The successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes that together form partly redundant pathways, depending on the cell type. The biochemical analysis of this process is challenging due to its dynamic and rapid nature. Here, we systematically compared monopolar and bipolar cytokinesis and demonstrated that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-suited system for global biochemical analysis in mammalian cells. Based on this, we established a phosphoproteomic signature of cytokinesis. More than 10,000 phosphorylation sites were systematically monitored; around 800 of those were up-regulated during cytokinesis. Reconstructing the kinase-substrate interaction network revealed 31 potentially active kinases during cytokinesis. The kinase-substrate network connects proteins between cytoskeleton, membrane and cell cycle machinery. We also found consensus motifs of phosphorylation sites that can serve as biochemical markers specific to cytokinesis. Beyond the kinase-substrate network, our reconstructed signaling network suggests that combination of sumoylation and phosphorylation may regulate monopolar cytokinesis specific signaling pathways. Our analysis provides a systematic approach to the comparison of different cytokinesis types to reveal alternative ways and a global overview, in which conserved genes work together and organize chromatin and cytoplasm during cytokinesis. Cytokinesis requires the coordinated action of multiple cellular processes including membrane trafficking, cell adhesion and cell contractility, actin filaments as well as core cell division components such as microtubules and chromosomes3. The signal to cleave the cell at the correct time and in the correct place is provided by the midzone, a structure consisting of overlapping bundles of microtubules that form between the separated chromosomes during anaphase. Dynamic spindle microtubules get reorganized into the midzone regulated by conserved phospho-regulation pathways, notably the CDK1, Aurora B and PLK1 kinase signaling networks6. Communication from the midzone to the actomyosin cortex through Rho signaling spatially and temporally regulates the cleavage furrow formation and ensures that the furrow exactly bisects the segregating chromosomes7. Similarly, signals from the cleavage furrow to the midzone stabilize the midzone microtubules in a manner depending on the Aurora B kinase activity8.Many components of the cell division mechanism are ancient and evolutionarily conserved across eukaryotic cells. In comparison to the nuclear division, cytokinesis uses the most diverse cellular pathways11. Establishing a robust system to analyze redundant biochemical pathways by systematically comparing cytokinesis in different cell types is expected to yield a better understanding of cytokinesis. A major challenge to this goal is the difficulty of synchronizing different mammalian cells in cytokinesis. Synchronization in mitosis, by activating the spindle assembly checkpoint followed by a wash-out of microtubule-damaging drugs, only allows for partial synchronization, since recovery time after spindle damage varies between cells. A previously developed monopolar cytokinesis system, where cells are arrested in monopolar mitosis followed by cytokinesis induction using a CDK inhibitor12 yields an excellent synchronization efficiency. This approach makes a biochemical comparison between cell cycle stages and cell types possible. For example, we were previously able to identify even subtle differences in the binding partners of microtubules and Aurora B kinase during the mitosis-to-cytokinesis transition using this approach6. Numerous laboratories also take the advantage of this drug induced monopolar cytokinesis assay to study various aspects of cytokinesis21. However, pharmacological synchronization methods must be assessed with caution.Incorporation of all these diverse cellular components varies in different cell types. For example, cell adhesion systems have been shown to drive cytokinesis independently from myosin in certain cell typesIn this study, we aimed to systematically evaluate the differences between monopolar and normal cytokinesis using quantitative proteomics to provide insight into both the redundant and conservative pathways during cytokinesis. Our systematic comparison reveals specific signaling networks in monopolar and bipolar cytokinesis, which showed that monopolar cytokinesis specific phosphosites have more sumoylation sites in close proximity than the bipolar specific ones. Therefore, combination of sumoylation and phosphorylation may be part of monopolar cytokinesis specific regulatory mechanisms. Overall, the majority of the phosphoproteome is shared between monopolar and bipolar cytokinesis. Our analysis suggests that monopolar cytokinesis is a competent surrogate for bipolar cytokinesis. By taking the advantage of monopolar cytokinesis, we provide the largest cytokinesis phosphoproteome to our knowledge. We systematically analyzed the cytokinesis phosphoproteome and reconstructed the network of the interactions between kinases and phosphoproteins that revealed 31 different kinases active during cytokinesis.22. To synchronize HeLa cells at cytokinesis, we adopted the traditional approach: After double thymidine block, HeLa cells were arrested at G2/M phase with Nocodazole. Following the wash-off step, cells were released into cytokinesis. The synchronized cells were fixed and cytokinesis synchrony was evaluated using immunofluorescence. We optimized the parameters of the protocol for the best synchronization efficiency  and C-phase was induced by applying the CDK1 inhibitor Purvalanol A for 15–20 minutes  to 1, were calculated using Rate4Sitesis Fig. .Figure 327. SUMO2 modification and monopolar specific phosphorylation cross talks may have a unique role in monopolar cytokinesis. To tackle this, we used a comprehensive sumoylation database that is compiled from multiple proteomic studies28. We tested whether the monopolar specific phosphopeptides are exclusively sumoylated. Strikingly, when phosphorylation sites are centered to 0 and the number of sumoylation sites within +/−14 amino acids of monopolar and bipolar specific phosphorylation sites are visualized, we observed a significant difference between monopolar and bipolar cytokinesis. Monopolar cytokinesis specific phosphorylations have 73 sumoylation sites near the phosphorylation site, whereas bipolar cytokinesis specific phosphorylations have only 26 sumoylation sites . All peptide fractions were analyzed on a high-resolution Orbitrap instrument coupled with online nanoscale reverse phase HPLC. The experimental workflow is shown in Fig. Because monopolar cytokinesis is a good biochemical surrogate for studying phosphorylation events in cytokinesis, we employed the above mentioned synchronization method to globally investigate protein phosphorylation dynamics during cytokinesis. HeLa cells were tightly synchronized in monopolar cytokinesis, interphase and mitosis. After tryptic digestion, the resulting peptides were labeled using stable isotope dimethyl labeling protocol as light, intermediate and heavy, respectively, and were combined in equal peptide amounts. Global phosphopeptide analysis was performed by peptide fractionation using SCX-HPLC, followed by phosphopeptide enrichment using titanium dioxide TiO. All pephttp://www.phosphosite.org/)29 and recent global cell cycle based phosphoproteomic studies35, 175 out of 9,279 unique phosphorylation sites have not been reported yet  and 5,975 phosphopeptides with at least two ratios  versus mitotic clusters  did not reveal distinct pathways Fig. . ProteinWe used the motif finder tool motif-x to identify numerically dominant consensus phosphopeptide sequences for significantly regulated phosphopeptides in cytokinesis selective clusters. We found seven distinct motifs in the cytokinesis selective clusters, where pS-P and pT-P were dominant Fig. , in addiNext, we searched our data set for the known kinase-substrate relationships annotated in PhosphoSitePlus. Only about 10% of the phosphopeptides 146) from the cytokinesis selective clusters, have at least one known upstream kinase (Supplementary Table 6 from th37. We focused on the significant phosphosites located in cytokinesis-selective clusters to further predict the kinases that play a role in cytokinesis. We identified highly confident interactions between 31 kinases and their substrates (NetworKIN score >5). We propose that these kinases are among the main drivers of cytokinesis  as one of the proteins that became highly phosphorylated during cytokinesis. Ki-67 is a proliferation marker that is widely used in basic research and in clinical diagnostics-67 Fig. . Among tWhen we monitored the Ki-67 phosphorylation dynamics in a cell cycle dependent manner, 15 of 36 phosphosites fell into cluster 4 and 5  as the potential kinases regulating the phosphorylation of the protein during cytokinesis Fig. . Five poein Fig. . We notiein Fig. . BecauseWe used quantitative proteomics to analyze how the phosphoproteome changes as the cell progresses into cytokinesis. We identified over 14,000 phosphopeptides and quantified about 10,000 of them in a cell cycle dependent manner. To our knowledge, this is one of the largest datasets that characterizes the phosphorylation events during cytokinesis (C-phase).26). Among them, cytokinesis (C-phase) is the shortest (~30–60 minutes)46 and the least studied cell cycle stage26. The major challenge in cytokinesis is the difficulty of obtaining a homogenous cell population due to the lack of the robust synchronization methods. As a cell enters into cytokinesis, protein phosphorylation may rapidly occur and reach its maximum level followed by a rapid dephosphorylation stage. Tracing such dynamic phosphorylation levels of the sites that peak in cytokinesis in heterogonous cell populations is therefore not feasible. Synchronizing cells in monopolar cytokinesis overcomes the problem12. All the characteristic changes of cytokinesis also take place in monopolar cytokinesis; e.g., bundling of midzone microtubules, accumulation of contractile ring effectors to the furrow-like cortex and initiation of cytokinesis specific protein-protein and protein-microtubule interactions16. However, the pharmacological perturbation in the monopolar cytokinesis system raises some concerns over the physiological relevance of the identified changes. In this work, we systematically investigated to what extent monopolar cytokinesis mimics the biochemistry of cytokinesis. Our systematic comparison revealed that the phosphorylation pattern of monopolar and normal bipolar cytokinesis cells highly overlap and that only a small sub-group of phosphorylation sites (~10%) exhibits differential regulation. This result suggests that monopolar cytokinesis is a well-suited system for global biochemical analysis of cytokinesis.In the last decade, the changes in protein phosphorylation during the different stages of the cell cycle have been extensively monitored in multiple phosphoproteomic analyses  at the early mitotic exit. This results suggest that ordered dephosphorylation events by phosphatases regulate mitotic exit and cytokinesis36. Our data also supports the gradual de-phosphorylation of mitotic phosphorylation sites and the importance of dephosphorylation in the transition between mitosis and cytokinesis. 13% of mitotic phosphorylation sites get dephosphorylated at cytokinesis. Besides the dephosphorylation events, phosphorylation of around 2% of the sites quantified across three cell-cycle stages  are up-regulated only in cytokinesis in both biological replicates. Altogether, these results imply that there is cross talk between phosphorylation and dephosphorylation events, and coordination between kinases and phosphatases therefore drives cytokinesis. Consistent with this notion, cytokinesis related proteins possess both mitosis and cytokinesis specific phosphorylation sites which was also demonstrated in multiple other studies49. The underlying molecular mechanism of interaction between kinases and phosphatases in this complicated circuitry remains to be elucidated.A previous studyin vivo and in vitro51. Our analysis identified multiple possible cytokinesis selective Aurora kinase substrates including the CLASP1, NUMA1, MKI67, PSMD1, ARHGEF7, INCENP and GIT1 proteins. CLASP1 (CLIP-associating protein 1), a microtubule plus-end tracking protein52, has been identified as a cytokinesis selective microtubule binding protein in our previous study6. Phosphorylation of CLASP1 by Aurora kinases promotes microtubule binding activity of CLASP16. Upon its interaction with PRC1 at the onset of anaphase, CLASP1 localizes to the antiparallel microtubules at the central spindle and the PRC1-CLASP1 interaction is required for a stable anti-parallel microtubule organization53. Cytokinesis specific phosphorylation of CLASP1 by Aurora B may regulate this interaction. Our analysis predicts cytokinesis selective phosphorylation of ARHGEF7  and GIT1 (ARF6 GTPase activating protein GIT) by Aurora kinase. PIX and GIT form a complex, which regulates Rac1 activity54. It is plausible that their cytokinesis selective phosphorylation by Aurora plays a role at the spatial and temporal regulation of Rac1 or potentially Rho A during cytokinesis58 , GSKB are most likely active during mitotic exit. Aurora kinase is a master controller of the mitosis-to-cytokinesis transition. Its activity has been previously shown to organize the antiparallel microtubules at the cleavage furrow both 59. The fission yeast ortholog of Casein Kinase1 triggers the signaling module that monitors the mitotic exit checkpoint, and hence controls the timing of cytokinesis60. Malik et al. also observed increased Casein kinase activity during late mitosis, where the phosphorylation profile of proteins bound to the mitotic spindle was analyzed61. Further analysis of potential downstream targets may further shed light on the mechanistic role of the cytokinesis selective kinases.MAPKs (Mitogen-activated protein kinases) and Casein kinases are other notable active kinase family members identified in cytokinesis in comparison to mitosis and interphase. The literature suggests different functional roles of the activity of both kinase family members during cytokinesis. For example, the MAP kinases ERK1/2 are associated with the midbody and ERK/MAPK activation at the midbody is required for abscission in HeLa cells62. Extending this to the cytokinesis stage may have great value in assessing cell cycle progression and anti-mitotic cancer chemotherapy. The most striking candidate protein is the proliferation marker Ki-67, which is present in each cell cycle stage  of dividing cells but absent in resting cells (G0)38. Our analysis identified multiple phosphorylation sites of Ki-67 that peak in the cytokinesis phase of the cell cycle. Strikingly, multiple such sites in the K167/Chmadrin repeat domain of Ki-6763 show a distinct phosphorylation motif that does not match with any known kinase. Dissecting the molecular mechanism of this cytokinesis specific phosphorylation domain will shed new lights into the responsible kinase and the role of Ki-67 during mitotic exit. Furthermore, motif-based analysis suggested that Aurora and TTK (Mps1) kinases might phosphorylate Ki-67 during cytokinesis. The roles of both kinases in chromosomal organization are well established. We speculate that this signaling network regulates relevant chromosomal reorganizations as cytokinesis progresses. A recent study suggested that Ki-67 is localized on the surface of chromosomes, providing a physical and electrostatic barrier to keep mitotic chromosomes apart during mitosis39. Potentially, cytokinesis selective phosphorylation of Ki-67 triggers its dissociation from segregating chromosome peripheries into the nucleolus and temporally controls its unique chromosome segregation function , supplemented with 10% fetal bovine serum (Sigma), 1% penicillin/streptomycin (Sigma) and 1%w/v L-glutamine at 37 °C. For interphase arrest, cells were first treated with 2 mM thymidine , released for 8 hours and then treated again with 2 mM thymidine for 17 hours. For mitosis synchronization, cells were treated with 10 µM STC  for 12 hours. To induce monopolar cytokinesis, cells were subsequently treated with 30 µM Purvalanol A  for 15 minutes and then washed immediately and extensively with PBS.To induce synchronization in bipolar cytokinesis, following double thymidine blocking, the cells were released for 7 hours, then incubated with 0 ng/ml, 10 ng/ml and 25 ng/ml of Nocodazole  for 4 or 5 hours. Next, cells were extensively washed with PBS. Mitotic cells were collected by gentle shaking and incubated for 50 or 60 minutes in the new plate.For immunostaining or Western blotting, the following primary antibodies were used: anti-phospho-Histone H3 (Ser-10) , Histone H3 , α-Tubulin , Ki-67  and RhoA . A Nikon Eclipse 90i Confocal Microscope coupled with the EZ-C1 Imaging Software was used for imaging. For cytokinesis synchronization optimization, at least 10 shots were taken from different spots per slide in 60x magnification and images were quantified using the ImageJ  software.19. On-column dimethyl labeling was performed as described in22. Labeling reagents consisted of 4% (v/v) proper combinations of formaldehyde , 0.6 M of proper sodium cyanoborohydride (NaBH3CN and NaBD3CN) (Sigma) in 50 mM NaH2PO4 (Fisher), and 50 mM Na2HPO4 (Fisher). The labeled peptides were combined in equal ratios based on their average peptide intensities.In-solution digestion was performed as described previously2)  packaged micro columns pre-equilibrated with loading buffer, subsequently washed with 50% ACN/0.1% TFA. The bound peptides were eluted by 10% ammonia into 10% formic acid for LC-MS/MS analysis.Labeled peptide mixes were fractionated using either HyperSep™ SCX Strong Cation Exchange SPE columns or PolySULFOETHYL A 200 × 2.1 mm (PolyLC Inc.) column using an Agilent 1100 HPLC system . Then, pooled fractions in loading buffer  were loaded into Titanium dioxide  35; charge exclusion unassigned, 1, 7, 8, >8; dynamic exclusion 30, 0 s. The isolation window for MS/MS was 2 m/z and fixed first mass was 100 m/z. For MS2 analysis of phosphopeptide enriched samples, the AGC target and NCE were set to 5e4 and 25, respectively. Dynamic exclusion was 20 s and the isolation window for MS/MS was 1,5 m/z. The mass spectrometry data were deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD008451.The peptides were subjected to a reversed phase Nano LC-MS/MS  connected to a Q Exactive quadrupole Orbitrap mass spectrometer . Samples were directly loaded onto an in-house packed 100 μm i.d. × 17 cm C18 column  and separated at 300 nL/min with 75 or 97 minutes linear gradients, increasing from 5% to 40% or 30% acetonitrile in 0.1% formic acid, respectively. Survey spectra were acquired on the Orbitrap with the resolution of 70,000; mass range 350–1500 m/z; automatic gain control (AGC) target 1eAll raw data files were processed and quantified with Proteome Discoverer (PD) . The search was done against the Homo sapiens subset of the Swissprot database version 2014_08  using Mascot . Settings for PD search were as following: Mass tolerance of ±20 ppm for precursor masses, and ±0.05 Da for fragment ions, two missed cleavages were allowed, and cysteine carbamidomethylation was set as a fixed modification. Light, medium and heavy dimethylation of peptide N termini and lysine residues; methionine oxidation; and phosphorylation on serine, threonine and tyrosine were set as variable modifications. The identification of the phosphorylation site was done by the phosphoRS 3.0 node of Proteome Discoverer. A probability threshold of at least 0.75 was used for confident phosphosite localization. The 3plex or duplex dimethyl-based quantitation method was chosen in Proteome Discoverer with a mass precision requirement of 2 ppm. We allowed spectra with a maximum of 1 missing channel to be quantified with the integrated Percolator-based filter using a false discovery rate of 0.01.19. The ratio normalization of the phosphopeptides was based on the regular peptides. First, we centered the median intensity of the regular peptides on zero. Then, we corrected the intensity distribution of the phosphopeptides by the same factor as the regular peptides. To derive an empiric fold change (fc) threshold, we fitted a normal distribution to the fc distribution of the regular peptides. The normal distribution was estimated using the median and the median absolute deviation as unbiased estimators for the mean and standard deviation. At alpha = 5%, the 2.5% and 97.5% quantiles of the fitted distribution were further interpreted as significance thresholds for up- or down-regulated peptides. The derived significance thresholds on a log2-scale were defined as follows: monopolar/bipolar cytokinesis: t = |1.0| (replicate 1), t = |0.96| (replicate 2); Mitosis/Cytokinesis: t = |0.98|; Interphase/Cytokinesis: t = |1.14| and Mitosis/Interphase: t = |0.98|.Statistical analysis was performed on the resulting PD peptide tables as described in19, to derive a p-value based on the binomial, the number of trials was defined as the number of times, a peptide was quantified; the number of successes was defined as the number of times, a peptide passed the significance threshold; the probability of success was adjusted to the chosen alpha of 5%. Derived p-values were adjusted for multiple testing by Benjamini/Hochberg correction64.For the evaluation of the analysis of monopolar versus bipolar cytokinesis with label swaps, we treated the technical replicates as independent trials to assess the significance of the regulation by a binomial probability function. As described previously65. The E value cutoff was set to 0.0001 and a non-redundant protein database was used. Hypothetical and predicted proteins were removed from the results. The MAFFT-multiple alignment software- was used to align homologous proteins66. Evolution rate of each residue was calculated using Rate4Site from the aligned homologous proteins25. Evolution rates were scaled to conservation scores between 9 and 1 for each protein. Proteins with intensities lower than 8 in log10 scale were excluded. We compared medians of monopolar specific, bipolar specific and shared phosphoproteins’ conservation scores of phosphorylation sites using Mann-Whitney U test.We used BLASTp to search for homologous proteins of phosphoproteinshttps://david.ncifcrf.gov/)67 and Enrichr (http://amp.pharm.mssm.edu/Enrichr/)68 were used to reveal the enriched annotations for biological function and cellular component. GO subgroups that were significantly enriched with p-values below 0.05 were reported.The Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 and  was used to generate kinase-substrate networks72. The confidence weighted protein-protein interactions in the iRefWeb database73 were used as the global interactome and the peptides having significant changes in their phosphorylation status were used as the experimental hits. Then, each peptide was mapped to the corresponding protein. These proteins are called ‘terminals’ in Omics Integrator. If multiple peptides were mapped to the same protein, we took the maximum value of the fold changes. Instead of forcing to connect each terminal directly, the software allows adding intermediate hidden proteins that are not present in the phosphoproteomic set.Optimal protein interaction networks were reconstructed by using the Forest module of the Omics Integrator software10. Phosphopeptides with two pairwise ratios (Mitosis/Cytokinesis and Interphase/Cytokinesis) where at least one ratio is significantly different from the rest of its population were selected and clustered based on all possible combinations of phosphorylation changes during transition from one phase to the other. First, the phosphorylation status of a site can either be up-regulated or down-regulated during mitosis as compared to interphase. The same applies to the mitosis-cytokinesis transition. Second, the final abundance of a phosphopeptide in cytokinesis can be higher or lower than in interphase. By combining these two criteria, a total of six clusters were defined for the phosphopeptide abundance profiles.Interphase, mitosis and cytokinesis abundance values of each phosphopeptide were calculated from Mitosis/Cytokinesis and Interphase/Cytokinesis ratios by normalizing interphase to 1 and converting the relative ratios to log74 was used. The significance threshold was set to 0.0005 and the motif occurrence limit to 2. The background was set as “IPI human proteome” provided by the motif-x webserver. Weblogo75 was used to generate sequence logos.For motif analysis, motif-xSupplementary FiguresSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5


In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used.In this study we found that oligopeptidase OpdA of opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that The online version of this article (doi:10.1186/s12866-016-0900-7) contains supplementary material, which is available to authorized users. Salmonella enterica serovar Typhimurium as an enzyme capable of hydrolysing N-acetyl-L-alanyl-L-alanyl-L-alanyl-L-alanine (AcAla4) -L-norleucine (ALLN), 0.01 mM leupeptin, 1 mM PMSF, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 28 μM E-64 and 0.01 mg/ml phosphoramidon according to manufacturer’s protocol (G-Biosciences). In order to study effects of pH on the enzymatic activity, the pH of the test buffer was adjusted with hydrochloric acid.P value less than 0.05 was used to demonstrate statistically significant difference. Data were expressed as means ± SD of three readings from each of two independent experiments.Statistical analysis of the results was carried out using a one way ANOVA. Yersinia, the gene encoding OpdA was disrupted in Y. pseudotuberculosis IP32953  using casein as a substrate (data not shown). However, a very strong activity with Abz-NKPRRPQ-EDDnp substrate confirmed that the enzyme is an oligopeptidase. It was also found that, although EGTA inhibited the activity of the native enzyme , a full inhibition of activity could not be achieved . Zn2+, the proposed metal cofactor of OpdA, increased activity of the enzyme in the assay at low concentrations, but there appeared to be a limit to the concentration of Zn2+ the protein could tolerate, as the stimulation was abrogated at higher concentrations . As with Abz-NKPRRPQ-EDDnp, the hydrolysis of Abz-NKPRRPQ-EDDnp was stimulated at low, but inhibited at high concentrations of Zn2+ , whilst there was complete inhibition at 100 mM of this cation. As observed previously with OpdA from S. typhimurium [Previous reports demonstrated inhibition of t 0.1 mM , 17. Ourhimurium , 17, cobhimurium .E. coli having two active sites [2+ when hydrolysing Z-AALpNA, but slight inhibition when hydrolysing Boc-Val-Pro-Arg-NH-Mec. In addition, inhibition assays from the study showed that the hydrolysis of Z-AALpNA was not affected by antipain or 4-guanidino benzoic acid 4- tert-butylphenyl ester, whereas the hydrolysis of Boc-Val-Pro-Arg-NH-Mec was completely inhibited by these two inhibitors.A previous study suggested a possibility of OpdA from ve sites . In partE. coli OpdA-like protein , in our experiments an addition of Co2+ to partially depleted OpdA of Y. pseudotuberculosis stimulated the hydrolysis of both Abz-NKPRRPQ-EDDnp and Abz-AAL-EDDnp. The discrepancies in the results may be due to different properties of OpdA proteins extracted from different sources. Neither our data with two different substrates, nor more recent studies provide any evidence for the presence of two active centers in OpdA and similar enzymes [Y. pseudotuberculosis OpdA by similarity to E. coli OpdA are showing in Additional file Following these data reported by Jiang et al. , in the  enzymes , 7, 8. P4 by OpdA [Previous studies showed the inhibitory effect of EDTA on the hydrolysis of AcAla by OpdA , 17. In 600SHIFAGGYAAGYYSY614. The flexible loop contains two serine residues that could be important for enzyme activity and may be the site of action by antipain and chymostatin. It is also noteworthy that the loop also contains the important substrate recognition residue Tyr607 [Despite confirming that OpdA is a metallopeptidase, unexpectedly we found inhibitory effects of antipain and chymostatin. This could be a result of disturbances to the flexible loop i.e. 3 and AcAla4 [E. coli bacterial lysate produced the Abz-GF product suggesting that OpdA is the major oligopeptidase in E. coli [OpdA and Dcp are thought to be the major peptidases involved in the hydrolysis of peptides in the cytoplasm since bacterial lysates lacking both of these enzymes have no catalytical activity against AcAlad AcAla4 . Incubat E. coli . Althoug E. coli , one canA role of OpdA protein in pathogenesis remains to be elucidated. One attractive hypothesis is a possibility of this enzyme destroying short positively charged antimicrobial peptides involved in innate host immunity. Remarkably, the substrate used in this study is derived from bradykinin peptide known for its antimicrobial activity . One of opdA gene with that encoding a modified protein (e.g. not inhibited by elevated Zn2+ concentrations) would have any effect on the virulence of Y. pseudotuberculosis.Overall, the current study has demonstrated the effect of various metal ions and inhibitors on the hydrolysis of two fluorogenic substrates by OpdA. The study showed concentration dependent inhibitory effect of zinc on OpdA activity. Further research could be aimed at determining the residues responsible for the zinc inhibition via site directed mutagenesis. Furthermore, it will be interesting to determine if replacement of the wild type copy of the Yersinia pseudotuberculosis oligopeptidase (OpdA) required for bacterial pathogenicity. The enzyme was found to be specific to short oligopeptides, with its peptidase activity dependent on the presence of particular divalent cations. The activity of this enzyme towards positively charged peptides mimicking those produced by host innate immune system may explain the reason for attenuation of the opdA mutant of this bacterium.In this study we report the identification and enzymatic properties of present results of extensive studies on the effects of different cations of the activity of this enzyme.explain unexpected limited effects of chelators on the native enzyme predicted to be a metallopeptidase.describe a novel approach used for ‘depletion’ of the native enzyme of divalent cation(s) by repeated treatment with a chelator and dialysis.2+ on the activity.show unusual and unexpected inhibitory effect of higher concentrations of Zn2+ concentrations.demonstrate that the latter could be observed even after initial activation of the enzyme at lower Znshow that the inhibitory effect is reversible due to unexpected restoration of activity after addition of EGTA.The data generated by this study are novel and important as they:In addition, this is the first study suggesting a role of OpdA enzyme in pathogenicity. The results reported provide valuable contribution to the investigation of bacterial metallopeptidases in general, and that of oligopeptidases in particular, and are important for better understanding of the role of OpdA-like enzymes in pathogenesis of bacterial infections. Furthermore, characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of its peptidase activity.


Crassostrea gigas was investigated with the aim to select individuals with different susceptibility to the infection. Naïve spat transferred to infected areas and juveniles currently being grown at those sites were analyzed using molecular and histology approaches. The survey period distinguishes itself by very warm temperatures reaching up to 3.5°C above the average. The virus was not detected in the virus free area although a spread of the disease could be expected due to high temperatures. Overall mortality, prevalence of infection and viral load was higher in spat confirming the higher susceptibility in early life stages. OsHV-1 and oyster mortality were detected in naïve spat after 15 days of cohabitation with infected animals. Although, infection was associated with mortality in spat, the high seawater temperatures could also be the direct cause of mortality at the warmest site. One stock of juveniles suffered an event of abnormal mortality that was significantly associated with OsHV-1 infection. Those animals were infected with a previously undescribed microvariant whereas the other stocks were infected with OsHV-1 μVar. Cell lesions due to the infection were observed by histology and true infections were corroborated by in situ hybridization. Survivors from the natural outbreak were exposed to OsHV-1 μVar by intramuscular injection and were compared to naïve animals. The survival rate in previously exposed animals was significantly higher than in naïve oysters. Results derived from this study allowed the selection of animals that might possess interesting characteristics for future analysis on OsHV-1 resistance.The occurrence of OsHV-1, a herpes virus causing mass mortality in the Pacific oyster  Crassostrea gigas has become the oyster of choice for cultivation in many regions of the world due to its rapid growth and wide tolerance to environmental conditions. Although, mortality outbreaks of this species have been reported worldwide since the 1950's  produced in Ireland (Site A) was tested by standard PCR to confirm the absence of the virus before being transferred to two infected sites (Site B and C). Primer pairs OHVA and OHVB , placed in the same area where the oysters were sampled, recorded temperature data every hour. Maximum temperature reached per day was also included in the analysis.Shell length and wet weight were measured before dissection. A piece of gill tissue in juveniles was immediately collected after dissection and kept frozen (−20°C) until use. A cross section including the gill, mantle and gonad was fixed in Davidson's solution for 48 h and preserved in Ethanol (70%) before being processed for histology. Depending on the size of the animal and in order to obtain enough DNA material, whole organism excluding the digestive gland or a mix of gill and mantle were collected in spat oysters.Genomic DNA was extracted from gills (juveniles) or mix of tissues (spat) using the DNeasy Blood and Tissue kit (Qiagen) following the manufacturer's instructions. DNA concentration and quality was assessed by spectrophotometer . The number of samples to be analyzed per sampling point was decided attending to the percentage of mortality, 15 animals were processed when normal mortality was observed and 30 in the case of abnormal mortality. A total of 375 juveniles were processed for DNA extraction in five sampling times and 176 spat oysters collected in three sampling times.n = 3). A selection of positive samples (40) was subsequently sequenced to identify the OsHV-1 microvariant. The amplicon obtained with C2-C6 primers  and CAP3 (http://doua.prabi.fr/software/cap3) were used for sequence assembly. Multiple sequence alignment were executed by Muscle . Searches of homology were performed on GenBank database using the Blast algorithm (http://ncbi.nlm.nih.gov/blast/). Sequences were deposited on GenBank with the accession number: KX147758.According to the method of Lynch et al.  standarde Edgar,  and visuhttp://www.eurl-mollusc.eu/SOPs) using the HVDP-F and HVDP-R primers .The quantification of OsHV-1 was carried out by real-time PCR following the Standard Operating Procedure “OsHV-1 detection and quantification by Real Time Polymerase Chain Reaction using OsHV-1 DNA polymerase sequence”  after being anesthetized using Magnesium Chloride  were settled in 4 tanks, 2 for virus injected animals and 2 for controls. Spat were injected in the adductor muscle with 50 μl of OsHV-1 μVar suspension (10in situ hybridization (ISH). Tissues were dehydrated and embedded in paraffin wax. Blocks were cut in 7 μm sections. Standard histology procedures were carried out before staining with haematoxylin and eosin (Sigma) and covered using DPX mounting medium (Sigma). A selection of juveniles confirmed positive by PCR were processed for ISH to detect OsHV-1 in tissues. DNA digoxigenin-labeled probes were synthesized by PCR with OHVC- OHVD primers , varying between 15.8°C and 28.1°C  and Site C (5.8 ± 0.9 cm). OsHV-1 was not detected in naïve oysters from Site A (8.5 ± 0.9 cm) during the survey. OsHV-1 was detected in both infected areas from sampling 2 to sampling 5 Figures . OverallMaximum values of mortality reached 34.6% at Site B and 14.2% at Site C. Although, overall mortality was not significantly different between the two locations, comparison per sampling point showed significantly higher mortality at Site B (28.6%) compared to Site C (6.5%) at sampling point 3. No abnormal mortality was observed at Site A.p = 0.03). However, a higher significance (p = 0.006) was obtained when temperature was included in the analysis. No significant correlation between mortality, temperature and prevalence was found at Site C.Episodes of high temperatures, considered in this study as a period of 14 consecutive days with highest maxima over 16°C, were not associated with mortality in juveniles in any of the infected sites. Prevalence of infection over 20% was significantly associated with abnormal mortalities at Site B  was significantly lower than spat from Site C (1.4 ± 0.3 cm) and spat that remained at Site A (1.5 ± 0.5 cm). Prevalence of infection reached 70% at Site B Figure  and 100%p = 0.01, Site B; p = 0.04, Site C). Episodes of high temperatures were also related to mortalities at Site C (p = 0.04) but not at Site B (p = 0.2).Overall mortality in spat was not significantly different between sites B (64.2%) and C (46.6%). Analysis per sampling point showed that mortality was significantly higher at Site B in samples 3 (49.9%) and 4 (62.3%). At sample 5, mortality increased significantly in spat from Site C. Overall prevalence of infection was significantly higher at Site C (83.3%) compared to Site B (33.3%). However, at sampling point 3 the prevalence of infection was higher at Site B and no significant differences were observed at sampling point 4 and 5. Abnormal mortality in spat was significantly related to the presence of OsHV-1 at both sites  compared to juveniles   and bidirectionally sequenced with specific primers. Chromatograms were carefully analyzed to discard any erroneous reading. All sequences from the same set of positive samples were identical. Multiple alignment grouped sequences in two main contigs obtaining two consensus sequences Table . One conp = 0.02). No significant differences were observed between virus and control exposed animals from Site B (p = 0.05). Comparison between the two stocks revealed that survival was significantly higher in virus injected animals from Site B compared to virus injected animals from Site A (p = 0.03). Prevalence on virus injected animals was observed from day 1 in Site A and from day 2 in Site B reaching a peak of 80 and 100%, respectively.Figure Cellular changes were predominantly observed in gills Figure . Hypertrin situ hybridization procedure was more evident in mantle . The study sites, with different records of oyster mortality and infectivity, were selected on the basis of previous information .Managements of stocks, environment and oyster source could contribute to the spread of the disease. Among environmental parameters, sea water temperature is considered a risk factor EFSA, . In ordeTo evaluate the effect of sea water temperature on disease development, we selected 16°C as a threshold for our analyses and periods of 2 weeks prior to samplings  was funded by the Seventh EU Framework Programme (FP7- PEOPLE 327932) under the Marie Curie Action.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CK19 mRNA, specific to breast epithelial cells. Inhibitors interfere with the PCR amplification process of PCR. Thus, OSNA, based on gene amplification without RNA purification, might be impacted by numerous factors persisting in a sample, and thereby potentially acting as PCR inhibitors. However, neither the characteristics of breast cancers showing inhibitory effects during OSNA, nor any of the possible inhibitors, have as yet been identified. Inhibitory effects detected during OSNA in 72 metastatic lymph nodes and the patients’ clinicopathological features were examined. Left-over OSNA samples were analyzed with mass spectrometry to identify proteins possibly acting as inhibitors. Most tumors showed inhibitory effects, though to varying degrees. Large tumor, young age and high tumor-infiltrating lymphocyte counts were related to stronger inhibitory effects. Proteome analysis revealed elevations in RPB9 protein and EIF2 signaling upregulation in samples showing strong inhibitory effects. Tumors showing strong inhibitory effects had clinically relevant characteristics, including large size and extensive tumor-infiltrating lymphocyte involvement. Identifying inhibitors in OSNA might provide new insights into breast cancer biology as well as advancing the current technology.One-step nucleic acid amplification (OSNA) is an established method for intraoperative diagnosis of breast cancer metastasis in sentinel lymph nodes, based on quantification of  This assay is performed based on quantification of cytokeratin 19 (CK19) mRNA and has been shown to be more diagnostically accurate than classical pathological diagnosis using frozen sections3. Prediction of metastasis in non-sentinel lymph nodes is also potentially feasible with this method7.The one-step nucleic acid amplification (OSNA®) assay  is an established method for intraoperative diagnosis of breast cancer metastasis in axillary sentinel lymph nodes (SLNs)9. Even with the recent preference for less radical surgical invasion for managing breast cancer, OSNA still has a role, e.g. in selecting patients in whom axillary dissection can be omitted, by providing more accurate evaluation of lymph node metastasis10.OSNA has been widely introduced throughout Japan and many other countries, mostly in Europe, and is now being applied to clinical practice. It is also applicable to colon and gastric cancersCK19 mRNA in sentinel lymph nodes. A node will be judged as harboring breast cancer metastasis when amplification of CK19 mRNA is detected, because CK19 expression is specific to breast epithelial cells and theoretically does not exist in normal lymph nodes. RTqPCR generally requires several hours with multiple steps, including mRNA extraction, purification and cDNA synthesis, making intraoperative evaluation time-consuming. To reduce the time required for examination, OSNA employs the LAMP  method. LAMP allows gene amplification without RNA purification and can be completed in a single step by incubating samples at a constant temperature with a mixture of primers and DNA polymerase11. Thereby, gene detection is completed within 30 to 40 minutes.OSNA detects 12 and that inhibition is one of the most common causes of PCR failure13. The mechanism underlying this phenomenon is poorly understood, though multiple steps during the PCR process have been suggested to be involved 12. Generally, researchers overcome inhibitory effects in their experiments by devising specific protocols, e.g. employing several reagents to remove contaminants in order to obtain samples that are as pure as possible. Dilution of the inhibitors below their effective concentration is also a common approach14 and, indeed, PCR is restored when samples are diluted. However, it should be noted that even in highly purified samples, the effects of inhibitors might persist without significantly altering the PCR reaction.It is widely known that the amplification process of PCR can be inhibited or prevented by several substances known as “inhibitors”15 and bile salts, to possibly exist in clinical samples17. However, to date, no inhibitors impacting OSNA analysis have been identified.With the LAMP method, samples can be analyzed without RNA purification and results are obtained very quickly. However, numerous factors, i.e. impurities including proteins and DNA remaining in a sample, might act as PCR inhibitors. Previous reports have suggested inhibitory factors, such as hemoglobinCK19 is generally adequate in most cases. However, if a lymph node has only a small cancer nest, such as micrometastasis, inhibition might directly affect the diagnosis. OSNA categorizes positive results into 2 groups according to copy numbers of CK19 . Details of the diagnostic strategy provided by the manufacturer are shown in Table CK19 copy number is smaller than 5000 and the reaction is inhibited, the result will be displayed as “plus I”  metastasis diagnosed by OSNA to elucidate the characteristics of breast cancers that exert inhibitory effects on PCR during OSNA analysis. We also endeavored to identify the causes of such inhibitory effects.Most samples showed an inhibitory effect during OSNA, though to varying degrees Fig. . The medNext, we divided all patients into two groups based on a cut-off value of 0.41, the median CK19-D/CK19 ratio Fig. . When thAs noted above, left-over OSNA samples from 8 patients, 4 cases each from Groups A and B, were analyzed with mass spectrometry. Among 1228 proteins examined, LIMMA analysis revealed 83 proteins which differed significantly in their expression levels between the two groups. Thirty-eight proteins were highly expressed and 45 showed lower expressions in Group B than in Group A , although the number of patients examined was too small to allow meaningful conclusions to be drawn.Strong inhibitory effects were more common in younger patients. We speculate that differences in serum estradiol levels according to age might be one of the factors impacting these effects, though the mechanism is unknown. We further investigated premenopausal patients in Groups A and B  to determine whether they were in the luteal phase on the day of surgery, since we previously observed a Ki67 labelling index increase in patients who were menstruating21, and would thus unusually express a variety of proteins. We might have detected novel inhibitory factors among the large variety of proteins examined in this study.Also, large tumor burden appears to be associated with an inhibitory effect, as Group B had larger tumors and more lymph node metastases. Tumor size was an independent factor predicting a strong inhibitory effect (p = 0.004). Large tumors may have more heterogeneous tumor cells, as compared to relatively monotonous breast cancersAs to the malignant propensity of the tumor itself, tumors in Group B were higher grade and had higher Ki67 labelling indices, although the differences were not statistically significant. Extensive TIL was also an independent factor predicting a strong inhibitory effect (p = 0.024). We speculate that a tumor with high malignancy might have more turnover of cancer cells and would thereby trigger immune responses against metastatic disease. EIF2 signaling particularly involves the first step of protein synthesis in mRNA translation. Upregulation of this signaling in Group B might simply explain this high cell turnover, not only of cancer cells but also of a variety of immune cells present in lymph nodes.CK19 cDNA during OSNA. Target-specific analyses should be conducted in the future to identify materials causing inhibitory effects. Experiments to test whether removal of RBP9 protein diminish inhibitory effects are eagerly awaited and we plan to use such an approach in future studies.Highly expressed proteins in Group B were related to transcriptional regulation, cell cycle regulation, and metabolism. Substances exerting an inhibitory effect are not necessarily proteins. Furthermore, several substances may exist in an OSNA sample. However, it is very interesting that RPB9, the most highly expressed protein in Group B, is a component of RNA polymerase. It might interfere with DNA polymerase in the process of synthesizing We could not determine whether the possible inhibitors identified herein might act generally in PCR or be specific to the LAMP method. We speculate that the latter is more likely, considering that there are several specific processes comprising the LAMP method, including the extension reaction using double stranded DNA as a template after cDNA has been synthesized and formed a loop structure.CK19). Also, identifying inhibitors might provide new insights, for future research, into breast cancer biology.Our data revealed that a tumor showing a strong inhibitory effect has clinically relevant characteristics, such as large size and extensive TIL involvement. Tumor heterogeneity might also be associated with inhibitory effects. Further investigation with more materials is needed in the future to identify inhibitors impacting OSNA, to conduct more precise quantification of LN metastasis and advance the current technology  who underwent surgery with OSNA at our institution during the January 2013 through December 2015 period. All patients were diagnosed clinically as N0 prior to surgery. Seventy-two patients  were eventually diagnosed as having LN metastasis by OSNA. We enrolled these 72 cases and investigated the inhibitory effects observed during OSNA in detail. Clinicopathological features of the 72 cases are shown in Supplementary Table CK19. Diluted samples (1 in 10) were also simultaneously measured, indicated as CK19-D. Theoretically, the rate of CK19-D/CK19 should be 0.1. The value would exceed 0.1 if even very slight inhibition is exerted by inhibitory factors. In assessing Plus I cases, a cut-off value of 1.0 was automatically used as a default setting by the manufacturer. The CK19-D copy number in such cases is the inverse of that of CK19, meaning that there is an apparent inhibitory reaction during OSNA analysis. In this study, we defined a case with a CK19-D/CK19 value of 0.1 or less as showing “no” inhibitory effect. We also calculated the CK19-D/CK19 ratio for all samples. However, absolute CK19-D/CK19 values could not be calculated in Plus I cases because the CK19-D value is available only as “250 copies or less”, displayed as the result  and indigo carmine (blue dye) into the skin of the breast. OSNA was intraoperatively performed according to the manufacturer’s instructions and the results were assessed, as shown in Table in situ hybridization. As to Ki67, a hot spot was chosen under 200× magnification and cells positive for nuclear Ki67 were then assessed semi-quantitatively.Pathological examinations for primary tumors were carried out at Juntendo University Hospital by two experienced pathologists. Nuclear grade (NG) was judged based on the modified Bloom-Richardson histological grades. Estrogen receptor (ER) status and progesterone receptor (PgR) status were assessed semi-quantitatively and reported as positive when more than 1% of the nuclei of cancer cells showed staining. HER2 was judged to be positive if more than 10% of tumor cells showed strong staining of the entire cell membrane, or HER2/neu gene amplification was confirmed by fluorescence 22. In the current study, “high” TIL was defined as a tumor with more than 30% stromal TIL.To reveal the characteristics of breast cancers exerting an inhibitory effect on PCR, we also assessed TILs in the primary lesion, speculating that stromal reactions including TILs might produce more numerous reactions including immune responses involving SLN. The TIL evaluation was performed under 200× magnification at the edge of the primary tumor and the percentage of stromal TIL was assessed, following the guidelines established by the International TIL Working Group 2014www.qiagen.com/ingenuity).Left-over OSNA samples from 8 patients, 4 samples each showing no and strong inhibitory effects, were analyzed with mass spectrometry. For comprehensive analysis, we employed the iTRAQ®  system. After purification with dialysis, 100 μg of proteins in remnant OSNA samples from 8 patients in total were digested with trypsin and then labelled with an iTRAQ reagents-8plex kit according to the manufacturer’s protocol. The digested peptides of 4 samples showing no inhibitory effect in OSNA analysis were labelled with 113, 114, 115, and 116 iTRAQ reagents, respectively, while the other 4 samples that showed a strong inhibitory effect were labelled with 117, 118, 119, and 121. Samples were then fractionated into 8 fractions using a Pierce High pH Reversed-phase peptide fractionation kit . Each fraction was dried with a vacuum centrifuge concentrator and dissolved in 0.1% TFA. Mass spectrometric analysis was performed with the Triple TOF 5600 operated with Analyst TF 1.7 software (AB Sciex). The Triple TOF 5600 was connected to a Eksigent nano LC system with attached nano cHiPLC trap column chrom XP C18-CL (200 μm × 0.5 mm) and nano cHiPLC column chrom XP C18-CL (75 μm × 15 cm). The solvent system consisted of solution A: 0.1% (v/v) formic acid, and solution B: 80% (v/v) acetonitrile containing 0.1% (v/v) formic acid. The column was eluted with a linear gradient from 0% (v/v) to 40% (v/v) of B in 100 min. The flowrate was 300 nl/min. Mass spectrometric data were analyzed with Protein Pilot 5.0 software (AB Sciex) using the Uniprot database (release 8/30/2016). A confidence cut-off value of 1% FDR  was applied for protein identification. Data were then analyzed employing QIAGEN’s Ingenuity® Pathway Analysis . Associations between clinicopathological parameters and inhibitory effects were evaluated using Fisher’s exact test. For comparisons of mean values, such as those for age and tumor size, the two-sided Welch’s t-test was carried out. A p-value < 0.05 was considered to indicate a statistically significant difference. We applied a logistic regression model to our data and used the backward procedure with the minimum Akaike information criterion (AIC). We conducted a preliminary principal component analysis that allows grouping of all variables. Based on the results obtained, age, tumor size, NG, and TIL were selected as full-model variables for the present analysis. For comparison of protein expressions obtained from mass spectrometry, LIMMA analysis was performed employing R softwareNo datasets were generated or analyzed during the current study.Supplementary figures and tables


Clostridium thermocellum solubilization and bioconversion are investigated.Efficient deconstruction and bioconversion of solids at high mass loadings is necessary to produce industrially relevant titers of biofuels from lignocellulosic biomass. To date, only a few studies have investigated the effect of solids loadings on microorganisms of interest for consolidated bioprocessing. Here, the effects that various switchgrass loadings have on Clostridium thermocellum was grown for 10 days on 10, 25, or 50 g/L switchgrass or Avicel at equivalent glucan loadings. Avicel was completely consumed at all loadings, but total cellulose solubilization decreased from 63 to 37% as switchgrass loadings increased from 10 to 50 g/L. Washed, spent switchgrass could be additionally hydrolyzed and fermented in second-round fermentations suggesting that access to fermentable substrates was not the limiting factor at higher feedstock loadings. Results from fermentations on Avicel or cellobiose using culture medium supplemented with 50% spent fermentation broth demonstrated that compounds present in the supernatants from the 25 or 50 g/L switchgrass loadings were the most inhibitory to continued fermentation.C. thermocellum’s hydrolytic machinery is more vulnerable to inhibition from switchgrass-derived compounds than its fermentative metabolism.Recalcitrance alone cannot fully account for differences in solubilization and end-product formation between switchgrass and Avicel at increased substrate loadings. Experiments aimed at separating metabolic inhibition from inhibition of hydrolysis suggest that  Clostridium (Ruminiclostridium) thermocellum have identified this bacterium as a particularly capable organism for CBP [Efficient plant cell-wall deconstruction and solubilization is a major challenge to overcome when converting lignocellulosic feedstocks to renewable fuels and chemicals. One promising low-cost strategy to produce cellulosic ethanol through bioconversion is consolidated bioprocessing (CBP), which relies on the simultaneous solubilization and fermentation of lignocellulose carbohydrate polymers without additional enzymes . The hyd for CBP , 3. In a for CBP  as well  for CBP , 5.C. thermocellum-mediated conversion to ethanol. However, little has been reported regarding the effect that substrate loading has on C. thermocellum solubilization and biofuel production despite the realization that feedstock loadings in excess of > 100 g/L carbohydrate are considered essential for industrialization and economic viability of cellulosic ethanol [Differences in feedstock type and composition –8, time  ethanol , 13. Fur ethanol , 14, 15.Clostridium phytofermentans, decreased sugar conversion efficiencies were observed as loadings of washed, pretreated corn stover increased [Caldicellulosiruptor bescii has been reported to grow on unpretreated switchgrass at concentrations as high as 200 g/L [High-solid fermentations of real-world biomass are known to produce a variety of challenges to biocatalysts. For example, soluble sugar accumulation , 17, redncreased . The red 200 g/L , 22. Fur 200 g/L .C. thermocellum’s solubilization and conversion capabilities. It further seeks to provide insight into what processes are most vulnerable to inhibition at increased loadings.The recalcitrance barrier is one that all bioconversion strategies face, though the magnitude of this barrier is known to vary widely . SimilarC. thermocellum. The glucan contents were normalized between comparator fermentations based on a reported glucan content of 35% cellulose in the Alamo cultivar [C. thermocellum M1570 ranged from 50 to 60% of the theoretical maximum, which is consistent with previous reports for the strain [Batch fermentations of minimally pretreated switchgrass or Avicel were run in parallel to compare solubilization and biofuel production by cultivar –24. At 3e strain . In the C. thermocellum growth [Near complete glucan utilization was observed in the Avicel fermentations Table . Five-tom growth , 27. OnlC. thermocellum suggested that the basis for inhibition in the switchgrass experiments was not related to end-product inhibition, nutrient availability, or pH limitation  fully account for the differences in end-product titers observed in the initial switchgrass and Avicel fermentations or (ii) explain why ethanol titers plateaued during the first-round fermentations when glucans were still available for solubilization and conversion Fig. . The poted Table . Furthered Table , but theNext, inhibition of hydrolysis was examined using fermentation medium containing 50% spent broth (as above), but using 17.5 g/L Avicel instead of cellobiose. The molar ethanol yields remained relatively consistent across all conditions with one exception Fig. . Once agUnlike the cellobiose fermentations, however, there was significant variation in the glucose equivalents remaining. Total Avicel solubilization was less for cultures containing supernatant from the initial switchgrass fermentations relative to those containing supernatant from the initial Avicel fermentations. The greatest reduction in solubilization was observed in cultures containing supernatant from the initial 50 g/L switchgrass fermentations. Here, ~ 22% less Avicel was hydrolyzed after 10 days of incubation than was observed in the control condition.C. thermocellum has been well established with glucan utilization efficiencies up to 60–70% on multiple potential bioenergy crops, including switchgrass [C. phytofermentans [The plant cell-wall solubilization efficacy of tchgrass , 9, 28. tchgrass , 18 as wrmentans  under hiC. thermocellum’s hydrolytic machinery was still capable of accessing fermentable substrates in the insoluble portion of the residual biomass. Despite the continued fermentation, however, the ethanol titers achieved during second-round fermentations  inhibition of total Avicel solubilization relative to the control. As cultures containing Avicel-derived supernatants would have no lignocellulose-derived inhibitors, the most plausible explanation is that C. thermocellum fermentation products inhibited cellulase activity. Ethanol and other fermentation end-products have been reported to non-competitively inhibit fungal cellulases with concentrations as low as 24 mM (1.09 g/L) ethanol leading to significant reductions in activity [C. thermocellum cellulase activity became inhibited. Multiple ethanol tolerant strains have been reported, which in some cases have improved total solubilization capabilities, showing that this barrier can be overcome for C. thermocellum [Hydrolysis was another process shown to be vulnerable to inhibition. Specifically, at the initial 25 and 50 g/L switchgrass loadings, the solubilized, but unfermented glucose equivalents represent 12 and 11%, respectively, of the initial glucan provided. In those same fermentations, however, the solubilization efficiency decreased by 17 and 26% relative to the 10 g/L loading  and allow the development of new strategies to overcome inhibition.Fungal cellulases have also been reportedly inhibited by mixed xylo-glucan oligomers . In thatcumented , 9, 43, C. thermocellum or other bioconversion strategies for lignocellulosic biofuel production. Efforts to reduce the recalcitrance of minimally pretreated feedstocks are well underway through the use of genetically engineered or natural plant variants that have altered cell wall compositions. For continued improvements to microbial bioconversion, however, the data presented here suggest that inhibition of hydrolysis plays a greater role in reducing biofuel production at higher biomass loadings than does metabolic inhibition. Accordingly, successful efforts to reduce inhibition of hydrolysis may allow significant steps forward in applying CBP with C. thermocellum to convert industrially relevant biomass loadings to fuels and chemicals.Overcoming hurdles related to the recalcitrance barrier, metabolic inhibition and addressing inhibition of hydrolysis are likely required for the industrialization of C. thermocellum M1570 [C. thermocellum was grown on 3.5 g/L Avicel for 48 h. All experiments were run for 10 days at 55 °C with orbital shaking at 100 rpm unless otherwise noted.Lab stocks of um M1570  were useum M1570  with the2, 10% CO2, and 85% N2. At the end of fermentation, cultures were centrifuged at 8000×g for 15 min and the supernatants and pellets analyzed. Fermentation end-products in the supernatant were measured using a Waters Breeze 2 high performance liquid chromatography (HPLC) system  equipped with an Aminex HPX-87H column (Bio-Rad Laboratories) and a refractive index detector as described previously [2SO4 flowing at a rate of 0.6 mL/min. Soluble carbohydrate content was determined via quantitative saccharification assay NREL/TP-510-42618 and HPLC method NREL/TP-510-42623 essentially as described [During first-round fermentations, 1 mL of liquid was removed every 24 h for pH and fermentation end-product analyses. After each sampling, headspace pressure was removed by venting the bottles for 15 s inside an anaerobic chamber filled with an inlet gas of 5% Heviously , 45. Theescribed  using an2 headspace and an equal volume of 2X MTC medium was added (as described above). Second-round fermentations were then performed identically to first-round fermentations with the following exceptions: (i) 6 mL cultures were used instead of 50 mL cultures and (ii) samples for end-product analyses were taken only immediately after inoculation and after 10 days of fermentation.Residual switchgrass samples from equivalent first-round loading concentrations were pooled together. The solids were washed with ultrapure water at a ratio of 1 L for every 2 g of solids and then again dried at 60 °C until a decrease in weight was no longer observed. The dried, spent switchgrass was then autoclaved in water under an N2 headspace. After cooling, 3 mL of spent supernatant plus 1.5 mL of 4X filter-sterilized MTC medium was added to each bottle. All bottles were once again gassed:degassed with N2. Inoculum for the cellobiose containing cultures was grown for 24 h in MTC medium containing cellobiose or for 48 h in medium with Avicel as described above. Fermentations were run for 5 days (cellobiose) or 10 days (Avicel) and end-product and mass-balance analyses were conducted at t = 0 and at the end of fermentation. To avoid substrate losses for the t = 0 measurements, replicate bottles were prepared and sacrificed. The t = 0 analyses of the sacrificed cultures were assumed to be equivalent to those allowed to incubate for the duration of the experiment.Residual supernatants from the first-round fermentations were combined and neutralized to pH = 7.2 using 8 M NaOH. Milli-Q water was once again used to normalize the liquid addition to the supernatants and maintain a consistent dilution across samples. The neutralized supernatants were sterilized via vacuum filtration through at 0.22 µm filter. For the metabolic inhibition studies, 3 mL of sterilized spent supernatant was combined with 3 mL of filter-sterilized fresh 2X MTC medium containing cellobiose. For the hydrolysis inhibition studies, Avicel was first autoclaved in 1.5 mL of water under an Nn = 6).For the second-round Avicel experiments, total solubilization was calculated as a function of the residual dry weight measurements plus detectable glucose and cellobiose as measured by HPLC. Values were measured in reference to a control condition that contained water instead of spent supernatant from the initial fermentations and are expressed as a percent reduction in total solubilization observed. Second-round fermentations for both cellobiose and Avicel conditions were run using biological triplicates with whole experiments duplicated (


G-protein-coupled bile acid receptor, Gpbar1 (TGR5), is a member of G-protein-coupled receptor (GPCR) superfamily. High levels of TGR5 mRNA were detected in several tissues such as small intestine, stomach, liver, lung, especially in placenta and spleen. TGR5 is not only the receptor for bile acids, but also the receptor for multiple selective synthetic agonists such as 6α-ethyl-23(S)-methyl-cholic acid  and a series of 4-benzofuranyloxynicotinamde derivatives to regulate different signaling pathways such as nuclear factor κB (NF-κB), AKT, and extracellular signal-regulated kinases (ERK). TGR5, as a metabolic regulator, is involved in energy homeostasis, bile acid homeostasis, as well as glucose metabolism. More recently, our group and others have extended the functions of TGR5 to more than metabolic regulation, which include inflammatory response, cancer and liver regeneration. These findings highlight TGR5 as a potential drug target for different diseases. This review summarizes the basic information of TGR5 and its new functions. G-protein-coupled receptors (GPCRs) are large family of receptors, playing important roles in multiple pathways  and obesity. Its activation also mediates novel effects on inflammation and cancer in different organs. In this review, we summarize the basic properties of TGR5 including its ligands and basic functions. Specifically, we will discuss the new findings about TGR5 in different signaling pathways and diseases.As a plasma membrane-bound GPCR, the endogenous natural agonists of TGR5 are bile acids. Taurolithocholic acid (TLCA), lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), and cholic acid (CA) can dose-dependently induce cAMP production in human TGR5-transfected CHO cells. The rank order of potency (EC50) is TLCA (0.33 μM) >LCA (0.53 μM) >DCA 1.01 μM) >CDCA (4.43 μM) >CA (7.72 μM) . Zambad et al.  are not only the activators of TGR5 but also the activators of farnesoid X receptor (FXR)  is one of the key downstream effectors for the AKT signaling  stimulated hepatocytes. It is an enhancer element in the promoter region of acute-phase genes  can be activated by 8-pCT-2′-O-Me-cAMP, a cAMP analog, and stimulates phosphoinositide (PI) hydrolysis. As the result of the effect, insulin releases from pancreatic β cells  and Peptide YY (PYY) release  and muscarinic receptor 2 (Chrm2) (Zhou and Hylemon, TGR5, as an important membrane receptor, is activated by bile acids and multiple compounds. The novel roles of TGR5 in different diseases make it become a new drug target. Further investigation of TGR5 will provide novel insights into the complex mechanism of metabolic diseases and cancer.CG wrote the manuscript, WC and YW revised and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Observations and model experiments highlight the importance of ocean heat in forcing ice sheet retreat during the present and geological past, but past ocean temperature data are virtually missing in ice sheet proximal locations. Here we document paleoceanographic conditions and the (in)stability of the Wilkes Land subglacial basin (East Antarctica) during the mid-Miocene (~17–13.4 million years ago) by studying sediment cores from offshore Adélie Coast. Inland retreat of the ice sheet, temperate vegetation, and warm oligotrophic waters characterise the mid-Miocene Climatic Optimum . After the MCO, expansion of a marine-based ice sheet occurs, but remains sensitive to melting upon episodic warm water incursions. Our results suggest that the mid-Miocene latitudinal temperature gradient across the Southern Ocean never resembled that of the present day. We demonstrate that a strong coupling of oceanic climate and Antarctic continental conditions existed and that the East Antarctic subglacial basins were highly sensitive to ocean warming. Ocean heat is important in forcing ice sheet retreat, yet past ocean temperature data from proximal ice sheet locations are sparse. Here, the authors present temperature reconstructions from the Wilkes Land subglacial basin during the mid-Miocene, and show that warm waters sustained ice sheet retreat 17–14.8 Ma. Satellite monitoring demonstrates that the rate of basal melting (warm ocean melting the marine-terminated ice sheet margins from below) has exceeded that of surface melting (due to radiative forcing on the ice sheet surface)2. About 74% of the glaciers covering the Wilkes Land sector of East Antarctica have progressively retreated since 2000 ad due to ingression of warm waters, likely as a consequence of a reduction in sea–ice production and changes in ocean stratification4. East Antarctica marine-based subglacial basins have the potential of ~14 m sea level rise and are vulnerable to marine ice sheet instability5. Cryosphere–-ocean interactions, therefore, play a critical role in the current ice sheet mass imbalance. Observational data7 rarely cover a period of time longer than few decades6, and are therefore insufficient to provide a record of cryosphere (in)stability at adequately varying CO2 concentrations and temperatures as those predicted for the near future. Studying ice sheet instability and ice–ocean interactions during past warm geologic episodes, when atmospheric CO2 was analogous to present day or higher, can shed light on the long-term stability of continental cryosphere for our future.Assessment of the stability and dynamics of the Antarctic ice sheet in a changing climate is fundamental given its role in the climate system including global sea-level change. Observational data and modelling studies suggest consistently that influx of warm waters onto the Antarctic continental shelf invigorates ice retreat11. The Miocene Climatic Optimum  represents one of the warmest intervals since the inception of Antarctic glaciation12, with atmospheric CO2 concentrations as high as 500–600 parts per million by volume (ppmv)17, analogous to those expected for the end of the century given unabated carbon emissions. Global surface-ocean and deep-sea temperatures were ~3–6 °C and 5–6 °C above present-day values, respectively18. The termination of the MCO, widely referred to as mid-Miocene Climatic Transition , was characterised by progressive cooling and an expansion of global ice volume10; it coincided with a CO2 decline to close to pre-industrial values (200–300 ppmv17). To date, the variability in Antarctic ice sheet volume during the Miocene has mostly been inferred from far-field deep-sea oxygen isotope data11 and numerical modelling18, and attributed to the combined forcing of atmospheric greenhouse-gas concentrations and orbital variations. However, far-field sedimentary records leave ambiguity as to how much ice volume change is involved in deep-sea oxygen isotope variability, while numerical modelling have difficulties replicating the warm polar climates as derived from proxy data. The few available ice-proximal records, such as from the Ross Sea Antarctic Geological Drilling project (ANDRILL)21, have provided first evidence for a dynamic ice sheet during the Miocene and suggest extensive melting during peak MCO warmth21. Modelling experiments22 infer substantial ice-mass loss at boundary conditions of atmospheric ~500 CO2 ppmv, astronomical configuration favourable for deglaciation and 2 °C of surface-ocean warming. Ice sheet advances onto the continental shelves, with grounded ice extending into the Ross Sea, are simulated at 280 CO2 ppmv and a cold orbit. Hence, both the available field data and modelling efforts indicate that the mid-Miocene Antarctic ice sheets were highly sensitive to relatively small changes in atmospheric CO2 concentrations22. Such sensitivity can be triggered by changes in the ocean dynamics, which are complex, include several feedbacks, and have not yet been considered in most model simulations23.For the mid-Miocene epoch, geological records show major variations in Antarctic ice sheet volume, global sea level, ocean temperatures, and marine fauna and flora24 , subantarctic (SAF) and subtropical (STF) Fronts25  retrieved sediments from Site U1356  at the continental rise/abyssal plain boundary, at ~4000 m water depth, ~350 km offshore the Adélie Coast along the Wilkes Land margin, East Antarctica27, the Wilkes Land Site U1356, and the Ocean Drilling Program (ODP) Site 1171 south of Tasmania 9 contain mid-Miocene sediments and are ideally situated along a north–south latitudinal gradient 28 records 24 in the context of available Miocene data from the Ross Sea AND-2A record21 and ODP Site 11719. We demonstrate that marine-based ice sheets are absent or extremely reduced compared to present day during the MCO at the Wilkes Land. Surface waters similar to those found today close to the subtropical front bath this area and sustain ice sheet melting. A much greater ocean temperature gradient between the Ross Sea and the Wilkes Land sites exists compared to the present day, suggesting a different oceanographic structure. Continental conditions at the margin sustain growth of temperate vegetation and soil formation. After the MCO, sea ice occurs and ocean temperatures generally cool at the margin. However, episodic reoccurrence of warm waters destabilises the marine-based ice sheet and the continental cryosphere along the margin is substantially reduced compared to the present day even at pre-industrial atmospheric CO2 values. A larger-than-today ocean temperature gradient between the Ross Sea and the Wilkes Land site still exists.Here we document the environmental dynamics characterising both the circum-Antarctic ocean and the Antarctic continent during the early to mid-Miocene based on new palynological , organic geochemical, and sedimentological data from Wilkes Land Site U135624. The analysed sediment cores are well dated29. We herein use novel techniques of constrained optimisation (CONOP)30 to further improve the age model  found in the recovered part of the record  are dominated by comparatively higher concentrations of clay and dispersed gravel. Clast-poor diatom-rich beds locally preserve an interlaminated structure of clay-rich diatom ooze and diatom-rich silty clay. Clast-rich mudstones are interbedded with laminated silty claystones. Laminae are predominately sub-mm in thickness, and have sharp upper and lower contacts, suggesting localised reworking of the turbidite levee deposits by bottom currents32  in the Pacific sector of the Southern Ocean25, a setting consistent with the available tectonic reconstructions and core lithology24 for the site during the Miocene. A common to abundant component of the assemblages is the genus Operculodinium, which today rarely occurs in assemblages south of the Subantarctic Front33 , which occurs in the Ross Sea AND-2A record exclusively in the two warmest pulses of the MCO21, is here present throughout the record  oceanic environments occur. I. pallidum is the only species presently found in Antarctic environments in the vicinity of the polar front, where sea ice occurs, salinity can be seasonally reduced due to ice melting, and seasonality is high33. However, this is a species for which a clear tolerance to high temperatures has been demonstrated35, and its value as sea ice indicator can hence be questioned. Protoperidinioid heterotrophic dinocysts are present, but never abundant except at 15.5 Ma when a single peak (13.5%) of Selenopemphix antarctica  sea ice. Moreover, the absence or low percentages of reworked dinocysts encountered in this interval  and conifers (Podocarpidites spp.), both of which indicate woody subantarctic or sub-alpine vegetation , shrubs of the family Haloragaceae , grasses (Graminidites spp.), Caryophyllaceae (Colobanthus-type), and Sphagnum-moss (Stereisporites sp.) are abundant, particularly after the MCO (Supplementary Data Myricipites (nearest living relative (NLR): Casuarinaceae) as well as regularly occurring tree ferns (NLR: Cyathea) suggest that even after the MCT a cold-temperate woody vegetation with shrubs and trees still existed in sheltered areas of the coastal lowlands. Temperature reconstructions derived from the fossil pollen assemblages suggest mean annual temperatures (MATs) between 5.8 and 13 °C, and summer temperatures >10 °C  of the Wilkes Land sector of East Antarcticaion Fig. . Element41. We are also confident that most of the pollen, as well as any terrestrial organic matter (including soil), must have originated from close to the depositional site, as most pollen grains are well preserved and Antarctica was already isolated from surrounding landmasses at the Miocene time.Comparison of Miocene pollen assemblages with Eocene and Oligocene species from the same Site U1356 reveals a different taxonomic composition in all samples. We therefore exclude the possibility of reworked Palaeogene pollen being re-deposited into these sediments42) suggest temperatures of 11.2–16.7 °C   were fundamentally different from that of today. Deposition occurred in an open-water environment where only occasional erosion or reworking of older marine sediments and transport to the core location took place. Open-marine surface waters were warm temperate, lacked a strong sea ice component, and were relatively low in nutrients compared to today. Specifically, dinocyst assemblages resemble those found today in the Pacific sector of the Southern Ocean at around or north of the Subtropical Fronts, where sea-surface temperatures vary between 8 °C and 17 °C7–14.8 Ma46 during the MCO, i.e., 4° more to the north than today , humid, locally ice-free coastal zone along the Wilkes Land margin covered by woody vegetation dominated by southern beech and Podocarpaceae conifers. Plant communities similar to that reconstructed from the Wilkes Land record have also been derived from Miocene pollen records near McMurdo Sound/Ross Sea49, McMurdo Dry Valleys50, and the Antarctic Peninsula51. However, in contrast to previously published records, the pollen assemblages here contain exceptionally high percentages of Podocarpaceae conifers and a greater diversity of woody taxa, suggesting warmer conditions at the Adélie Coast than at other Antarctic locations, with maximum temperatures around the MCO. Temperature based on branched tetraether lipids, likely reflecting mean annual temperature or mean temperature of growing degree days above freezing52, are 10–12 °C . An additional palaeotemperature proxy based on inorganic chemical weathering indices53 indicates comparable, albeit lower mean annual temperatures of 6–8 °C to its terrestrial margin at peak warmth and ocean conditions warmer than those of today.46, Fig. Selenopemphix antarctica ) at 14–13.8 and 10.8 Ma suggest that nearby glaciers were terminating into the marine system, much in contrast to the MCO for which such evidence is entirely lacking. Few icebergs reach the latitude of Site U1356 today, despite abundant marine-based ice sheets nearby due to deflection by the strong Polar Current. The episodic presence of extensive IRD at Site U1356 after the MCO thus requires the presence of marine-based ice sheets and an ocean circulation different from present. The location of Site U1356 at ~13 Ma was at 60°S, close to the present-day position of the Antarctic Divergence. If the ocean structure were similar to today, high surface water productivity would be expected, and heterotrophic protoperidinioid dinocysts would dominate the assemblages. However, episodic high abundance of gonyaulacoids temperature cysts and N. labyrinthus, which is at present associated to northern oceanic fronts, suggest a different oceanographic structure. A weakened circulation compared to that of present day is indicated allowing episodic southward transport of warm waters at Wilkes Land Site and icebergs to escape northward out of the Polar Current.Our reconstructions for the post-MCO interval show a dynamic ice sheet and a fundamentally different environment compared to that of MCO. Wilkes Land Site U1356  in these sediments is generally low compared to those of the MCO, which is consistent with a colder, more productive ocean and less soil formation due to continental ice cover compared to the MCO. However, the pollen flora remains broadly unchanged, although higher abundance of the southern beech (Nothofagus spp.) and mosses  and sedimentological proxy data indicate erosion of older sediments from the shelf, likely during ice advance and stronger current activity. A hiatus between 13.4 and 11 Ma and seismic profiles suggesting the migration of the channel levee systems depocentres in the region point to ice sheet expansion on the continental shelf46) with existing Miocene reconstructions from the Ross Sea AND-2A record21  south of Tasmania  Circumpolar Deep Waters55 (CDW), if circulation was similar to that of today, or south displacement of warm surface waters. A weak (proto-) Antarctic polar front could possibly explain the movement of warmer waters from the north. The Southern Ocean circulation, the Antarctic Circumpolar Current (ACC), the front developments, and the upwelling of warm CDW depend on the wind patterns and topography, conditions for which there are poor constraints for the Miocene.To further investigate the importance of the Southern Pacific Ocean surface oceanographic structure and temperature patterns in relation to the continental cryosphere dynamics during and after the MCO, we integrated our Wilkes Land record , the latitudinal temperature gradient between both sites appears strongly reduced, with the Wilkes Land site recording exceptionally warm surface seawater. A much-reduced temperature gradient between the two sites could exist if the ACC and its oceanic fronts were weaker compared to today and/or the ACC was displaced southwards.A comparison between the Wilkes Land and the Southern Tasmanian ODP Site 1171 records shows comparable surface seawater temperatures  during the MCO Fig.  that are59, although younger ages have been proposed60. As the modern Southern Ocean circulation was possibly developed during the MCO, oceanic fronts were in place. The ACC and associated fronts are very sensitive to seafloor topography61, and a different topography may have had consequences for latitudinal heat transport. During the MCO the Tasmanian Gateway was narrower than at present . In conclusion, our data infer a fundamentally different oceanographic and environmental setting across the Pacific sector of the Southern Ocean during the MCO compared to the modern day.The exact timing of a modern-strength fully developed ACC is still uncertain, but usually indicated between 41 and <23 Ma28) show mean annual values of 16.5–20 °C  were derived using Constrained Optimisation methodology as presented in Crampton et al.30 for diatom turnover events in the Southern Ocean. We apply the same methodology but use both radiolarian and diatom data. CONOP generates a parsimonious, best-fit composite sequence of biostratigraphic events of radiolarian and diatom derived from 36 cores in the Southern Ocean, to which observed biostratigraphic events in U1356 could be placed. This paper uses the “hybrid range” model, which was shown by Cody et al.66 to be most suitable for sites close to the Antarctic continental margin. We use two versions of this model, a “strict” and “relaxed” version, which prohibits or allows for FAD and LAD contraction to move out of its observed range as a correlation is made between U1356 observations and the composite sequence. Full details on this methodology are discussed in Cody et al.66 and Crampton et al.30. To represent uncertainties, we apply the spread of the ages from all CONOP-placed FAD and LAD data in these two models at every depth where there is an observed biostratigraphic event. This is in contrast to the approach used by Tauxe et al.29, which assigned absolute values for FADs and LADs as were presented in the CONOP Average age range model of Cody et al.67. However, Cody et al.66 showed that the Hybrid age model converged upon a robust age model earlier than the Average age range model and thus is more appropriate to use. The magnetostratigraphy presented in Tauxe et al.29 is revised on the basis of these new CONOP constraints. We also include palynology-based FADs presented in Tauxe et al.29, and note there is now an improved fit between the various chronological datasets.We developed the age model to U1356, using an integrated biostratigraphic and magnetostratigraphic methodology similar to that presented in Tauxe et al.Lycopodium clavatum tablet with a known amount of marker spores  was added for quantification of palynomorph abundances68.Sample processing was performed at Utrecht University, following standard techniques of the Laboratory of Palaeobotany and Palynology. Samples were oven-dried and weighed (~15 g dry weight sediment each), and one 69 and informally and formally described species in the literature70. Of the 31 palynological samples analysed for dinocysts, 11 were either totally or almost barren (yielding only 15–25 dinocysts). These samples are still considered in our dataset; however, because of the low dinocyst yield, careful interpretation is required for these samples. Dinocyst percentages were calculated based on the total in situ dinocysts counted, excluding reworked specimens  dinocysts mostly include Impagidinium spp., Operculodinium spp., Batiacasphaera spp., Nematosphaeropsis labyrinthus, and Spiniferites/Achomosphaera spp. Protoperidinioid cyst percentages were calculated to identify productivity trends, as P dinocysts are likely produced by heterotrophic dinoflagellates71, while G are generally produced by phototrophic dinoflagellates. Reworked dinocysts include Eocene and Oligocene taxa . Reworked dinocyst percentages, calculated based on a sum comprising both in situ and reworked dinocysts, vary between 0% and 34%. In situ dinocyst absolute abundance  and cold 38% HF (Hydrofluoric acid), and sieved over a 10 µm mesh with occasional mild ultrasonic treatment. To avoid any potential processing-related preservation bias, no oxidation was carried out. The processed residue was transferred to microscope slides using glycerine jelly as a mounting medium, and 2–3 slides were analysed per sample at 400× magnification. Slides were used for both marine  and terrestrial (pollen and spores) palynological analyses. Dinocysts were identified based on a taxonomical index73. Percentages of reworked pollen and spores were calculated based on the sum of total pollen and spores. For calculation of in situ palynomorph percentages, reworked pollen and spore counts were excluded from the total sum. Reworked pollen and spores have been identified using visual colour and fluorescence microscopy41. Permian to Palaeogene reworked pollen and spores have a dark, yellowish colour indicating geothermal maturity, and were clearly distinguishable from well-preserved in situ palynomorphs.Of the 31 samples analysed for pollen and spores, 15 were productive, and total counts range between 80 and 210 pollen and spores 33. Prebble et al.32 augmented the information available from the Southern Ocean by increasing the number of samples analysed to 311. We use their data to derive present-day dinocyst assemblages across the Southwest Pacific fronts and produced the data presented in Fig. 32 analysed the relationships between assemblages and sea-surface temperature, water column depth, and productivity by means of ordination techniques and identified seven clusters. One of these clusters (Cluster 7) groups the South Atlantic samples only, north of the STF and is not considered here. Data from the other six clusters are presented in Supplementary Selenopemphix antarctica (SA), other protoperidinioids (oP), Impagidinium spp. (I), Nematosphaeropsis labyrinthus (Nl), Operculodinium spp. (O), and other Gonyaulacoids (oG) and are presented in percent relative to the total assemblage. These six groups are chosen either based on specific ecological preferences or because of their cosmopolitan occurrence33. Importantly, dinocysts in our Miocene record can also be categorised in the same six groups. In this way, we aimed to minimise the uncertainties in the environmental reconstructions potentially caused by extinct Miocene species, although we cannot discard the possibility that some taxa may have partly changed their preferred ecological niche through time35. However, we defer from quantitatively interpreting the changes observed and use the modern dinocyst distribution only as a qualitative tool. The percentages presented in Fig. Present-day ecological preference of dinocysts is based on the analyses of more than 2400 globally distributed surface sediment samples2O3 column chromatography using hexane/DCM (9:1), 100% DCM, and DCM/methanol (1:1) to yield the apolar, ketone, and polar fractions, respectively. The ketone fractions were checked for alkenones, but none of the samples were found to contain them. The polar fractions were analysed for tetraether lipids and were used to calculate the 75 the branched vs. isoprenoid tetraether BIT43, and the relative distribution of branched tetraether lipids used to estimate mean annual temperature45. The TEX86 ratio is based on isoprenoidal–glycerol dialkyl glycerol tetraether (GDGTs)76, which are assumed to derive mainly from Thaumarchaeota, formerly known as Crenarchaeota group 177, which is an abundant group of marine Archaea. The polar fractions were dissolved in a 99:1 hexane/propanol solvent, and were filtered using a 0.45 μm, 4 mm diameter polytetrafluoroethylene filter, before being analysed using a high-performance liquid chromatography/atmospheric pressure positive ion chemical ionisation mass spectrometry (HPLC/APCI-MS) as described by Schouten et al.78. Extractions were performed at Utrecht University, and HPLC/MS analyses were carried out at the NIOZ .Twenty-nine powdered and freeze-dried sediments (~15 g dry weight) were extracted with dichloromethane (DCM)/methanol (9:1) using the Dionex accelerated solvent extraction (ASE) technique. The extracts were separated by Al42 for water depth of 0–200 m. The analytical error is 0.3 °C, while the uncertainty introduced by the calibration error for 42  values based 42 and for the Bayesian calibration of TEX8681. These two calibrations produce similar values compared to each other, but are up to 8.5 °C degrees higher than the 82 due to changes in the GDGT 2/3 ratio. However, similar trends between The uncertainty introduced by the calibration error is in this case ±4 °C. We calculated the SST based on the 43 is used as indicator for continental organic matter input. A large input of organic matter from the continent also carries isoprenoid GDGTs, which can mask the autochthonous TEX86 signal83. We discarded 44.BIT indexConsequently, 10 of the 29 samples measured in the Wilkes Land record were excluded from the 86 can be further biased by an input of GDGTs derived from methane-utilising Archaea85. Furthermore, methane-generating Archaea, which mainly produce GDGT-0 but also minor amounts of GDGTs 1–3, can alter the TEX86 signal. The “Methane Index” can be calculated86, which evaluates the contribution of methanotrophic archaea to the total GDGT pool:TEXValues of 0.3–0.5 of MI mark the boundary between “normal marine sediments” and methane-impacted sediments. Calculations of this MI index for the studied Wilkes Land record vary between 0.05 and 0.23, in the samples for which temperatures are calculated and used, and between 0.03 and 0.06 for the AND-2A record , annual MAT can be estimated in samples where high BIT index values (>0.3). For this, the calibration of Peterse et al.52. For this, samples with high BIT indices were rerun using improved chromatography of brGDGTs89. Temperature were estimated using the calibration of De Jonge et al.45:However, newer methods and calibrations have recently become availableThe uncertainty introduced by the calibration error is ±4.6 °C . Original raw data GDGTs concentrations and chromatograms) are available from the corresponding author upon request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5