analogues on the viability of three different clonal cell lines, HeLa cells and the neuronal cell lines PC12 and SH-SY5Y. Viability assays showed that treatment with up to 10 μM AnnH75 for 3 days caused minimal cytotoxicity in HeLa and PC12 cells , whereasTo evaluate the new harmine analogues for their capacity of inhibiting DYRK1A in cell culture experiments, we compared the effect of AnnH75 and other select compounds on the DYRK1A-catalysed phosphorylation of splicing factor 3b1 (SF3B1). Phosphorylation of SF3B1 on Thr434 closely reflects the cellular activity of endogenous DYRK1A and provides a useful measure for the cellular efficacy of DYRK1A inhibitors . HeLa ce50 values determined in the in vitro-assays  by DYRK1A has been considered potentially relevant in the early Alzheimer’s disease-like neurodegeneration in Down syndrome and in spontaneous Alzheimer’s disease [50 of 1 μM . In conChemical probes serve as invaluable small molecule tools to functionally annotate signalling proteins, to understand their role in physiological and pathological processes and to validate them as potential drug targets. Here we report the evaluation of new harmine analogs with regard to their suitability as chemical probes for the protein kinase DYRK1A. To support the conclusion that AnnH31 and AnnH75 meet the key requirements of a useful tool for this purpose, we discuss our results with respect of the “fitness factors” of chemical probes that were proposed by Workman & Collins : selectiA key criterion for the use of a kinase inhibitor as a chemical probe is that its selectivity profile is well defined ,6. The kRegarding off-targets outside the protein kinase family, we reached the goal of eliminating the MAO-A inhibitory activity of harmine. AnnH31 displays already 40 fold selectivity for DYRK1A over MAO-A and complies with the requirements for a high quality chemical probe . AnnH75 The availability of inactive analogs and active probes from a different chemical class can help to prove target-dependency in a biological experiment ,6. When Several mechanisms can account for discrepancies between inhibitor potency in biochemical and in the cellular context ,48. ManyThe procedures for the synthesis and the structural characterization of the new β-carboline compounds will be fully described in a separate manuscript, and the structure of the AnnH75/DYRK1A cocrystal has been solved (PDB ID 4YU2). The physicochemical properties of AnnH31 and AnnH75 comply with Lipinski’s “rules of 5”, which predict oral bioavailability of drugs ) Table . The effIt is to be emphasized that the appropriateness of a chemical probe depends on the hypothesis under investigation and our knowledge of the biological context . Thus, tStrep-Tactin HRP conjugate  were purchased commercially. The rabbit antibody for detecting phosphorylated Thr434 in SF3B1 has been described [Mouse monoclonal antibodies against phosphotyrosine , SF3B1 , and FLAG-tag , goat antibody against GFP , a rabbit antibody for phosphorylated Thr212 in the tau protein  as well as escribed .in vitro translation reactions and cell assays or 1% DMSO in kinase activity assays if not indicated otherwise.Harmine was obtained from Fluka, Buchs, Switzerland and 5-iodotubercidin from Tocris Bioscience, Minneapolis, MN, USA. A report on the synthesis of the harmine analogues will be presented elsewhere. All inhibitors were dissolved in dimethylsulfoxide (DMSO), except for harmine which was dissolved in ethanol. Further dilutions of all inhibitors were prepared in water to get final concentrations of 3% DMSO in via engineered BamHI and EcoRI sites. Expression in logarithmically growing E. coli culture was performed for 3 h at 37°C (GST-DYRK1A-ΔC and GST-DYRK2), 3 h at 26°C (GST-HIPK2cat), over night at 28°C (GST-CLK1cat) or over night at room temperature (GST-DYRK1Bcat). GST fusion proteins were partially purified by affinity adsorption to glutathione-Sepharose and stored in the elution buffer  at -80°C until use.Bacterial expression plasmids for GST-DYRK1A-ΔC and GST-DYRK2 have been described earlier –53. To gInhibition of monoamine oxidase A (MAO-A) was determined with the MAO-GLO Assay kit from Promega according to the manufacturer’s instructions using 12 μU human recombinant MAO-A and 25 μM MAO-A substrate in a total volume of 20 μL.2, 5 μM ATP) with appropriate peptide substrates . Assays were started with this amount of a kinase preparation that consumed approximately 90% of the ATP in the assay (linear range of the kinase titration curve). The reactions were run at room temperature for 30 min before 10 μL Kinase-GLO reagent was added. After incubation at room temperature for additional 10 min, luminescence was recorded for 1 s.Compounds were screened for inhibitory effects against GST-DYRK1A-ΔC, GST-DYRK1Bcat, GST-DYRK2, GST-CLK1cat or GST-HIPK2cat using the Kinase-GLO Luminescent Kinase Assay from Promega. Assays were performed in a total volume of 10 μL in kinase-buffer  after automatic outlier elimination.Radioactive kinase assays were used to determine ICThe profiling of AnnH75 against a 300-kinase panel was performed by ProQinase  using radiometric protein kinase assays in a duplicate measurement. Gini coefficients were calculated and the Lorenz curve  was plotCytotoxicity was evaluated in HeLa, PC12 , originaE. coli-based in vitro transcription-translation system, was used to express a DYRK1A construct comprising the catalytic domain of DYRK1A fused to an N-terminal Strep-tag II sequence. Reactions were run in a total volume of 10 μL with 10 ng/μL pET-ST2-DYRK1A [The PURExpress In Vitro Protein Synthesis Kit , which is a reconstituted 2-DYRK1A  and 80 n2-DYRK1A  at 37°C HeLa cells were seeded in 6-well plates and transfected (400 ng/well) with expression vectors for GFP-SF3B1NT (containing amino acids 1–492 of human SF3B1 ) or FLAG50 determination was done with the help of the GRAPHPAD PRISM 5.0 program .In all assays, cells were lysed with 100 μL SDS lysis buffer , samples were sonicated and cleared by centrifugation before SDS-PAGE and immunoblotting. Immunoreactivities were detected by enhanced chemiluminescence using HRP-coupled secondary antibodies and quantified using the AIDA Image Analyzer 5.0 program . Relative phosphorylation of SF3B1 and tau was calculated by normalisation to total protein levels. To calculate relative DYRK1A activity in tau assays, the basal pT212 signal in control cells not treated with doxycyclin was subtracted from all values, and the phosphorylation in DYRK1A expressing cells not treated with inhibitors was set to 100%. SEPT4 phosphorylation was determined as the ratio of intensities of the phosphorylated upper band to the lower band. Curve fitting for ICS1 FileTable B. Drug like properties of AnnH31 and AnnH75. Fig A. Inhibition of DYRK1A and MAO-A by selected β-carbolines. Fig B. Predicted binding mode of AnnH75 at DYRK1B (homology model). Fig C. Inhibition of DYRK1A by β-carbolines after prolonged incubation in aqueous solution. Fig D. Chemical structures of DYRK inhibitors. Supplementary Methods. Computational methods applied for docking studies.(PDF)Click here for additional data file.


The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction.729 phosphorylation were detected with Western blot.In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε.The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε SerRho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge. AR)-mediated interaction with the cytoskeletonAR, leading to the hyperpolarisation of the neuronGeneral anaesthesia is a standard procedure for most surgery, used routinely on patients of all ages. The exact mechanism(s) on how anaesthesia is(are) achieved on the cellular level is not known but increasingly evidence shows that the crosstalk between different brain regions are reduced1 and orexin2 (OXR1 and OXR2)Recent data demonstrate that the induction and emergence paths through which anaesthetic-induced unconsciousness arise and dissipate are not identical The aim of this study is to investigate whether OA interferes with neurite retraction induced by the intravenous anaesthetic propofol in cultured rat brain cells and its signalling pathway.The study was approved by the Linköping Ethics Committee for Animal Research, Dnr 113/11. Primary cultures of mixed rat neurons/glial cells were obtained essentially as described by Hansson and Rönnbäcki.e with at least one long cellular protrusion (a neurite) - most often with vesicles in the protrusion, lying on a glial cell layer. The neurite had to be visible for the entire experiment. Time-lapse series were obtained at 1 min intervals, with application of drugs 15 sec before the measurement image was captured. Neurite length was measured manuallyhttp://rsb.info.nih.gov/ij/, 1997–2005). Measurement obtained from a single neurite is defined as (n = 1) and the neurons were obtained from at least 3 different rat litters in each group. The length of the neurite at time (-1) was used as the reference point (100%). After the experiment, the area around the oil drop was marked on some coverslips used for live cell imaging. To identify neuronal cells, they were immunolabelled for β3 tubulin. The cover-slips were fixed for 30 min in 4% paraformaldehyde in phosphate buffered saline (PBS), rinsed, and mouse anti-tubulin-β3 antibody  followed by Alexa-546-conjugated antibody . All antibodies were diluted in 1% bovine serum albumine (BSA)/0.1% saponin/PBS and incubated for 45 min, rinsed and mounted on object glass. Thereafter the cell used in the live-cell imaging was identified with a 63x oil- fluorescence objective, numeric aperture (NA) 1.4  equipped for DIC light microscopy, and thereafter evaluated for fluorescence.The coverslip was rinsed twice in calcium-containing medium (CCM) and mounted in a closed bath imaging chamber placed in a heated stage to reach 37°C. Cells were observed by light microscopy  with a 40x [numeric aperture 1.3] oil immersion objective). Differential interference contrast images of cells were taken, processed and stored as previously describedNeurite length was measured after CCM for 5 min to establish the steady state, followed by propofol (2 µM) administration . Each coin vitro experiments and the cells in time-lapse experiments were chosen based on our judgment that their morphology resembled that of neurons. This is a subjective choice, and we can only confirm that they were β3 -tubulin-positive afterwards.A limitation of our study was that our cultures consisted of mixed neuron-glial cells, and we analysed only superficial cells lying on a glial cell layer. The findings in this study were obtained from 2 culture flasks were washed twice in CCM, then incubated with either CCM, OA (10 nM) or propofol (2 µM) for 10 minutes in CCM at 37°C in a waterbath. The CCM and propofol treated flasks received AE 0.001% (added 1 min before propofol). The effect of PLD was assessed by pretreatment for 1 h with FIPI (100 nM) in cell culture media, followed by washing twice in CCM-FIPI. The experiment was thereafter done as described above, with drugs added to CCM-FIPI. After removal of stimulation medium, ice-cold lysis buffer with phosphatase inhibitors (250 µl) was added and subsequent procedures were carried out at 4°C or on iceg) to remove remaining intact cells and nuclei. Protein concentration was measured by spectrophotometry and samples were diluted with the lysis buffer to equal relative protein concentration in each experiment. 70 µl of lysate was mixed with sample buffer729 antibodies  (1∶1000 in 1% PBS-BSA) for (2 h (rt) or overnight (4°C)). The membrane was washed six times (PBS-Tween 0.05 %) and incubated with peroxidase-linked goat anti-rabbit antibodies . After extensive washing in PBS-Tween, the membrane was incubated with enhanced chemiluminiscence (ECL) Western blotting detection reagents and visualized using a chemiluminiscence sensitive camera.Cells in 25-cmOverall significant differences between conditions were determined by two-way analysis of variance (ANOVA) with repeated measures. Post hoc comparisons were performed using the Bonferroni test for multiple comparisons. A p value of <0.05 was considered statistically significant. The values were expressed as the mean ± standard error of the mean (SEM). All statistical analyses and graphing were carried out using Prism 4.0 software .The anaesthetic propofol (2 µM) caused a time-dependent neurite retraction to 74.4±5.6% of the initial value (n = 10) after 10 min of stimulation . To explOA (10 nM), the regulator of wakefulness, was added 1 min before the anaesthetic propofol to evaluate if OA could interfere with propofol. The propofol-induced retraction was blocked . The signal cascade of OA includes PLD and PKC. Pretreatment of the cells with a PLD inhibitor  prevented the inhibitory effect of OA on the propofol-induced retraction of the neurites, allowing propofol to retract the neurite length to  at 15 min . PropofoIn the signalling cascade of OA, PLD activates PKC, which induce phosphorylation of several proteins. In this experiment, OA (10 nM) added 1 min before propofol, blocked the propofol-induced retraction . The PKC inhibitor staurosporine, 3 nM, prevented this inhibitory effect of OA on the propofol-induced retraction of the neurites after 10 min, and thus the neurite length was 78.3±9.9%, n = 7, p<0.001. Staurosporine on its own had no effect on the neurite length  and did not affect the response of AE/P2 after 10 min of propofol treatment . Activation of PKC by PMA (100 nM) inhibits propofol-induced neurite retraction, where a brief 3 min pretreatment abolished the retraction after 15 min , this PKC is not available for the signal cascade. No effect was seen with PKCεI alone . When the translocation inhibitor peptide was pre-incubated for 45 min before addition of OA/propofol, the cell retracted . Cells treated with the translocation inhibitor and propofol, retracted , similar to propofol-treated cells , RhoA, ROK, an increase in intracellular calcium concentrationThe signal cascade used by propofol, to cause changes in cytoskeletal actin organisation and retraction of the neurites, involve the GABAOA is a neuropeptide that regulates wakefulness, but it has also been implicated in reducing anaesthetic effects2+ and DAG, novel PKCs  activated only by DAG and atypical PKCs , whose activation is not dependent on Ca2+ or DAG. PKCε are abundant in the nervous system and promote neurite outgrowth729, which makes the catalytic site more active. This is followed by translocation of the PKCε to the cell membrane and activation of the actin-binding motif729, and this phosphorylation is markedly reduced when PLD is blocked by FIPI (729 compared with unstimulated cells. This is contrast to previous data, where propofol increased phosphorylation of PKCε Ser729 in dorsal root ganglions729 to translocate to the membrane. We did not see any effect on the propofol-induced retraction when translocation was inhibited by PKCε I (729 phosphorylation (The PKC family consists of 10 different isoforms by FIPI . Propofoy PKCε I . Our datrylation . Propofo729 phosphorylation, where the activation of PKCε Ser729 is crucial, as both propofol and OA changed the phosphorylation in opposite ways. For OA, translocation of activated PKCε to the cell membrane is important, where it may interact with a target structure as a kinase. It has been shown that PKCε reduces the sensitivity of GABAAR to barbiturates, benzodiazepines, neurosteroids and ethanolAR at the cell surface and attenuates GABAAR currentsAR β3 subunit and increase receptor activity, whereas activators of PKC enhance β3 phosphorylation, leading to a decrease in channel activityAR modulation of sensitivity to propofol, and is a possible pathway by which OA could prevent propofol-induced neurite retraction. However, the activated PKCε also expose its actin-binding motif, where it helps stabilising F-actinThe intracellular signalling used by OA to block neurite retraction caused by propofol is not fully understood. Our data suggest a PLD/PKCε SerAR sensitivity to propofol and a decrease in the amount of GABAAR at the cell surface, which would in turn inhibit activation of the RhoA/ROK/MLC pathways. The other possibility is that PKC activation interferes directly with actomyosin-mediated contractility. The dominant pathway is still to be determined.Taken together, at least two options for the mechanism of the OA inhibition of propofol-induced neurite retraction could be considered: One is a signalling pathway by which OA inducing PLD/PKC activation might lead to the reduction of GABA729 and translocation of the enzyme.In conclusion, the results from our study suggest that the RhoA-ROK- signalling pathway has an essential role in the regulation of propofol-induced neurite retraction, most probably by interaction with the actomyosin complex. The results further indicated that PLD-PKCε activation is important for the OA inhibition of propofol-induced neurite retraction, via increased phosphorylation of PKCε Ser


Bile acids (BA) are signaling molecules with pleiotropic paracrine and endocrine functions  is a ligand activated transcription factor responsive to different bile acids and highly expressed in hepatocytes. FXR plays an important role in the regulation of bile acid synthesis, detoxification and secretion. Activation of FXR contributes to liver regeneration following partial hepatectomy  and alleH4 is a lTGR5 Gpbar-1, M-Bar) is a GPCR responsive to various unconjugated and conjugated bile acids with taurine-conjugated secondary bile acids, such as taurolithocholic acid (TLC) and taurodeoxycholic acid (TDC), being the most potent TGR5 ligands , M-Bar i. ExpressAim of our studies is to elucidate the role of TGR5 in bile acid induced liver damage and regeneration. TGR5 knockout and wildtype mice were fed ad libitum with a bile acid containing diet for 7 days. Liver injury was assessed by serum biochemistry and liver histology. Cellular proliferation was made visible using immunohistochemistry of liver sections and an antibody against proliferating cell nuclear antigen (PCNA). Ductular proliferation was assessed and quantified by cytokeratin (CK)-19 immunofluorescence staining and confocal laser scanning microscopy. Cholangiocytes were cultivated from isolated ducts from livers of TGR5 wildtype and knockout mice and cell proliferation was measured by BrdU incorporation after stimulation with bile acids or specific TGR5 agonists. The role of TGR5 downstream signalling pathways was analyzed with different kinase inhibitors. The generation of reactive oxygen species (ROS) was measured using a fluorescent dye and shedding of EGF was determined by an ELISA assay. Western blotting was carried out to confirm the phosphorylation of the EGFR and ERK1/2. Bile acid feeding resulted in more elevated liver function tests in TGR5 knockout mice as compared to their wildtype littermates on the same diet. Livers from TGR5 knockout mice fed with bile acids for 4 days showed larger areas of hepatocytes necrosis as livers from wildtype animals on the same diet. In contrast, livers from bile acid-fed wildtype animals displayed increased PCNA-positive hepatocytes and cholangiocytes, as compared to the livers from the respective knockout animals, indicating a rise in cell proliferation following bile acid feeding in wildtype livers. Since TGR5 is highly expressed in cholangiocytes, we focused on bile acid mediated cholangiocyte proliferation. While the amount of CK-19 positive bile ducts was comparable between TGR5 wildtype and knockout mice on chow diet, a significant increase of bile duct proliferation as measured by CK-19 staining was detected in livers from wildtype mice following 7 days on CA diet as compared to livers from TGR5 knockout mice on the same diet.Incubation of isolated cultivated cholangiocytes from TGR5 wildtype and knockout mice with taurolithocholic acid (TLC 10 and 25 µM) or TGR5 specific agonists led to a significantly increased cholangiocyte proliferation exclusively in wildtype derived cells. Preincubation of wildtype derived cholangiocytes with inhibitors of Src kinases, epidermal growth factor receptor (EGFR) kinase or ERK1/2 kinases significantly reduced TLC and TGR5 agonist mediated cell proliferation as measured by BrDU incorporation. Treatment with inhibitors of ROS formation, such as N-acetylcystein or apocynin also suppressed TLC dependent BrdU incorporation. In contrast, inhibition of adenylate cyclase by SQ22536 or dideoxyadenosine showed no effect on TLC or TGR5 agonist mediated cholangiocyte proliferation. Stimulation of wildtype derived cholangiocytes with TLC and a TGR5 agonist significantly elevated ROS formation. Increased shedding of EGF was also detected after incubation of wildtype cholangiocytes with TLC or the TGR5 agonist. Furthermore, TGR5 activation significantly induced tyrosine phosphorylation of the EGFR at amino acid positions 845 and 1045 and increased ERK1 and ERK2 phosphorylation. Thus, bile acids mediate cholangiocyte proliferation through a TGR5-ROS-Src-EGFR-ERK signaling pathway, which is independent of adenylate cyclase activation.In summary, TGR5 knockout mice show reduced hepatocyte and cholangiocyte proliferation and more pronounced liver injury in response to bile acid feeding. This is in line with a recent publication, demonstrating that liver regeneration is impaired in TGR5 knockout mice following partial hepatectomy .


Saccharomyces cerevisiae. Maf1 is a RNA polymerase III (Pol III) inhibitor that tailors 5S rRNA and tRNA production in response to various environmental cues. Both PKA and Sch9 have been shown to phosphorylate Maf1 in vitro at similar amino acids, suggesting a redundancy in Maf1 regulation. However, here we find that activating PKA by bcy1 deletion cannot replace Sch9 for Maf1 phosphorylation and cytoplasmic retention; instead, such modulation lowers Maf1 protein levels. Consistently, loss of MAF1 or constitutive PKA activity reverses the stress resistance and the extended lifespan of sch9Δ cells. Overexpression of MAF1 partially rescues the extended lifespan of sch9Δ in bcy1Δsch9Δ mutant, suggesting that PKA suppresses sch9Δ longevity at least partly through Maf1 abundance. Constitutive PKA activity also reverses the reduced tRNA synthesis and slow growth of sch9Δ, which, however, is not attributed to Maf1 protein abundance. Therefore, regulation of lifespan and growth can be decoupled. Together, we reveal that lifespan regulation by PKA and Sch9 are mediated by Maf1 through distinct mechanisms.The Protein kinase A (PKA) and Sch9 regulates cell growth as well as lifespan in  Protein kinase A (PKA) and Sch9 belong to the ACG family of Serine/Threonine kinase . These tPKA and Sch9 are also well known to modulate lifespan in various organisms . In yeasDrosophila fat body boosts the development and increases the larvae body size [in vitro kinase assay demonstrate that both phosphorylate Maf1 at similar amino acids [in vivo remain unclear. If so, whether these two kinases regulate Pol III-dependent transcription through redundant function or through distinct mechanisms is unknown. Furthermore, since PKA and Sch9 are critical to lifespan regulation, whether Maf1 can mediate such biological process is an interesting question yet to be answered.Recently, Maf1 has been identified to be a substrate for both PKA and Sch9 kinases. Maf1 is a key regulator of Pol III-dependent transcription conserved from yeast to mammal , 21. It ody size , demonstno acids , 35–37, in vivo. Upon further investigation, we found that Maf1 protein amount is cooperatively controlled by both PKA and Sch9 for lifespan regulation and stress resistance, but not Pol III-dependent transcription and growth. We have therefore identified Maf1 as an important modulator of lifespan and revealed distinct mechanisms of regulation by PKA and Sch9.To address these issues, we have modulated the activity of PKA and Sch9 in yeast cells in order to compare their roles in regulating Maf1 phosphorylation, subcellular localization, protein levels and Pol III-dependent transcription of tRNA. We found that Sch9 but not PKA is majorly responsible for Maf1 phosphorylation and cytoplasmic localization, demonstrating that these two kinases do not function redundantly in vitro at similar amino acids [in vivo to regulate Maf1 and Pol III, hence cell growth. We tested this possibility by asking whether constitutively activating PKA could override the loss of Sch9 for Maf1 phosphorylation. In yeast strain lacking Sch9, Maf1 has reduced phosphorylation [sch9Δ cells. Maf1 phosphorylation can be revealed by a retarded species through western blot [sch9Δ  with those of wild-type (WT), sch9Δ alone or maf1Δ alone. Chronological lifespan is defined by the time of cells surviving in stationary phase. By using colony assay, we calculated the percentage of living cell over 3 weeks. Our results showed that MAF1 deletion significantly decreased the CLS of sch9Δ, but only slightly decreased that of WT . We asked if Maf1 could also contribute to such growth suppression since Maf1 is decreased in protein levels in sch9Δbcy1Δ. If so, loss of Maf1 should mimic PKA hyper-activation and suppresses the slow growth of sch9Δ cells. Interestingly, although MAF1 deletion partially suppressed stress resistance of sch9Δ cells  does not affect its phosphorylation at all [in vitro assay. Care should be taken to interpret the in vitro kinas assay, as many kinases are promiscuous, but become specific in vivo due to restricted subcellular localization or/and defined regulatory subunits. To finally address this issue, we constitutively activate PKA in sch9Δ mutant by deleing BCY1 and ask if PKA can compensate for the loss of Sch9 for Maf1 regulation in vivo. The results demonstrate that this is not the case, as neither the reduced phosphorylation nor the nuclear localization of Maf1 in sch9Δ is reversed by hyper-activated PKA. Together with evidence from other groups, our data demonstrate that PKA and Sch9 do not function redundantly as previously thought in Maf1 regulation.Since both PKA and Sch9 can phosphorylate Maf1 at similar amino acids in vitro , 37, an in vitro . Later, in vitro . Therefoin vitro , suggestin vitro –37, lossn at all . In addin at all , 35. TheDespite the fact that PKA and Sch9 are distinct in Maf1 regulation and probably many other biological processes, they have common targets . For exasch9Δbcy1Δ double mutant where Maf1 protein is reduced, overexpression of Maf1 rescues the stress resistance and lifespan of sch9Δ mutant, albeit partially. On the other hand, this model is not impeccable with respect to growth, because rescued expression of Maf1 in sch9Δbcy1Δ double mutant does not rescue the reduced tRNA synthesis and the slow growth phenotypes of sch9Δ mutant. Growth and longevity are two major traits of biology and it has been noticed over decades to be inversely correlated [The finding that PKA decreases Maf1 protein stability in growth inhibiting condition could have suggested a model to explain the compensation for Sch9 depletion by PKA hyper-activation: loss of Sch9 activates Maf1 through phosphorylation and nuclear enrichment while hyper-activation of PKA antagonizes such activation by down-regulating Maf1 protein amount. However, this model is only partially supported by our data. On the one hand, this model is plausible in that in rrelated . Part ofrrelated , proposerrelated . Our datleu 2-3,112 his3Δ1 trp1-289 ura3-52 GAL+) and sch9D  strains are originally from Dr. Longo lab at University of Southern California. All genetic manipulations are based on these two strains. bcy1Δ ; sch9 Δ bcy1Δ ; maf1Δ ; sch9Δ maf1Δ . To detect Maf1 protein at endogenous levels, MAF1-MYC9 with its native promoter was inserted into low copy centromeric plasmid pRS313. To obtain high levels of Maf1 protein, MAF1-MYC9 with its native promoter was inserted into high copy centromeric plasmid pRS423.Yeast cells were grown in either standard YPD  or synthetic defined (SD) medium with appropriate amino acid dropouts. DBY746 . Crude lysates were cleared by centrifugation and the supernatants were boiled in loading buffer. Protein samples were subjected to SDS-PAGE and transferred to membrane. For Western blotting, membrane was blocked by 1X TBST with 5% of non-fat milk, followed by incubating with mouse monoclonal anti-myc (9E10) antibody at the dilution of 1:5000 for at least 1 hour. Membrane was washed 3 times, 5 min each time by 1X TBST to remove non-specific binding. Membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min in 1X TBST with 5% of non-fat milk. Membrane was washed again by 1X TBST 3 times, 5 min each time before detection by enhanced chemiluminescence (ECL).600 = 0.2) were fixed by 3.7% formaldehyde for 1 hour. Cells were collected and washed with phosphate buffer , re-suspended in potassium buffer containing 1.2M sorbitol, and treated with 50 μg/mL zymolyase to obtain spheroplasts. Spheroplasts were immobilized on glass slide with cold methanol followed by acetone. Glass slides were dried and blotted with 1X PBS buffer containing 5% BSA then incubated with primary antibody  for 1 hour. Surplus primary antibody was washed away by with 0.5% of Triton X-100 for 3 times. Glass slides were then incubated with secondary antibody for 30 min followed by wash with 0.5% of Triton X-100 for 3 times. Yeast cells were covered with cover slides and sealed with cytoseal 60 before imaging.Yeast cells cultured to early log phase  were calculated and plotted.Overnight yeast cultures (3 replicates for each sample) were diluted to OD600 = 0.4) and collected by centrifuge. Total mRNA was extracted by hot phenol method [ACT1) and expressed as the percentage of wild type. Primers for MAF1: Forward, ttacaatgctacccttcagcaa; Rreverse, gagcagggtgattggtttgt. Primers for ACT1: forward, agctccaatgaaccctaaatca; reverse, acgacgtgagtaacaccatcac.Yeast cells were cultured to early log phase  was used for statistical analysis.


Polarstern Expedition PS87/2014, we discovered multiple submarine landslides along Lomonosov Ridge. Removal of younger sediments from steep headwalls has led to exhumation of Miocene sediments close to the seafloor. Here we document the presence of IP25 as a proxy for spring sea-ice cover and alkenone-based summer sea-surface temperatures >4 °C that support a seasonal sea-ice cover with an ice-free summer season being predominant during the late Miocene in the central Arctic Ocean. A comparison of our proxy data with Miocene climate simulations seems to favour either relatively high late Miocene atmospheric CO2 concentrations and/or a weak sensitivity of the model to simulate the magnitude of high-latitude warming in a warmer than modern climate.Although the permanently to seasonally ice-covered Arctic Ocean is a unique and sensitive component in the Earth's climate system, the knowledge of its long-term climate history remains very limited due to the restricted number of pre-Quaternary sedimentary records. During  Despite the importance of Arctic sea-ice in the global climate system, a paucity of pre-Quaternary sedimentary archives limits our understanding of its long-term history. Here, based on ancient sediments revealed by submarine landslides, the authors reconstruct Arctic sea-ice conditions during the Miocene. There is a general consensus that the polar regions—and in particular the Arctic Ocean and surrounding areas—are at present, and were over historic and geologic time scales, subject to rapid and dramatic environmental changes. Owing to complex feedback processes, collectively known as ‘polar amplification', the Arctic is both a contributor to climate change and a region that will be most affected by global warming12452, and to test the reliability of climate models by evaluating their simulations for conditions very different from the modern climate. This type of records giving detailed information about past Arctic sea-ice conditions are still very rare, especially due to the lack of precise proxies for sea-ice reconstructions 1225 is absent under a permanent sea-ice cover limiting light penetration and, in consequence, sea-ice algal growth . The same consequence applies to totally ice-free conditions . In general, the PIP25 values determined in Arctic Ocean surface sediments correlate reasonably well with the modern sea-ice distribution based on satellite data, although regional differences in the correlation patterns seem to be obvious1425 values of most of the surface sediments are between 0.65 and 1 8The ability to (semi-)quantitatively reconstruct paleo-sea-ice distributions has been significantly improved by a biomarker approach based on the determination of a highly branched isoprenoid (HBI) with 25 carbons  to sediment cores most recently recovered during S87 ref. , to recoPolarstern Expedition PS87 was scheduled for August–September 2014 to explore the Lomonosov Ridge area  deposits, some contain prominent unconformities with lower/pre-Quaternary sediments underneath  sea ice in the late Miocene central Arctic Ocean  and low (278 p.p.m.) CO2 levels  indicateevels  spr (ref. The AOGCM-based monthly mean sea-ice concentrations for 450 and 278 p.p.m. CO.5) ref.  are show.5) ref. . These s2 levels have been relatively high or, alternatively, the applied model has an overly weak sensitivity especially in the northern high latitudes, as in the simulation with a relatively low CO2 level of 278 p.p.m. summer SSTs are too low to explain our sedimentary SST proxy indicative of 4–7 °C. , as well as maximum input of terrigenous biomarkers (and ice-rafted debris (IRD))  Arctic Ocean sea-ice cover. Within this debate, the distinction between seasonal and perennial sea ice is critical, because year-round sea ice in the central Arctic implies very different climate feedback mechanisms, that is, Earth's albedo and heat exchange conditions, than an environment with ice-free conditions during summer. As outlined above, our new proxy data and modelling data clearly indicate a late Miocene seasonal sea-ice cover. Similar or even warmer climatic conditions also occurred in the Middle Miocene Arctic Ocean when looking at the alkenone-based summer SSTs of 10–13 °C reconstructed from ACEX sediments . Abundan24et al.et al.Using a ‘coupled sea-ice slab-ocean model including sediment transport by sea ice', Tremblay http://www.iodp.org/expeditions). Based on the new PS87 seismic data, ∼200 m of Plio–Pleistocene, >600 m of Miocene and >300 m of Oligocene–Eocene may be recovered at the proposed drill sitesOur proxy and model reconstruction of late Miocene Arctic climate is certainly a substantial step forward to improve the understanding of the pre-Quaternary Arctic Ocean sea-ice and SST history. However, to decipher the pre-Quaternary climate history of this unique and sensitive but still not well-known region on Earth in more detail, long continuous sedimentary records to be obtained only by scientific drilling are needed. These records are planned to be recovered within a new IODP drilling campaign scheduled for 2018 , expandable CTD (XCTD) and Valeport Sound Velocity Profiler data.−1, which explains minor differences in water depth between PARASOUND and swath-sonar data.Sub-bottom profiling data were acquired using the parametric hull-mounted system ATLAS Hydrographic PARASOUND DS III-P70. Primary operating frequencies were 18.75 and 22.95 kHz with a secondary sediment-penetrating frequency of 4.2 kHz, a beam angle of 4° and a pulse length of 2. The vertical resolution is ∼0.2 m. As a result of the narrow beam angle, reflections from strata dipping by >4° cannot be received by the vessel. This explains why the thin veneer of post-slide sediments covering the slump scar is not resolved as it is above and below the headwall . The heaFor the MCS data acquisition, a 3,000-m-long streamer  and an air gun array of four G-Guns  were used. Processing included sorting, that is, common depth point sorting with 25 m spacing, frequency filtering (20–180 Hz), velocity analysis, multiple suppression and stacking.Vp) and loop-sensor MS at 10 mm intervals, using a standard Multi-Sensor Core Logger . The principle of logging cores is described in more detail in the GEOTEK manual ‘Multi-Sensor Core Logging', which can be downloaded from the web (http://www.geotek.co.uk).Whole-core measurements included non-destructive, continuous determinations of core geometry (diameter), WBD, P-wave velocity , the core catcher samples from the sediment cores were barren or give a Pleistocene age . The onlD. martinheadii is accompanied by low numbers of the dinoflagellate cyst N. labyrinthus. Such co-occurrence has also been observed within the upper part of its stratigraphic range in IODP Hole M2AB. micropapillata complex has not been recorded in PS87/106 samples. This dinoflagellate cyst dominates assemblages in the late Serravallian of IODP/ACEX Hole M2A, decreases significantly across the Tortonian and disappears close to the Tortonian/Messinian boundaryB. micropapillata complex dominates the assamblage until ca. 8.2 Ma after which it only occurrs sporadically until its highest common occurrence at ca. 4.5 Ma26D. martinheadii and N. labyrinthus in combination with the absence of B. micropapillata complex may allow to place the analysed interval of Core PS87/106 into the upper Tortonian to lower Messinian. However, we note that the highest occurrence derived from ODP Site 907 may represent a minimum age for this bioevent in the Central Arctic Ocean, as successive Neogene cooling may led to an earlier disappearance of species in the higher latitudes.In Core PS87/106, the consistent occurrence of D. martinheadii and N. labyrinthus are both very delicate species that bear processes and trabeculae (ribbon-like bars), which tend to crumple easily. All specimens encountered during palynological analyses, however, are well preserved indicating in situ deposition.25 quantification), squalane (2.4 μg per sample) and cholesterol-d6  were added before analytical treatment. Separation of the hydrocarbon and sterol fractions was carried out via open column chromatography ). The latter fraction was silylated with 500 ml BSTFA (bis-trimethylsilyl-trifluoroacet-amide) . IP25 and sterols were analysed by gas chromatography (GC)/mass spectrometry. Component assignment was based on comparison of GC retention times with those of reference compounds and published mass spectra  most recently found in ancient Arctic sediments and characterized by very similar chromatographic and mass spectral properties25  is 0.005 ng μl−1 in SIM (selected ion monitoring) mode. To obtain mass spectra in TIC  the limit is 0.05 ng μl−1. The retention indices for brassicasterol 3), campesterol (as 24-methylcholest-5-en-3β-O-Si(CH3)3) and β-sitosterol (as 24-ethylcholest-5-en-3β-O-Si(CH3)3) were calculated to be 1.018, 1.042 and 1.077 , respectively.Extraction of 5–10 g of freeze-dried sediments was carried out using an accelerated solvent extractor  with dichloromethane:methanol  as the solvent. For quantification internal standards, 7-hexylnonadecane  in relation to the abundant fragment ion m/z 266 of the internal standard (7-HND) was used (SIM mode). The different responses of these ions were balanced by an external calibration (3)3), campesterol (24-methylcholest-5-en-3β-O-Si(CH3)3) and β-sitosterol (24-ethylcholest-5-en-3β-O-Si(CH3)3) were quantified as trimethylsilyl ethers using the molecular ions m/z 470, m/z 472 and m/z 486, respectively, in relation to the molecular ion m/z 464 of cholesterol-D6.For the quantification of IPibration . Brassic25 and the sterols are described elsewhere82829585960More details about the identification and quantification of IPet al.25 and phytoplankton biomarkers in a phytoplankton-IP25 index, the so-called ‘PIP25 index' . Most recently, Smik et al.25 calculation. This modified PIP25 approach is far less dependent on the balance factor c and based on biomarkers from the same group of compounds  with more similar diagenetic sensitivity, certainly an important improvement for paleo-sea-ice reconstructions and comparison of records from different Arctic areas.with 37 alkenones synthesized by haptophyte algae37:3- and C37:2-alkenones were present in all samples, whereas the C37:4-alkenone was not found. For alkenone (C37:2 and C37:3) analysis, extraction of additional 6 g of freeze-dried sediment was carried out using the ASE method under same conditions as decribed above but with dichloromethane as the solvent. The separation of compounds was carried out by open column chromatography using 5 ml n-hexane, followed by 5 ml n-hexane:dichloromethane  and 5 ml dichloromethane for eluation of the alkenones. As internal standard n-C36:0 (10 μg per sample) was added before any analytical treatment. The alkenones were analysed by GC. Individual alkenone (C37:3 and C37:2) identification is based on retention time and the comparison with an external standard (Emiliania huxleyi with known growth temperature) during the analytical sequences. The range of the total analytical error calculated by replicate analyses is <0.4 °C.Our reconstruction of SST is based on long-chain Cstandard . To exclFor calculation of SST, we used the simplified cf., et al.et al.et al.on  ref. , the calon  ref. . For the25 and PIP25) carried out on samples from various PS87 sediment cores, are listed in −1 and μg gSediment−1) are available online at http://dx.doi.org/10.1594/PANGAEA.855509.The results of the biomarker analyses  we have re-analysed Miocene climate simulations3232The preconsolidation stress of a geological sample experienced in the past can be assessed by incrementally loading the specimen in a uniaxial deformation apparatus , where deviations in the settling behaviour can be converted to the thickness of the missing overburden. We used this approach to estimate the thickness of sediment removed at prominent unconformities observed in some of our studied sediment cores .We conducted our experiments using a combined GIESA oedometer—direct shear apparatus in which both uniaxial compression tests and shear tests can be conducted in consecutive stepsin situ stress the samples had experienced before. After 24 h, the normal load is doubled. Tests were run over many days until the desired maximum normal load was reached . The initial water content of each sample was taken from an aliquot before the specimen was mounted into the GIESA oedometer. We measured the wet weight of the aliquot, subjected it to 48 h of gentle drying in an oven (60 °C) and then measured the dry weight, to calculate the void ratio e (where e=volume of voids/volume of solids).For the experiments in this study, we only used the consolidation function of the system. As is done routinely in such testse is plotted against normal stress, the latter on a logarithmic axis  after each loading increment. The graph of each experiment show a smooth function when mic axis . In an uh) of the overburden based on the effective load (h=preconsolidation stress/(bulk density of the sample−density of seawater) × g). The degree of overconsolidation of a given sample is then calculated as the ratio between preconsolidation stress and normal stress at that depth below the seafloor.The stress measurements are then used to the thickness  and mean sedimentation rates of 3.2 cm ky−1 refs , 71, res−1 refs .How to cite this article: Stein, R. et al. Evidence for ice-free summers in the late Miocene central Arctic Ocean. Nat. Commun. 7:11148 doi: 10.1038/ncomms11148 (2016).Supplementary Figures 1-8, Supplementary Tables 1-5, Supplementary Note 1 and Supplementary References.