Following TORC1 downregulation by rapamycin treatment or nutrient limitation, clearance of G1 cyclins and C-terminal phosphorylation of Sic1 by unknown protein kinases are both required for Sic1 to escape ubiquitin-dependent proteolysis prompted by its flagging via the SCFCdc4 (Skp1/Cul1/F-box protein) ubiquitin ligase complex. Here we show that the stabilizing phosphorylation event within the C-terminus of Sic1 requires stimulation of the mitogen-activated protein kinase, Mpk1, and inhibition of the Cdc55 protein phosphatase 2A (PP2ACdc55) by greatwall kinase-activated endosulfines. Thus, Mpk1 and the greatwall kinase pathway serve TORC1 to coordinate the phosphorylation status of Sic1 and consequently START with nutrient availability.The target of rapamycin complex 1 (TORC1) pathway couples nutrient, energy and hormonal signals with eukaryotic cell growth and division. In yeast, TORC1 coordinates growth with G The target of rapamycin complex 1 (TORC1) pathway couples nutrient availability with cell growth and division by destabilizing the cyclin-dependent kinase (CDK) inhibitor Sic1. Here the authors show that TORC1 downregulation leads to stabilization of Sic1 via phosphorylation by the MAP kinase Mpk1 and inhibition of dephosphorylation via the greatwall kinase pathway. A well-studied example in yeast indicates that the Cln3 G1 cyclin levels, and consequently START-promoting G1 cyclin-dependent protein kinase  activity, are specifically sustained by TORC1-mediated stimulation of translation initiation. The latter is required for ribosomes to bypass a translational repressive upstream open reading frame and reach the start codon of the 5′-untranslated region within the CLN3 messenger RNA (mRNA)71 cyclin expression, TORC1 further couples cell growth with cell cycle progression by antagonizing the expression and/or function of CDK inhibitors (CDKIs) that restrain CDK-mediated G1–S transition1 CDK-dependent multi-site phosphorylation, the F-box protein Cdc4 of the SCFCdc4 ubiquitin ligase complex that flags it for ubiquitin-dependent proteolysis1011121 CDK activation but also by confining the phosphorylation of specific residue(s)  in Sic1  to stimulate anabolic, growth-related processes  in concert with cell cycle transition events11 of the cell cycle and enter a quiescent state that is characterized by a distinct array of physiological, biochemical and morphological traits141 arrest) when released from inhibition by the AGC family kinase, Sch9, which requires, analogously to mammalian S6 kinase (S6K), activation by TORC1 2021Cdc55) mediates the activation of a quiescence-specific gene expression programme in part via the transcriptional activator Gis1 and likely additional factors that protect specific mRNAs from degradation via the 5′–3′ mRNA decay pathway2223241 via Igo1/2-PP2ACdc55 is currently not known. Interestingly, in this context, Xenopus, Drosophila and likely human cells employ their respective greatwall kinase pathway (Gwl-endosulfine-B55) to maintain high-level phosphorylation of cyclin B-CDK1 substrates, thereby promoting mitotic entry2628Attenuation of signalling through TORC1  incites yeast cells to arrest in GRC1 refs , 18, 19.Cdc55 and prevent it from dephosphorylating pThr173 within the CDKI Sic1. This specific phosphorylation event depends on the mitogen-activated protein kinase (MAPK) Mpk1 and ensures protection of Sic1 from SCFCdc4-mediated ubiquitination and subsequent proteolysis to enable it to grant proper G1 arrest when TORC1 is downregulated. Thus, TORC1 coordinates the phosphorylation status of Sic1 and consequently G1–S cell cycle progression with nutrient availability via Mpk1 and the greatwall kinase pathway.Here we show that TORC1 inhibition and consequently activation of Igo1/2 by the Gwl Rim15 serves to antagonize PP2A1 cell cycle arrest via activation of endosulfines and consequently inhibition of PP2ACdc55, we treated wild-type (WT) BY4741 cells with rapamycin and examined the cells by standard fluorescence-activated cell sorting (FACS) analyses. Unexpectedly, we found that BY4741 WT cells, like the ones from other commonly used WT strains such as W303-1A and SP1  is in part responsible for their expedited rapamycin-induced G1 arrest (1 cyclin gene expression and has been assigned a specific role in preventing premature entry of cells into an off-cycle stationary phase (at G1) in response to nutrient limitation1 arrest in JK9-3D cells to address our question whether Rim15 mediates G1 cell cycle arrest via activation of endosulfines. Accordingly, we found that a large fraction of rim15Δ and igo1/2Δ cells was significantly impaired in proper G1 arrest following rapamycin treatment when compared with their isogenic JK9-3D WT cells  or of the CDKI Sic1 was altered in rapamycin-treated rim15Δ or igo1/2Δ mutant cells. In agreement with previous reports6CLN1–3 transcripts and their corresponding proteins were progressively depleted in rapamycin-treated WT cells  that is not directly related to G1 cyclin expression control.Since our results suggested a role for Rim15/Igo1/2 in cell cycle control, we next examined whether the expression of GWT cells . In paraWT cells . In addiA levels , severelA levels . This la mutants , may in Cdc55. Supporting this notion, we also found that loss of the regulatory Cdc55 subunit of the heterotrimeric PP2ACdc55 complex rescued the Sic1 stabilization defect in rapamycin-treated rim15Δ and igo1/2Δ cells  were detectable in WT cell extracts. These were either absent (isoform 6) or reduced in intensity (isoform 5) in rapamycin-treated and in nitrogen-starved rim15Δ and igo1/2Δ cells, which likely explains the relative increase in the intensity of the faster migrating isoforms in the extracts of the respective strains  at levels comparably (or even higher) to the ones in WT cells under the same conditions. Of note, in exponentially growing, rapamycin-treated and nitrogen-starved cells, and independently of the presence or absence of Rim15 or Igo1/2, Sic1-myc13 preferentially migrated as isoforms 4, 5 and 6 when Cdc55 was absent. Together, these results indicate that PP2ACdc55 targets at least two Sic1 phosphoresidues (to various degrees), and that activation of Rim15/Igo1/2 following rapamycin treatment or nitrogen starvation restrains the respective PP2ACdc55 activity. To examine whether one of the respective residues corresponded to phosphorylated Thr173 (pThr173), which is critical for Sic1 stability in rapamycin-treated cellsT173A mutant allele via phosphate affinity gel electrophoresis in extracts of rapamycin-treated or nitrogen-starved cells. The Sic1T173A-myc13 migration pattern specifically lacked isoform 6 and was overall very similar to the one observed for Sic1-myc13 in extracts of rapamycin-treated or nitrogen-starved rim15Δ and igo1/2Δ mutant cells  bands in rapamycin-treated cells; isoforms 1–3; 173 to Ala in Sic1 destabilized Sic1 and compromised proper G1 arrest in rapamycin-treated cells, and both of these defects were marginally enhanced by combined mutation of Thr173 and Ser191 to Ala in Sic1 . To further verify this assumption, we decided to focus our subsequent analyses on Thr173 in Sic1. Using phospho-specific antibodies against pThr173 in Sic1 (see below), we found that the Sic1-pThr173 signal strongly increased in rapamycin-treated WT cells, but not in Cdc55 overproducing nor in rim15Δ, or igo1/2Δ cells, unless the latter two mutant strains were additionally deleted for CDC55 (in vivo specificity of the anti-Sic1-pThr173 antibodies), mutation of Thr173 to Ala in Sic1 totally abolished the Sic1-pThr173 signal, and eliminated the Sic1-myc13 isoform 6 on phos-tag gels, even when Cdc55 was absent  in Sic1, which presumably exposes Sic1 to a proteolytic degradation mechanism.To begin to study how many residues in Sic1, if any, are targeted by PP2A strains . Loss ofnt cells , indicatic1 ref.  was requ in Sic1 . The Sic in vivo . The staor CDC55 . In conts absent . Notablys tested . Togethe173 in Sic1 may serve to protect Sic1 from SCFCdc4-mediated ubiquitination and subsequent proteolysis, we introduced the temperature-sensitive cdc4-2ts allele in our WT, rim15Δ, igo1/2Δ and sic1T173A strains, and measured their capacity to accumulate Sic1 during exponential growth or following rapamycin treatment at the permissive (24 °C) and the non-permissive temperature (37 °C). At 24 °C, the cdc4-2ts allele did not noticeably alter the Sic1 expression pattern in any of the strains studied, whether they were grown exponentially or subjected to rapamycin treatment , however, prompted Sic1 accumulation to a similarly strong extent in all strains, independently of the presence or absence of rapamycin, and could thus override the defect in Sic1 accumulation, but not in Sic1-Thr173 phosphorylation  in Sic1, thereby preventing SCFCdc4-mediated ubiquitination and subsequent proteolysis of Sic1. To address the possibility that Sic1-Thr173 phosphorylation plays an additional role in nutrient-regulated nucleo-cytoplasmic distribution of Sic1 . In this context, the MAPK Hog1 has previously been proposed to mediate Sic1-Thr173 phosphorylation following exposure of cells to osmotic stress173 phosphorylation, loss of Mpk1, but not of Hog1, significantly reduced the Sic1-pThr173 signal and rendered Sic1 unstable in rapamycin-treated cells  in extracts of rapamycin-treated WT, mpk1Δ and hog1Δ cells indicated that Mpk1, but not Hog1, might (directly or indirectly) target one major residue in Sic1 . In addition, rapamycin treatment not only stimulated the activity of Mpk1 towards Thr173 in Sic1 10.4-fold , nor in the presence of unrestricted PP2ACdc55 (in rim15Δ or igo1/2Δ cells), we expected the combination of mpk1Δ with either rim15Δ or igo1/2Δ to cause an additive G1 arrest defect in rapamycin-treated cells. This was indeed the case  its association with SCFCdc4. Of note, Cln-CDK downregulation following TORC1 inhibition, which transiently relies on Rim15 and Igo1/2  medium with 2% glucose and supplemented with the appropriate amino acids for maintenance of plasmids. Before the experiments, cells were diluted to an OD600 of 0.001 in SD and grown until they reached an OD600 of 0.4. Rapamycin was dissolved in 10% Tween-20/90% ethanol and used at a final concentration of 200 ng ml−1. Strains and plasmids used in this study are listed in 3 and Cdc55-HA3 that were expressed from plasmids (under the control of their own promoter) to be used for the in vitro protein kinase and phosphatase assays.2O, centrifuged, resuspended in 250 μl of RNAse solution (50 mM Tris (pH 7.4), 200 μg ml−1 RNAse A (Axonlab AG)) and incubated for 3 h at 37 °C. Subsequently, cells were centrifuged again, resuspended in 250 μl of propidium idodide solution (50 mM Na+-citrate (pH 7.0) and 10 μg ml−1 propidium idodide (Sigma)) and analysed in a CyFlow (PARTEC) flow cytometer. Data were processed using the FlowJo software.A measure of 1.5 ml samples were collected at the indicated time points after rapamycin treatment, centrifuged and resuspended in 1 ml 70% ethanol. Following overnight incubation at 4 °C, cells were washed once with H173 (produced by GenScript), anti-GFP (Roche), phospho-specific anti-Igo1-pSer64 . Lysates were prepared by disruption of frozen cells in lysis buffer (50 mM TRIS (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.5% NP40 and 1 × protease and phosphatase inhibitor cocktails (Roche)) with glass beads (0.5-mm diameter) using a Precellys cell disruptor and subsequent clarification by centrifugation . Mpk1-HA3 was immunoprecipated with anti-HA magnetic matrix (Pierce) and co-immunoprecipitated Sic1-myc13 was determined by immunoblot analysis using anti-c-Myc antibodies.For co-immunoprecipitation analyses, Sic1-myc3 or Mpk1K54R -HA3was immunopurified from yeast cells using anti-HA magnetic matrix (Pierce). The respective matrices were incubated for 30 min at 30 °C with 3 μl of bacterially purified GST-Sic1 or GST-Sic1T173A in 50 μl of kinase buffer mix (125 mM Tris (pH 7.5), 50 mM MgCl2, 2.5 mM dithiothreitol and 10 mM ATP). The reactions were stopped by addition of loading buffer, boiled at 95 °C and analysed by immunoblot analyses. For the Mpk1 kinase time-course experiment, Mpk1-HA3 was purified from exponentially growing or rapamycin-treated (1 h) cells. The protein kinase reactions  were stopped at the indicated time points by addition of loading buffer and subsequent boiling (5 min).Mpk1-HA3 was isolated from exponentially growing cdc55Δ cells carrying the pRS416-CDC55-HA3 plasmid. Cdc55-HA3 was immunoprecipitated from total extracts in lysis buffer (50 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.5% NP40 and 1 × protease and phosphatase inhibitor cocktails from Roche) using anti-HA magnetic matrix (Pierce). Igo1-GST and Igo1S64A-GST were isolated from bacteria using glutathione sepharose  and phosphorylated where indicated by yeast-purified GST-Rim15-HA3 using 1 mM adenosine 5'-[γ-thio] triphosphate17in vitro phosphatase assay (30 min at 30 °C) was performed in phosphatase buffer (10 mM Tris (pH 7.5), 5 mM MgCl2 and 1 mM EGTA) with purified PP2ACdc55, bacterially purified Sic1-GST that was phosphorylated by Mpk1 in vitro as substrate, and different concentrations of Igo1, which was, or was not, subjected to in vitro phosphorylation by Rim15 before the use. To assess PP2ACdc55 activity, the decrease in Sic1T173 phosphorylation was detected using phospho-specific anti-Sic1-pThr173 antibodies. Levels of immunoprecipitated Cdc55-HA3 were assessed using anti-HA antibodies.Cdc55-HAHow to cite this article: Moreno-Torres, M. et al. TORC1 controls G1–S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway. Nat. Commun. 6:8256 doi: 10.1038/ncomms9256 (2015).Supplementary Figures 1-25, Supplementary Tables 1-2 and Supplementary References


Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus  and PEVK (increases PT). Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15 min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively), and to Ser11878 and Ser 12022 in the PEVK region . Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length (SL) ranging from 1.9 to 2.4 μm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity. Samples were then repeatedly homogenized with an ultrathurrax at 750 rpm. The myocyte suspension was then centrifuged with 1000 rpm for 3 min., resuspended and permeabilized for 3 min in the relaxing solution additionally supplemented with 3% Triton-X-100. Myocytes were washed in 3–5 centrifugation steps at 4°C and 2000 rpm using relaxing solution. After each centrifugation step the supernatant was discarded and the myocyte pellet was resuspended in fresh relaxing solution without Triton-X-100. The final myocyte suspension was kept on ice until further experimental use.For passive force measurements a few microliters of the cardiomyocyte suspension were transferred to a cover slip mounted on an inverse phase contrast microscope (Nicon eclipse Ti). One single cell was selected and fixed between a piezoelectric motor and a force transducer  both covered with a mixture (ratio 2:1) of silicone glues (Dow Corning Glue 3140 and 3145-transparent). Cells were then stretched from slack SL  in five steps to a maximum sarcomere length (SL) of 2.4 μm. In between the stretches a 5 s holding period was performed to wait for stress relaxation. Following the last stretch-hold, cardiomyocytes were released back to slack SL to test for possible shifts of baseline force. During the stretch protocol SL was recorded using an IMPERX (CCD) camera (Aurora Scientific). From the recordings we analyzed the force at the end of each hold period (near steady-state force). Passive forces of each cell were calculated considering their individual length and depth at initial SL before tightening.n = 3 bands per experimental condition. For PKCα expression level analysis, tissue samples were homogenized in modified Laemmli buffer. Standard 10 and 12.5% SDS-PAGE gels were performed according to standard protocols . Proteins were then transferred onto a PVDF-membrane by semi-dry Western blot technique using the Biorad turbo blot system (1.5A for 25 min with 20 V). Transfer efficiency was checked using Coomassie-based PVDF-stain, and only membranes showing good transfer of titin were used for further analysis. Titin phosphorylation status was tested using phospho-site directed antibodies generated for pSer4010 (VRIEEGKpSLRFPC) and pSer4099 (QANLFpSEWLRNID) in the titin N2-Bus region , and pSer11878 (CEVVLKpSVLRKR) and pSer12022 (KLRPGpSGGEKP) in the PEVK-region. Antibodies to pSer 4010, pSer4099, and Ser11878 had been generated by Eurogentech (Belgium) as previously described  was used as a size standard. Proteins were then transferred onto a PVDF-membrane by semi-dry Western blot technique using the Biorad turbo blot system (1.3A for 7 min with 20V). To determine the expression levels antibodies were used against Phospho-PKCα ,  PKCα  and Phospho-Phospholamban . Mouse and anti-rabbit IgG conjugated with horseradish peroxidase served as secondary antibody. All antibodies were diluted in standard TBST solution supplemented with 0.5–3% BSA or 0.5–3% milk powder. Signal intensity was analyzed densitometrically with ImageJ software. The signal intensity for phosphospecific PKCα or Phospholamban-signal was related to the respective PKCα-total or GAPDH-signal. Afterwards the ratio of phospho:total/GAPDH was used to normalize the status of phosphorylation of exercised muscle samples to the control samples.t-test and Mann-Whitney U-test was used to test for statistically significant differences. P-values <0.05 and P(exact) ≤ 0.001 were taken as an indication of significant differences and, if not noted otherwise, are represented in figures by asterisks.Unpaired Student's In order to characterize the effects of acute physical exercise on cardiac passive myofilament stiffness we determined the I-band phosphorylation status of titin using phosphosite-directed antibodies to Ser4010 (PKA-targeted) and Ser4099 (PKG-targeted) in the cardiac-specific N2-Bus region, and to Ser11878 and Ser12022 (PKCα and CaMKIIδ-targeted) in the ubiquitously expressed part of the PEVK region.As indicated by the large standard errors phosphorylation of residue Ser4010 in the N2-Bus differed largely among the analyzed exercise samples, and with a mean relative phosphorylation of 1.26 ± 0.72 compared to the control group the observed changes were not statistically significantly different Figure . In contWe further tested for changes in phosphorylation at Ser11878 and Ser12022 in the PEVK region of titin. The Western blot analyses demonstrated that relative phosphorylation of Ser11878 was significantly increased in the exercised samples to 2.15 ± 0.16 compared to control samples, whereas relative phosphorylation of Ser12022 was significantly decreased to 0.62 ± 0.04 of the control levels Figures .To determine the effects of altered titin phosphorylation on the passive properties of the cardiac myofilaments we mechanically isolated single cardiomyocytes and measured PT in relation to SL. For this purpose, cardiomyocytes were stretched in five steps to SLs ranging from 1.9 to 2.4 μm and passive forces were recorded for each step. The results demonstrate that PT was significantly higher in cardiomyocytes isolated from acutely exercised animals, compared to controls Figure . PT was We further investigated the effects of a single exercise bout on skeletal titin phosphorylation by analyzing the relative phosphorylation of Ser11878 and Ser12022 in the PEVK region of titin from the Musculus vastus lateralis (LAT). In order to determine putative differences in the response to concentric and eccentric exercise we analyzed LAT samples from animals after acute level and downhill running, respectively. Interestingly, unlike in cardiac tissue, Western blot analysis of the level-exercised skeletal muscles showed that the relative titin phosphorylation at Ser11878 was significantly reduced to 0.13 ± 0.02 compared to sedentary controls Figure . At the It has previously been shown that threonine 497 (Thr497) is a critical site for permissive activation of protein kinase Cα  in the cardiac-specific N2-Bus region, and a significantly increased phosphorylation of residue Ser11878 (PKCα-targeted) in the PEVK-region of titin. Decreased N2-Bus phosphorylation as well as increased PEVK phosphorylation have previously been shown to increase titin-based myofilament stiffness  that acute exercise is accompanied by activation of the beta-adrenergic system, followed by activation of cAMP-dependent protein kinase A (PKA) . This question is of great interest, since mechanical strains differ between concentric and eccentric muscle work with the consequence of muscle damage specifically after eccentric muscle contractions (Armstrong et al., An interesting and central finding of our study is that acute exercise induces divergent titin posttranslational modification patterns in cardiac and skeletal muscle tissues. While acute exercise increases titin PEVK phosphorylation at Ser11878 in cardiac muscle, the same intervention results in decreased titin PEVK Ser11878 phosphorylation in skeletal muscle. Opposite effects in cardiac and skeletal muscle could also be detected for titin PEVK Ser12022 phosphorylation. This result could indicate that cardiac and skeletal muscles sense exercise-induced mechanical stress by means of intrasarcomeric proteins, e.g., titin, in a different manner. Our data provide the first direct comparison between cardiac and skeletal muscle titin phosphorylation after a single exercise stimulation. Nonetheless these finding has to be proven by further studies to get a clear picture about titin's regulation in different striated muscle tissues.We conclude from these observations that acute exercise results in stiffer cardiomyocytes accompanied by increased titin PEVK phosphorylation. For skeletal muscle we postulate that the lowered PEVK phosphorylation leads to a decrease in titin based stiffness. According to this hypothesis, acute exercise induces divergent effects on titin posttranslational modifications and thus myocytes mechanical properties. Considering that the titin spring is a central regulator of the A-band position in the contracting sarcomere, the observed differences in titin stiffness between cardiac and skeletal muscles could be important to maintain their structural integrity, especially of the acutely exercised skeletal muscles For cardiac muscle, our data lead to the assumption that a single exercise bout induces rapid changes in titin-based myofilament stiffness, which may help to improve cardiac output by supporting the Frank-Starling mechanism, possibly crucial for cardiac functions during exercising conditions.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.


FoodAllergy (FA) has become a common problem in practice for the gastroenterologist and allergists, by increasing its frequency world wide. One cause of this increasing factor is the use of bottle of cow’s milk prior to breast milk. The aim of this work is to add new data for the inappropriate habit of offering a bottle of cow'smilk in the nursery before the breast Milk.130 patients with FA colected among the charts of the Brasilian Society of Food Allergy were studied regards the use of bottle fed prior to breast at the nursery station.Patients were classified according to their clinical and their laboratory tests in patients with mediated IgE FA, non-IgE mediated FA and mixed IgE and non-IgE FA.Of the 70 patients with IgE-mediated AA, 44 took the bottle in the nursery before human milk (HM) ; of 42 patients with non-IgE, 19 took the bottle in the nursery before the HM (45.23%), and 18 patientswith mixed FA, 8 took the cow's Milk before the HM (44.44%)The results show that the use in the nursery of the bottle of cow’s milk correlated with the cases of IgE-mediated FA (62.85% and 44.44% versus 45.23%) than the other types of allergies. This finding concurs with the literature data, with respect to that allergen stimuli in the first days of life, induce newborn to produce IgE Food Allergy.Early introduction of any other protein in the diet of the newborn before breast milk is associated, at high rates, to IgE FA.


TACI alleles increase susceptibility to autoimmune diseases. Taci−/− mice and BAFF transgenic mice both develop signs of human SLE. These findings that indicate inappropriate levels of TACI signaling may disrupt immune system balance, thereby promoting the development of autoimmune diseases. In this review, we summarize the basic characteristics of the TACI ligands BAFF and APRIL, and detail the research findings on the role of TACI in humoral immunity. We also discuss the possible mechanisms underlying the susceptibility of CVID patients with TACI mutations to autoimmune diseases and the role of TACI in the pathogenesis of SLE.Transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) is one of the receptors of B cell activating factor of the tumor necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL). TACI is a regulator in the immune responses. TACI inhibits B cell expansion and promotes the differentiation and survival of plasma cells. The mechanisms underlying these effects probably involve changed expressions of some crucial molecules, such as B lymphocyte induced maturation protein-1 (Blimp-1) and inducible T-cell costimulator ligand (ICOSL) in B cells and/or plasma cells. However, abnormal TACI signaling may relate to autoimmune disorders. Common variable immune deficiency (CVID) patients with heterozygous mutations in  Belimumab, a specific inhibitor of B cell activating factor (BAFF), was approved in 2011 by the US Food and Drug Administration (FDA) for the treatment of systemic lupus erythematosus (SLE). The FDA approval of belimumab not only represents the significant progress in the field of SLE therapeutics but also marks the success of BAFF research. BAFF and its homologue, a proliferation inducing ligand (APRIL), are recently discovered members of the tumor necrosis factor (TNF) superfamily , 36κB) pHSPGs bind to APRIL and TACI and play an important role in activating the APRIL-TACI pathway . For exa+ memory B cells, tonsillar and bone marrow plasma cells, a portion of activated CD27− non-GC cells, monocytes, DCs, and a portion of naive B cells in the blood plasma and tonsils [In mice, TACI is mainly expressed by mature B cells. High TACI expression has been detected in innate immune B cells, such as MZ B cells and B1 B cells . In huma tonsils . Additio tonsils .The TACI expression level is regulated by a variety of factors and constantly exhibits dynamic changes. For example, activation of TLR9, TLR7, or TLR4 results in elevated TACI expression in B cells , 40–42. + B cells is positively correlated with age in adolescents. TACI expression is reduced in the cord blood B cells of premature babies [TACI expression level is related to age. Schatorjé et al.  found the babies . In newbe babies . Newborne babies . Howevere babies , TACI exAlthough TACI is mainly present on the cell surface, a small amount of TACI is shed from the cell surface by a disintegrin and metalloproteinase (ADAM) 10 and converted into soluble TACI (sTACI) , 48. TheIn vitro studies have shown that activation of TACI decreases the numbers of human and mouse B cells [ Taci−/− mice develop hyperplasia in lymphoid organs, and the number of B cells increases drastically, indicating that TACI also exerts an inhibitory effect on B cells in vivo [ B cells , 3. Furt in vivo .TACI partially inhibits B cell number by promoting the sustained expression of B lymphocyte induced maturation protein-1 (Blimp-1) in GC B cells . Blimp-1 Taci−/− mouse, which strengthens the costimulatory signals received by inducible T-cell costimulator (ICOS) on TFH cells and promotes the activation and proliferation of TFH cells. The increase in the TFH number enhances the auxiliary effect of TFH cells on GC B cells and promotes B cell activation and proliferation, thereby increasing the number of B cells [Additionally, the expression of inducible T-cell costimulator ligand (ICOSL) is enhanced in the GC B cells of B cells . These fκB-inducing kinase (NIK) for degradation by ubiquitin-proteasome system, thereby inhibiting the BR3-mediated noncanonical NF-κB signaling pathway [The inhibitory effect of TACI on the B cell number is partially achieved through promoting B cell apoptosis. Tsuji et al.  reported pathway . The pro pathway . Taci−/− mice compared with WT mice, which may be because the B cells in Taci−/− mice are unable to bind to BAFF through TACI and reduce the concentration of BAFF [It is noteworthy that TACI may exert its inhibitory effect indirectly by regulating the BAFF concentration. The plasma BAFF level is significantly higher in of BAFF , 34, 53. Taci−/− mice undergo an increased number of cell cycles resulting in an increased level of somatic hypermutation. A high degree of somatic hypermutation causes an increase in the number of B cell clones that express high-affinity BCR, which eventually results in the production of antigen-specific antibodies with increased affinity in Taci−/− mice compared with WT mice. These results indicate that TACI exerts a regulatory effect on somatic hypermutation and Ig affinity maturation.Somatic hypermutation of immunoglobulin (Ig) genes is not only one of the mechanisms leading to antibody diversity, but it is also the main mechanism underlying Ig affinity maturation. A study conducted by Tsuji et al.  showed tκB pathway, prompting the transcription of germline CH genes, and inducing the expression of activation induced cytidine deaminase (AID) [Antibody class switch recombination (CSR) is another mechanism that leads to antibody diversity. Mouse studies have demonstrated that TACI mediates T cell-independent CSR , 47, 54.se (AID) .κB pathways and/or the upregulation in TACI expression [TACI alone or TLRs alone are able to mediate T cell-independent CSR by binding to MyD88. TACI-mediated CSR does not require TLRs, but both TLR7 and TLR9 together with TACI have a synergistic effect in promoting CSR . Similarpression , 57, 58.In summary, it has been shown that TACI plays a role in both T cell-dependent and T cell-independent CSR by binding to MyD88. Consistent with these findings, mutations in the gene encoding THC obliterate the synergistic effect of TACI and CD40 and the synergistic effect of TACI and TLRs on inducing CSR . Taci−/− mice fail to produce an effective antibody response against TI-2 antigens [−/− mice produce the IgM and immunoglobulin G1 (IgG1) antibodies at significantly lower levels compared with WT mice [TACI plays an important role in the antibody response to TI-2 antigens. The antibody response to TI-2 antigens is mainly mediated by MZ B cells and B1 B cells, and TACI is expressed at the highest levels in these two B cell subsets , 52. Addantigens , 10. The WT mice .Additionally, TACI also plays a role in the immune response to TD antigens. Castigli et al.  reported in vitro studies described above were conducted under very different conditions. In the former study, mouse naive B cells were used and CD40 was stimulated with less than the optimal concentration of anti-CD40 antibody. In the latter study, human peripheral blood B cells were used and CD40 was allowed to interact with a relatively high concentration of soluble CD40 ligand (CD40L). The discrepancy between these two studies may be due to the difference in the CD40 signal strength and/or differences in the effect of TACI in the mouse and human.It should be noted that these two Taci−/− mice compared with WT mice [ Taci−/− mice. These decreased antibody titers are caused by defective Blimp-1 expression in the GC B cells, which inhibits differentiation toward plasma cells and results in a decreased number of plasma cells.Several recent studies have demonstrated that antibody concentrations are significantly lower following TD antigen stimulation in WT mice –6. These Taci−/− mice are similar to those generated in the WT mice. However, in the late stages of the immune responses, number of antibody-producing cells (ASCs) and the plasma antibody titers are significantly lower in the Taci−/− mice compared with the WT mice [ Taci−/− mice during the early stages of the immune response. The number of DNA double-strand breaks increases accordingly, leading to a transient expression of Blimp-1 [−/− mice appear normal. However, sustained Blimp-1 expression requires TACI activity. Therefore, the levels of ASCs and antibodies cannot be maintained in Taci−/− mice during the late stages of the immune response.In the early stages of the immune responses against influenza virus, the humoral immune responses generated in the WT mice . It is p Blimp-1 . Therefo Taci−/− mice. However, the reduction in antibody titers is due to increased plasma cell apoptosis. The proapoptotic protein B cell lymphoma 2 interacting mediator of cell death (BIM) promotes apoptosis in plasma cells. APRIL and BAFF downregulate BIM expression by interacting with TACI, thereby promoting the survival of plasma cells. Taci−/− mice are unable to downregulate BIM expression through APRIL and BAFF, resulting in increased plasma cell apoptosis.Ou et al.  also demThe two studies described above indicate that TACI regulates the number of plasma cells and the production of antibodies through at least two mechanisms. First, TACI promotes the differentiation of B cells into plasma cells by maintaining a continuous expression of Blimp-1 in B cells. Second, TACI inhibits plasma cell apoptosis by downregulating BIM expression in plasma cells . Higher Studies have shown that TACI binds to membrane-bound ligands and activates macrophages or DCs through reverse signaling –64. For γ-chain (FcRγ) on their surface. Therefore, the activating effect of TACI-Fc previously observed in DCs and macrophages is likely induced by the activating signals that are generated via the high-molecular-weight TACI-Fc multimer-mediated cross-linking of Fc receptors [A recent study has shown that there is no difference in the cellular responses between BAFF and APRIL knockout cells and WT cells under the activity of soluble TACI-Fc or soluble BR3-Fc . Therefoeceptors . Because TACI gene, primarily the heterozygous C104R and A181E mutations [ TACI mutations C104R and A181E are also found in approximately 2% of healthy individuals [ in vitro assays. For example, compared with healthy individuals who do not carry TACI gene mutations, individuals with heterozygous TACI mutations demonstrate decreased TACI expression on their B cells. Additionally, stimulation of B cells with TLR9 fails to upregulate the expression of TACI, and stimulation of TACI with activating antibodies is unable to effectively induce the expression of AID mRNA in B cells [Approximately 7% to 10% of CVID patients carry allelic mutations in theutations , 70, 71.ividuals . Althoug B cells . Taci−/− mice compared with WT mice, the Taci−/− mice are able to produce antigen-specific antibodies with a higher affinity for the antigens and are able to clear Citrobacter rodentium at a faster rate. The results indicate that the high frequencies of monoallelic or biallelic TACI mutations in human populations may be an adaptation to the environment. Defects in TACI expression and function may allow part of the human population to rapidly produce high-affinity IgG antibodies to clear intestinal pathogens. However, TACI mutations increase the risk of developing CVID [However, the decrease in TACI expression and defects in TACI functions do not always have a negative impact on the immune system. Tsuji et al.  demonstring CVID . TACI mutations in CVID correlate with susceptibility to autoimmune disorders. TACI regulates the function of BCR, TLR7, and TLR9. BCR, TLR7, and TLR9 may be involved in self-antigen recognition and central B cell tolerance [ TACI gene mutations impair central B cell tolerance, resulting in an inability to remove autoreactive B cells through central tolerance mechanisms [ TACI alleles, normal peripheral tolerance may compensate for defects in central tolerance. Therefore, these individuals are not prone to autoimmune diseases. CVID patients, however, exhibit abnormalities in peripheral tolerance mechanisms. The level of BAFF is elevated in CVID patients, and the level of regulatory T cells (Treg) is reduced [ TACI gene, peripheral tolerance is unable to compensate for defects in central tolerance. Therefore, CVID patients are susceptible to autoimmune diseases [Additionally,olerance . Therefochanisms . In heal reduced , 73, 74.diseases . TACI alleles are related to a high level of TFH and high titers of IgG antinuclear antibodies, the level of TFH is extremely low in CVID patients that are homozygous for mutant TACI alleles. Moreover, IgG antinuclear antibodies are undetectable in these patients [ TACI alleles. Impaired ICOSL expression leads to a loss of interactions between the ICOSL on B cells and the ICOS on TFH cells. As a result, both the B cells and TFH cells are unable to undergo efficient activation and proliferation, and GCs fail to form [ Taci−/− mice, however, the expression of ICOSL on B cells is not reduced and the number of TFH cells is increased [Although heterozygous mutations inpatients . These p to form –77. In Tncreased , indicat TACI alleles result in a complete loss of TACI function and an inability to maintain continuous production of autoreactive antibodies. In contrast, heterozygous mutations in TACI alleles only impair TACI function. TACI function is not completely lost and is sufficient to maintain the continuous production of autoreactive antibodies [ TACI alleles are prone to autoimmune diseases, whereas CVID patients homozygous for mutant TACI alleles appear to experience an autoimmune disease-preventive effect [Additionally, TACI promotes the differentiation and survival of plasma cells –6. Theretibodies . Therefoe effect –13. Taci−/− mice age, they display SLE-like symptoms [ TACI gene is not sufficient to prevent the incidence of SLE in NZM2328 mice [Assymptoms . Additio328 mice . TherefoBAFF transgenic mice exhibit SLE-like symptoms . Blockag Taci−/− mice. As Taci−/− mice age, the mice develop SLE-like symptoms [ Taci−/− NZM2328 and WT NZM2328 mice. However, the renal histology scores are significantly higher in 5-month-old and 8-month-old Taci−/− NZM2328 mice compared with the corresponding WT NZM2328 mice [The number of B cells is increased insymptoms , 9, 10. 328 mice , which s Taci−/− mice is mainly due to increased B cell proliferation [Additionally, the increased number of GC B cells inferation , 6. Whenferation . TherefoMoreover, TACI promotes the expression of AID . A study Br3−/− NZM2328 mice and WT NZM2328 mice. Although the numbers of follicular (Fo) B cells and MZ B cells are significantly reduced in Br3−/− NZM2328 mice, no difference has been found in the number of plasma cells between Br3−/− and WT NZM2328 mice [ Br3−/− and WT NZM2328 mice [However, other evidence suggests that the effect exerted by TACI on SLE is not simply inhibitory. There is no significant difference in the incidence of SLE between328 mice . The rea328 mice , 6. Ther328 mice . These fAdditionally, distinct types of autoantibodies contribute differently to the pathogenesis of SLE . Most ofAs described above, TACI may inhibit SLE, but an overly strong TACI signal may promote SLE. Will TACI promote or inhibit SLE then? Indeed, the so-called normal immune system is in a state of dynamic equilibrium, and autoimmune diseases are caused by an imbalance in the system. Therefore, we conjecture that TACI signaling at a normal intensity is conducive to maintaining the body's immune balance and exerts an inhibitory effect on SLE. However, too strong of a TAC1 signal is just as detrimental as too weak of a signal. An overly strong or very weak TACI signal will disrupt the immune system balance and promote SLE .The characteristics of TACI signaling may partially explain the paradox concerning the efficacy of the monoclonal antibodies belimumab and rituximab in treating SLE. Belimumab was successful in 2 independent phase III SLE trials , 85. HowTACI plays an important role in the antibody responses to not only TI-2 antigens but also TI-1 and TD antigens. The mechanisms by which TACI regulates the number of B cells and promotes plasma cell differentiation and survival provide a rather reasonable explanation for TACI activity. Certainly, these mechanisms are not necessarily comprehensive. Further studies need to be conducted to fully elucidate the mechanisms regulating TACI function.Based on the large amount of contradictory findings, it is impossible to conclude that TACI exerts a simple promoting or inhibitory effect on autoimmune diseases such as SLE. TACI is a factor that affects multiple events in the humoral immune responses. Therefore, the intensity and/or the background of TACI activity may determine the outcome. TACI signaling at an appropriate level suppresses autoimmunity and maintains immune balance and tolerance. In contrast, overly strong or very weak TACI signaling does not appear to be conducive to immune balance and tolerance and thus promote SLE. TACI alleles exert a preventive effect on autoimmune diseases. Conversely, heterozygous mutations in TACI alleles result in a decrease rather than complete loss of TACI function and promote autoimmune diseases [ Taci−/− mice develop SLE appears to be contradictory [However, the so-called “appropriate TACI signal strength” is different under different conditions. For example, homozygous mutations indiseases , 40. If adictory .Certainly, TACI is just one of the various factors that affects autoimmune diseases. The roles of the other factors, such as T cells, cannot be ignored. The pathogenesis of SLE in BAFF transgenic mice does not depend on T cells , but it


O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-  •The structure of WcbL in complex with ligand analogs is reported•WcbL switches its cooperativity toward ATP in the presence of sugars•The kinetics and mechanism of WcbL are elucidated•Screening of a fragment library identified a potent inhibitor of WcbL Vivoli et al. present the structure and mechanism of the bacterial sugar kinase WcbL. WcbL demonstrates unusual kinetics: its cooperativity for ATP switches on binding of the sugar substrate. Screening of a fragment library identified a potent inhibitor of WcbL. Bacterial cells display a variety of polysaccharides and glycoconjugates in their cell walls and external structures. The functions of these macromolecules include maintaining the structural integrity of the cell (e.g. peptidoglycan), defense against a diverse range of environmental insults, and providing virulence determinants for the invasion of hosts . The polShigella, Yersinia pseudotuberculosis, Campylobacter jejuni O antigen; Burkholderia pseudomallei CPS-I  is the inhibitor concentration; and Ki is the inhibition constant.To determine the inhibition constant for 3-cyanochromone, compound was used at 0, 10, 100, and 500 μM as described above. The ATP concentration used in the reaction was varied from 0 to 1 mM, and enzyme activity was detected as described above. The Ki for 6-bromo-3-cyanochromone was extrapolated using the following equation, assuming a competitive inhibition:The 2 value, and Akaike's Information Criteria.Kinetic studies of the reaction mechanism of WcbL were performed by product inhibition experiments using the assay methodology outlined above. For these experiments, ADP was used as the product inhibitor. In brief, ADP at concentrations of 100, 500, and 1,000 μM was placed into plates containing 12 μg/ml WcbL, 80 μg/ml WcbN, 1 mM ATP and 0–250 μM M7P, or 250 μM M7P and 0–1 mM ATP, and residual enzyme activity monitored using the Pi Colorlock reagent as described above. The initial rates derived from these inhibition experiments were fitted to equations representative of competitive, non-competitive, mixed, or uncompetitive inhibition by a non-linear least-squares approach using GraphPad Prism version 6.0.2. The best fits of the data were compared using a combination of visual inspection, comparison of RB. pseudomallei wcbL− mutant  helper strain were inoculated as overlapping spots onto a nitrocellulose membrane previously placed on the surface of an L-agar plate. After an overnight incubation at 37°C, the bacterial growth was harvested into PBS (1 ml) and aliquots spread onto L-agar containing 50 μg/ml gentamicin and 750 μg/ml kanamycin.Complementation of the previously described − mutant  was compB. pseudomallei ΔwcbL isolates were incubated in L-broth containing 750 μg/ml kanamycin overnight at 37°C. Cells were pelleted, suspended in PBS, and inactivated by heating to 80°C for 4 hr. Once cooled, aliquots of each isolate (10 μl) were dried onto a nitrocellulose membrane and the membranes used for western hybridization as previously described   or two AMP-PMP molecules were prepared by adding the AMP-PNP molecules and ions from each molecule to the relevant molecule in the mannose complex. Explicit hydrogens were added to the structure, and the structure was optimized using the online version of YASARA . Normal elnemo/) , using selnemo/) . HydrogeR.N. and N.H. performed the molecular biology; M.V. performed the enzymatic assays; M.V., M.I., and N.H. undertook the crystallization and crystallography; A.H. and N.H. performed the fragment screening; A.S. and J.P. performed the in vivo assays; P.K. synthesized M7P; M.V., M.I., A.S., P.K., and N.H. wrote the manuscript.


CD23, the low affinity receptor for IgE, is mainly expressed on B cells and plays an important role in allergy. Besides its possible role in the regulation of IgE production, CD23 plays an important role in IgE-facilitated allergen presentation to T cells and subsequent activation of allergen-specific T cells.Four CD23 protein versions were recombinantly expressed in SF9 insect cells, human monoclonal IgE was isolated from a hybridoma cell line.The characterisation of CD23 via circular dichroism (CD) spectra and gel filtration showed folded, monomeric proteins. Binding of isolated monomeric human monoclonal IgE and of isolated polyclonal serum IgE as well as of both forms of IgE in complex with birch pollen allergen Bet v 1 to recombinant CD23 was demonstrated by ELISA and by surface plasmon resonance analysis.Next, we performed negative stain electron microscopy of the three molecules alone  and after complex formation. After addition of Bet v 1 allergen (17 kDa) to monoclonal IgE (190 kDa) we could observe an extension of one or both Fab arms of the antibody. Interestingly, further addition of recombinant CD23 molecules (35 kDa) to the IgE-allergen complex resulted in thickening of the antibody’s Fc structure, possibly because CD23 lies in the same plane as the IgE molecule in these pictures.in vitro formation of a trimolecular complex consisting of recombinant CD23, a monoclonal human allergen-specific IgE and the corresponding Bet v 1 allergen and take a first step towards the visualization of this complex using negative stain electron microscopy. Furthermore, the in vitro trimolecular interaction model may be useful for the screening of drugs and compounds for their potential to inhibit the IgE CD23 interaction with the goal to develop new therapeutic strategies for allergy.In summary, we report the Supported by grants 4605, 4613 and in part by 4604 and P23350-B11 of the Austrian Science Fund (FWF).


The results indicated N&P addition was capable to ameliorate the quality of the two sites in the Tibetan Plateau steppe. Increasing N application level resulted in significant increment in Gramineae and total biomass in the two sites. P addition significantly improved the quantity of Compositae, total biomass and the biomasss of other species in site II, while it only significantly improved the total biomass in site I. Gramineae was much more sensitive to N-induced changes than P-induced changes, and this indicated N addition was better to ameliorate the quality of plateau steppe than P-induced changes. No strong evidence was found for critical threshold within 15 g N m-2 yr-1, and there was decreasing tendency when P addition rate was above 15 g m-2 yr-1. N&P has the potential to accelerate soil acidification, which improved the content of available K, likely as a result of nonsignificant correlation between biomass and soil moisture. This work highlights the the tradeoffs that exist in N and P addition in recovering degraded steppe.In the alpine steppe zone on the Central Tibetan Plateau, a large amount of area has been degraded due to natural and artificial factors. N & P fertilization is widely accepted to recover degraded pastures in other regions all over the world. However, it is not clear how alpine steppe communities respond to N & P fertilization, and what is the optimal application rate, in the perspective of forage production. To attempt to explore these questions, in July 2013, two fencing sites were designed in Baingoin County with 12 treatments of different levels of nitrogen (N DespiteAmerica) –5, the pAmerica) . Though  plateau , most pr plateau . RecoverDegradation has reduced the proportion of forage grasses , contribCurrently, N & P fertilization is widely accepted to recover degraded pastures in other regions all over the world –29. SomeStipa purpurea and the other is dormnated by L. leontopodioide. We aim to answer the following questions: 1) How do alpine steppe communities respond to N & P fertilization? 2) What dosage is effective and economical, in the perspective of forage production? 3) Whether there exist any difference between the two typical steppes after N & P fertilizer addition? 4) And whether there are interaction between N and P fertilization? The findings of this study will shed light on the efficiency of fertilization in the recovery of the Tibetan Plateau steppe.In this study, we focus on two typical steppe, one is dominated by The field experiment was conducted in alpine steppe at a mean altitude of 4678 m above sea level, which is located in Baingoin County  in northern Tibet. The location is semiarid cold alpine steppe, and the soil is alpine steppe soil . AccordiStipa purpurea, accompanied with Leontopodium leontopodioide and Heteropappus bowerii. In site II, the dominant species is L. leontopodioide, accompanied with H. bowerii and S. purpurea. Site I and II were fenced with an area of 100 ×100 m to exclude large animals in July 2013.On a 5.5° gentle slope, two sites were chosen. No fertilizer had been applied in either site before this study. Site I is dominated by the species of 0: 0; N1: 7.5 gN.m-2.yr-1; N2: 15 gN.m-2.yr-1) and 4 levels of P  as commercial fertilizers of urea and triple super-phosphate. Fertilizers were applied evenly at different times: 1) initial growth season in July; 2) vigorous growth season in August.Identical treatments were applied to site I and site II. At each site, 60 5×5 m plots were laid out in a randomized design, and plots were separated by 2-meter buffer zones. To avoid edge effects, each plot was placed at least 3 m inside each site. There were 5 replicates for each of the 12 treatments which included 3 levels of N , Compositae , Cyperaceae , Rosaceae  and forbs . Since our preliminary analysis found no significant effects of N&P addition existed on the responses of Cyperaceae, Rosaceae and forbs, so we classified them together as other species in this study.In accordance with earlier studies, the total species were divided into 5 communities: Gramineae  concentration was measured by colorimetry on a SMARTCHEM 140 . Concentration of extractable soil NH4+-N was expressed as milligrams per kilogram on the basis of dry soil mass. Soil moisture was determined using the gravimetric method. The soil samples were weighed before and after being oven-dried at 105°C for 48 h. All the properties of soil before fertolized are as below (Table 1).Soil pH was measured with a soil to water ratio of 1:2.5; soil organic matter was determined by potassium dichromate oxidation ; total nAll statistical analyses were performed using SPSS version 16.0  and Origin 8.0 . ANCOVAs were performed to examine the significance of factors’ effects and their interactions on the observed parameters. Duncan’s protected least significant difference test was applied to examine the quantitative differences between treatments. Analyses across sites were performed using General Linear Model for N addition rate, P addition rate and their interaction as fixed-effects. After that, no significant difference were found between the biomass among the 12 treatments in July, while the interaction of N addition and sampling sites had significant influence on biomass. Thus, we ran additional analyses by ANOVA of the biomass in September for each site to determine the response of pattern and magnitude. Regression models with N or P as a continuous variable were used to determine the general relationship between N or P addition and various response (i.e. to estimate threshold levels). Those ANOVAS were followed by a Duncan’s multiple-ranges test to compare the N-addition or P-addition effects for each rate.ANCOVAS of aboveground biomass, using sites, N addition, P addition and all their interactions as fixed factors, and biomass in July as covariate, showed that the effects of different sites was highly significant on biomass of Compositae, other species and total biomass . The aveAboveground biomass tended to increase significantly at all N addition rate in site II, while significant increase only exist in Gramineae and total biomass in site I. However, no significant increase was found in various species with different levels of P addition whereas the total biomass increase significantly .L. leontopodioide, has higher aboveground biomass of plant functional groups than site I except Gramineae (9.41% lower than site I). The interactions between N, P and different sites were nonsignificant except the interaction between different sites and N addition rate for Compositae , dominant species is mpositae . In sitempositae .2 = 0.218; total biomass: t2 = 0.199). For different rate of P addition, only total biomass showed significant linear correlation with P rate  , with the increasing of N addition rate, the biomass of Gramineae and total biomass showed highly significant increase (Gramineae: tificant) .-2 yr-1, the effects showed significant compared to the control, and when added N rate at 15 g m-2 yr-1, the effects were same to the rate of 7.5 g m-2 yr-1. There was nonsignificant effects for P addition in site I for most plant except the total biomass, while the effects of P addition in site II was more significant, and the threshold was 7.5 g m-2 yr-1 for Compositae and total biomass, 15 g m-2 yr-1 for other species . Thus, throughout the study period, the Site II showed a more sensitiveresponse than site I.Our experiment in the Tibetan Plateau demonstrated that the qualitative effects of N&P addition were similar between the two sites, whereas the quantitative effects in site II was more significant. When added N rate till 7.5 g m-2 yr-1, and other species in two sites and the total biomass in site II when N addition rate was 15 g m-2 yr-1). Except the above treatment, the other treatment of N or P added alone also had nonsignificant effect compared to the control  like to feed on Gramineaerather than Compositae. In site I wherethe biomass of Gramineae was more than Compositae, we didn’t find any significant difference between different N or P level, while wefound there was a higher ratio when N addition rate was 7.5 g m-2 yr-1 and P addition rate was 7.5 or 15 g m-2 yr-1 compared to the control .In site II where the biomass ofGramineae was less than Compositae, our experiment results showed that the increament of P addition rate could decrease the relative quality in the total biomass significantly. And the N level of 15 g m-2 yr-1 could increase the ratio of Gramineae, though it was nonsignificant .4+-N tended to increase significangtly at all rates of N addition compared to the control. Soil AP also had a highly significance at different P application levels compared to control, while P3 has significantly difference with P1 and P2. Our results also indicated that there was a decreasing tendency for pH with the increasing of N addition rate  for site I. Our experiment showed that there were nonsignificant differences in the interaction of N and P for the soil physical and chemical properties based on our analyses. Soil NHion rate .We applied further analysis of multiple linear regression (MLR) between TB , GB (Gramineae biomass), CB (Compositae biomass) and different properities of soil. Three MLR models were developed for the predication of TB, GB and CB, respectively .The models obtained were the following:4+-N had a significant influence on the biomass of Gramineae. The same to total biomass, content of AP had a sigificant influence on the biomass of Compositae.For these models, only the explanatory variables which presented parameters with statistical significance were considered. Forthe total biomass, the AP content showed significant influence, while other properities had nonsignificant influence on the total biomass. The content of NH4+-N and pH. We could also conclude that there was a higher content of AK with the increase of SOM.For the other soil properities, we could conclude that there were highly significant positive correlation between SOM and moisture, while there was a negative correlation between pH and AP. There were significant correlation for moisture with AK and pH. There was negative correlation between content of NHIn spite of numerous efforts that have been undertaken to arrest land desertification in China, grassland degradation is advancing over wide areas through overgrazing, climate change, cropland misuse and unregulated collection of fuel and medical plants .-2 yr-1) or P (30 g m-2 yr-1) alone had toxic effects. Fifth, the difference observed between the two sites suggest that site-species dynamics modulate the impact of input of N or P [Our results showed that the N or P addition could significantly increase the total biomass of the Tibetan Plateau steppe, while there were different responses among various plant functional groups. In site I and site II, any application rates of N showed significantly increase of the biomass of Gramineae, while no significant increase were found with P addition. Compositae showed nonsignificant difference at any addition rate of N or P in site I, while it was significant difference in site II. The other species also has the same tendency with Compositae. We can conclude that Gramineae tend to be more sensitive to N addition than P addition in spite of the dominant species. This finding can also be demonstrated by the MLR’s results. Compositae only showed significant difference in site II where it is dominated. The other species showed significant difference in site II, while it was nonsignificantin site I. These patterns suggest five conclusions. First, N addition could significantly increase the biomass of Gramineae, which could improve the Tibetan Plateau steppe’s quality.However, we didn’t find strong evidence for critical threshold within the range tested here for Gramineae, maybe N addition rate was below this threshold and Gramineae need more nitrogen input, orhis may due to microbial and abiotic processes that outcompeted plants for excess N. Second,f N or P .Fertilization experiments provide effective ways of examining the nutritional status of ecosystems and have been conducted to test the effects of N addition on biomass . Our resOur results found that the combination of N and P could enhance the recovery of degraded grassland. However, the interaction between N and P was not significant. This results was consistent with the results of other studies in Alpine meadow . It coul+ release is initiated by a low K+ concentration in the soil solution and not by cation exchange [There was nonsignificant influence on biomass for moisture, this tendency is not consistent with the findings of an ecosystem above 3750 m and otheexchange . With in4+-N, soil moisture with pH, which indicated the application of fertilization initially acidified the soil. This results showed that application of N or P fertilizer alonel decrease the quality and quantity of our experiment site. It is well known that N fertilisers acidify soils [Our results showed there was negatively correlation between AP, NHfy soils , while tfy soils ,49.-2 yr-1 and 15 g P2O5 m-2 yr-1 may be the optimal chioce, since higher addition rate could decrease the ratio of Gramineae to Compositae. While 15 g N m-2 yr-1 additon was effective where Compositae is the dominate species. And any application rate of P could decrease the quality of steppe in site II. Compared to other communities, Gramineae had a more sensitive response and greater relative dominance because it has a higher nutrient use efficiency when there are sufficient nutrients, but a lower nutrient use efficiency when there are fewer nutrients available [To ameliorate the quality of steppe where Gramineae is the dominate species, 7.5 g N mvailable . Our res-2 yr-1, and there was decreasing tendency when P addition rate was above 15 g m-2 yr-1. The interaction between N and P was nonsignificant for most functional groups.Our study showed that N&P addition had all ameliorated the quality of the two sites in the Tibetan Plateau steppe. N addition led to a large increment in Gramineae and total biomass in the two sites. The addition of phosphorus fertiliser had different influence on the two sites due to the site-species. We did not find strong evidence for critical threshold within 15 g.N m


These cellular enzymes are metabolic sensors sensitive to NAD+ levels that maintain physiological homeostasis in the animal and plant cells.Sirtuins are evolutionarily conserved nicotinamide adenine dinucleotide (NAD Acetylation, catalyzed by acetyltransferases that transfer an acetyl residue from acetyl-CoA to the ε-amino group of specific lysine residues in histones and other proteins, is responsible for chromatin activation and regulation of metabolic pathways. A reverse process, the removal of acetyl group from the lysine of acetylated proteins, requires the participation of enzymes known as lysine deacetylases (KDACs). In general, the lysine deacetylases were divided into four classes: class I, II, III, and IV . Since a+) [Sirtuins constitute a highly conserved family of deacetylases that depend on the oxidized form of nicotinamide adenine dinucleotide (NAD+) . Seven h+) . Sirtuin+) . SIRT3, +) .Table 1SSirtuins play an important role in the regulation of cellular homeostasis, in particular metabolism , inflamm+-binding domain, consisting of about 275 amino acids, which is flanked by the N- and C-terminal sequences of variable length [+-binding proteins while a smaller domain includes a zinc-binding module containing three-stranded antiparallel β-sheet and a variable α-helical region [+)-binding loop region, connecting the small domain to the Rossmann-fold structure, consists of four loops forming an extended cleft that acts as the enzyme active site. Both NAD+ and acetylated lysine-containing substrates bind to this pocket [+-binding site can be divided into three regions: site A, binding site for adenine-ribose moiety; site B, nicotinamide-ribose binding region; and site C, nicotinamide moiety binding site [+ can undergo a conformational change bringing the nicotinamide group in the proximity to the C site where it can be cleaved. After nicotinamide cleavage, the acetyl carbonyl oxygen of the acetyl-lysine nucleophilically attacks the carbon C1′ of the ribose to form a first intermediate between the two substrates which is the 1′-O-alkylamidate. Then, the intermediate is hydrolyzed to produce a deacetylated polypeptide and 2′-O-acetyl-ADP-ribose [All sirtuins possess a conserved catalytic NADe length . The N- e length . A largel region . Cofactos pocket . The NADing site . In the P-ribose , 56  in various proteins to form N- or O-glycosidic bonds, depending on a nucleophilic group in the amino acid side chain [In addition to the deacetylation of nucleosomal histones and metabolic enzymes, the sirtuins may also exhibit other activities. Although SIRT1 and SIRT2 could decrotonylate histone peptides in vitro , SIRT3 ide chain  de chain , 30 or ode chain  and fattde chain , 41.Fig.+ due to the upregulation of the gene encoding an NAD+ synthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) while SIRT1 activates AMPK by deacetylation of its activator, liver kinase B1 (LBK1).This section only briefly discusses the broad functional diversity of sirtuins, and much more information on the topic can be found in the following reviews , 50, 55.2 to M, SIRT2 translocates to the nucleus to deacetylate histone H4 at lysine 16 leading to chromatin condensation during metaphase. SIRT2 also deacetylates the transcription factors FOXO1 and FOXO3 and lysine residues in the catalytic domain of histone acetyltransferase p300 [SIRT2 which is present predominantly in the cytoplasm colocalizes with microtubules and deacetylates the main component of microtubules, α-tubulin, at lysine 40 . During ase p300 .SIRT3 positively regulates the activity of mitochondria by deacetylation and activation of several components of the electron transport chain complexes I and II and acetyl-CoA synthetase (ACS) . SIRT3 aSIRT4, located in the mitochondrial matrix, is ubiquitously expressed in kidney, heart, brain, liver, and pancreatic β cells. SIRT4 suppresses the secretion of insulin in response to glucose and interacts with insulin-degrading enzyme (IDE) . By tranSIRT5 is localized in the mitochondrial matrix, mainly in brain, heart, liver, and kidney. SIRT5 deacetylates carbamoyl synthetase 1 (CPS1) which catalyzes the first rate-limiting step of the urea cycle. The CPS1 deacetylation by SIRT5 increases the activity of the enzyme. It has been shown , 50 thatPoly-(ADP-ribose) polymerase 1 (PARP1) that stimulates the repair of DNA damage in response to oxidative stress is ADP-ribosylated by SIRT6 to promote its poly-ADP-ribosylation activity . SIRT6 iSIRT7 participates in the transcriptional activation catalyzed by RNA polymerase I and III , 58 and Investigations conducted in mice have shown  that actPolyphenols are plant secondary metabolites and represent a large group of compounds of variable structural complexity with aromatic rings containing one or more hydroxyl groups. A growing number of reports suggests  that polinter alia in red wine and grape skins, increases the affinity of SIRT1 for acetylated peptide substrates. This sirtuin activator binds to the enzyme-substrate complex and lower Km for the acetylated substrate without affecting the Km for NAD+ or Vmax [It has been shown  that res or Vmax . Resvera+-dependent sirtuin activity has also been shown to increase when cells or animals are treated with NAD+ precursors such as niacin, nicotinamide, nicotinamide riboside or nicotinamide mononucleotide [In addition to resveratrol, several other small molecules  that activate SIRT1 were described . These c+ causes that they are less toxic [+ and the acetylated lysine of the substrate [It has been shown  that sirss toxic . Suraminubstrate . Howeverubstrate .+ binding site of sirtuins. Other example of inhibitory indole derivative is oxyindole (selective for SIRT2 in vitro) that inhibits α-tubulin deacetylation in MCF-7 mammary cells [Indole derivative EX-527, a selective SIRT1 inhibitor, easily penetrates into cells. Administration of this compound strongly increased the acetylation of p53 protein at K382 following the induction of DNA damage in human mammary epithelial cells and some tumor cell lines [ry cells . A SIRT2ry cells .+ binds and the hydrolysis takes place, binding the inhibitor to the C-pocket would block the transformation of NAD+ to productive conformation and thus inhibit the deacetylase activity. An analogue with hydroxyls at ortho and meta positions, (5-hydroxy-1-benzofuran-3-yl)methanone, which is a non-competitive inhibitor for acetylated peptides and a mixed competitive inhibitor for NAD+, is a more potent SIRT1 inhibitor than nicotinamide [Thiourea-based compounds called tenovins can also attenuate the activity of sirtuins. Tenovins are highly hydrophobic, and this property hampers or even precludes their use in vivo. However, a promising prospect involves synthesis of tenovin-6, a more water-soluble analogue , which etinamide . A diffetinamide .SIR2 genes in plants is strongly reduced and, in most cases, two genes were detected [Arabidopsis, 18 KDACs, including two sirtuins—SRT1 and SRT2  were identified in tomato flowers and cotyledons . While tnt roles .VvSRT1 and VvSRT2 are members of the SIR2 family in the grape vine genome (V. vinifera). The VvSRT1 transcripts in roots, leaves, flowers, and fruit and VvSRT2 transcripts in leaves, flowers, and fruits were revealed [revealed  that VvSrevealed .Over the last several years, a lot of efforts have been made to better understand the mechanisms of sirtuin actions. Disturbances in energy metabolism, genome stability, response to cellular stresses, and lifespan shortening in mice lacking specific sirtuins demonstrate that these enzymes could contribute to the maintenance of cellular homeostasis in mammals. A modification of the sirtuin activity by small molecule activators or suppressors may provide new opportunities for the treatment of type II diabetes, obesity, and neurodegenerative diseases associated with aging or to clarify the role of sirtuins in the carcinogenesis. To achieve these goals, a progress should be made in understanding the cellular effects of sirtuins as well as in identifying additional targets and modulators for these enzymes. Similarly, the exact function of plant SIR2 lysine deacetylases is not fully understood and further research is needed to explain their role.


A 27-year-old man with progressive bilateral visual decline was diagnosed to have Bietti's crystalline dystrophy (BCD). Fluorescein angiography revealed bilateral petaloid type late hyperfluorescence implicating concurrent cystoid macular edema (CME). Optical coherence tomography exhibited cystoid foveal lacunas OU. During the follow-up of six years, intraretinal crystals reduced in amount but CME persisted angiographically and tomographically. CME is among the rare macular features of BCD including subfoveal sensorial detachment, subretinal neovascular membrane, and macular hole. In 1937, Bietti [Bietti's crystalline dystrophy (BCD) is likely related to aberrant oxidation of cellular lipid metabolism  which isMacular complications are rarely encountered in patients with BCD. We hereby report a case with BCD and bilateral persistent cystoid macular edema with a follow-up of six years.A 27-year-old otherwise healthy man was examined by us for bilateral progressive visual loss of long duration. There was no family history of consanguinity or similarly affected family members. On examination, best-corrected visual acuity was 20/40 OD and 20/50 OS. Examination of the anterior segment was normal with no concurrent corneal crystals. Intraocular pressures were 16 mm Hg OU. Fundus examination showed refractile intraretinal crystals in the posterior pole and the midperiphery associated with slight RPE atrophy Figures . Fundus The diagnosis was BCD with bilateral cystoid macular edema. No treatment was given. The patient was reexamined six years later. This time, visual acuities were 20/80 OD and 20/100 OS. Ophthalmologic findings were more pronounced when compared to initial ophthalmological examination with marked chorioretinal atrophy, reduced numbers of crystals, and the occurrence of patchy retinal pigment epithelial hyperplasia. Figures  CME was Cone and rod photoreceptors are equally affected in many cases with BCD and therefore central vision drops even at early stages of the disease in some cases . In addiCystoid macular edema (CME) represents a known complication in patients with some hereditary retinal degenerations such as retinitis pigmentosa, X-linked retinoschisis, enhanced cone syndrome, choroideremia, and gyrate atrophy and is characterized by a localised expansion of the macular intracellular and/or extracellular space , 19. VarIn light of our observation, we believe that CME should be looked for in eyes with BCD besides the other rarely reported macular changes. However, therapy of CME in BCD is uncertain.


N-acetyl-pentapeptides, Ac-P1, Ac-P2, Ac-P3, and Ac-P4, regarding inhibition of mushroom tyrosinase activity. The peptides sequences of Ac-P1, Ac-P2, Ac-P3, and Ac-P4 were Ac-RSRFK, Ac-KSRFR, Ac-KSSFR, and Ac-RSRFS, respectively. The 4N-acetyl-pentapeptides were able to reduce the oxidation of l-DOPA by tyrosinase in a dose-dependent manner. Of the 4N-acetyl-pentapeptides, only Ac-P4 exhibited lag time (80 s) at a concentration of 0.5 mg/mL. The tyrosinase inhibitory effects of Ac-P4 (IC50 0.29 mg/mL) were more effective than those of Ac-P1, Ac-P2, and Ac-P3, in which IC50s were 0.75 mg/mL, 0.78 mg/mL, and 0.81 mg/mL, respectively. Kinetic analysis demonstrated that all 4N-acetyl-pentapeptides were mixed-type tyrosinase inhibitors. Furthermore, 0.1 mg/mL of Ac-P4 exhibited significant melanogenesis inhibition on B16F10 melanoma cells and was more effective than kojic acid. The melanogenesis inhibition of Ac-P4 was dose-dependent and did not induce any cytotoxicity on B16F10 melanoma cells.We investigated the kinetics of 4 The IC50 value was determined using regression of a constructing dose-response curve at which 50% target activity is lost.In a 96-well plate, 20  N-acetyl-pentapeptides  in the inhibition of tyrosinase (20 μL of 212.65 μg/mL) were determined using various concentrations of l-DOPA  as substrates. The reaction mixture was measured using an ELISA reader at 475 nm for 10 min. All experiments were conducted in triplicate. The inhibition mechanism was assessed using Lineweaver-Burk plots, and the inhibition constants KIS and KI were obtained from second plots of the apparent 1/Vmax and apparent Km/Vmax against the inhibitor concentration, respectively, as described by Liao et al.  were linearly fitted. The KI and KIS of Ac-P1 to Ac-P4 were determined as 0.276, 1.278; 0.281, 2.003; 0.276, 1,685; and 0.053, 0.517   . The inhcontrol) . The inhtenuated . Howevertenuated . A combi N-acetyl-pentapeptides can not only inhibit mushroom tyrosinase activity but also exhibit potent melanogenesis inhibition on B16F10 cells. It has been demonstrated that numerous N-acetyl-peptides exhibit potent biological activity, such as being chemoattractants or exerting an antifibrosis effect by binding to surface receptors [α-MSH-) induced melanogenesis. The other melanogenesis inhibition mechanism of Ac-P4 was through directly inhibiting intracellular tyrosinase activity .This study suggests thateceptors , 44. It It has been reported that aloesin and hesperidin can not only effectively inhibit tyrosinase activity but also inhibit melanin formation on B16F10 and on normal human melanocytes , 46. FurN-acetyl-pentapeptides exhibited potent inhibitory effects on mushroom tyrosinase, and the inhibition kinetics occurred through the same mechanism. Furthermore, melanogenesis inhibitory assays were performed using B16F10 cells. The results indicated that Ac-P4 might serve as a new potent depigmentation agent in cosmetics or food industries.In this study, 4α-MSH-induced intracellular tyrosinase activity in a dose-dependent manner. 1 mg/ml Ac-P4 significantly inhibited intracellular tyrosinase activity by 57.9% (P<0.05).Ac-P4 inhibited


Knowledge about the Holocene evolution of the Greenland ice sheet (GrIS) is important to put the recent observations of ice loss into a longer-term perspective. In this study, we use six new threshold lake records supplemented with two existing lake records to reconstruct the Holocene ice marginal fluctuations of the Qassimiut lobe (QL) – one of the most dynamic parts of the GrIS in South Greenland. Times when the ice margin was close to present extent are characterized by clastic input from the glacier meltwater, whereas periods when the ice margin was behind its present day extent comprise organic-rich sediments. We find that the overall pattern suggests that the central part of the ice lobe in low-lying areas experienced the most prolonged ice retreat from ~9–0.4 cal. ka BP, whereas the more distal parts of the ice lobe at higher elevation re-advanced and remained close to the present extent during the Neoglacial between ~4.4 and 1.8 cal. ka BP. These results demonstrate that the QL was primarily driven by Holocene climate changes, but also emphasises the role of local topography on the ice marginal fluctuations. In South Greenland the 100 km wide Qassimiut lobe (QL) is presently one of the most climate sensitive parts of the Greenland ice sheet (GrIS)568911In this study, we use proglacial threshold lakes to constrain the ice marginal fluctuations of the QL. Threshold lakes are located adjacent to the ice margin and by analysing the sediment cores it is possible to determine when the glaciers are present within the lake catchment (i.e. similar ice extent as today) and periods where the ice margin was retracted and significantly smaller than todayKingigtoq lake is located 550 m a.s.l. near the Ukassorssuaq glacier c. 30 km from the present ice margin. It presently receives meltwater from outlet glaciers that terminate on land and it has to retreat an unknown distance (km’s) before the meltwater inflow ceases . The lakThe clay units are interpreted as representing periods with glacial meltwater input to the lake whereas the clay-gyttja units reflect periods where the ice margin is retracted behind the present extent. The two age models are considered reliable although the timing of the middle clay unit, unit 3, is somewhat uncertain. Based on the age model, the lake was deglaciated before 9.5 cal. ka BP. Meltwater inflow occurred until ~9 cal. ka BP and again from ~4.5 to 3 cal. ka BP and was interrupted by a period of clay-gyttja deposition although the timing is uncertain because of the poorly constrained age model at this interval . The mosSermilik lake is located 100 m a.s.l. in a small depression, 4 km from the present ice margin at the central part of the QL . The lakThe clayey-silt units 1 and 3 are interpreted as representing periods with glacial meltwater input to the lake, whereas gyttja unit 2 reflects a period where the ice margin was retracted behind the present extent. The thin sand layers in gyttja unit 2 (1205 and 1211) are interpreted as debris or grain flows formed close to the inlets of the lake where deltas have formed and not as evidence of presence of glacier ice in the catchment. According to the well-constrained age models , the lakKangerdluatsiaup tasia (lake) is located in the foreland of the central part of the QL adjacent to the Qaleragdlit Sermia (glacier) that terminates in a fjord connected to Ikersuaq Fjord . During The lower unit is interpreted as a soil covering the valley floor whereas the laminated clay was deposited when the valley was dammed by the Qaleragdlit Sermia and meltwater flowed into the valley and deposited foresets and topsets in the proximal part of the lake and bottomsets in the distal part. The bulk and macrofossil ages of the soil are not in chronological order and range from ~2.1–6.7 cal. ka BP . HoweverAkuliarusseq lake is located adjacent to the outlet glacier that terminates in the Ikersuaq fjord in South Greenland . It receThe clayey-silt units are interpreted as representing periods of glacial meltwater input to the lake, whereas the gyttja units reflect periods where the ice margin was retracted behind the present extent. The distinct sand layers in the gyttja in core 1203, which is located close to a small inlet in the most distal part of the lake, are interpreted as sediment debris flow deposits and not as evidence of presence of glacier ice in the catchment. Based on visual inspection and the sediment proxies, there is good correlation between the upper and the lower clayey-silt unit in the two cores. Deglaciation of the lake basin is not recorded in the cores because the core bottoms out in gyttja with a basal age of ~5.5 cal. ka BP . MeltwatStoresø and Rundesø (lakes) are located 30 km north of Narsarsuaq and were overridden when the Nordgletscher and Kiagtût sermiat (glaciers) merged and formed the Narsarsuaq moraines in the east-west orientated valley draining into NordbosøThe lower clay unit 1 is interpreted as representing a period of glacial meltwater input to the lake whereas the gyttja reflects a period where the ice margin was retracted behind the Narsarsuaq stage extent. In Storesø the uppermost bulk age in the clay is ~0.6 cal. ka BP and in Rundesø the transition between clay and gyttja is fixed between ~0.7 and 0.4 cal. ka BP . Accordi10Be surface exposure ages proximal to the LIA moraines, suggesting that the ice margin retreated within the late Holocene maximum extent by ~11.2–10.6 ka in South Greenland10Be ages record when the ice retreated from the location proximal to the LIA moraines and thus it should be regarded as a maximum age of when the ice margin could have retreated behind the present extent, assuming that the early Holocene ice retreat occurred without any pauses. Accordingly, The QL ice margin reached the present extent by ~11.2–10.6 kaThe initial deglaciation is recorded in two of the six new threshold lakes showing that the area was deglaciated by ~9.6 cal. ka BP. Furthermore, the records reveal that the QL ice margin retreated outside the lake catchment areas between ~9.3–9.0 cal. ka BP . This agFollowing the initial deglaciation, the ice margin retreated behind its current margin until the Neoglacial when the ice margin on the flanks of the QL lobe  and 50 km north of the QL (Kingigtoq) reached its present extent between ~4.4 and 3.0 cal. ka BP. The ice remained at its present position from ~3.0 to1.9 cal. ka BP with a short period of ice retreat  and 8b. 141617181920142224252610Be surface exposure ages suggests that Narsarsuaq moraine was formed and abandoned by 1.5 ka and the ice margin has been within or at its LIA extent since 1.3 kaAfter the Neoglacial readvance that culminated between ~3 and 1.8 cal. ka BP the QL retreated in all sectors and gyttja was deposited in the lakes until ~0.5–0.2 cal. ka BP. Subsequently, the ice margin re-advanced to its LIA position  and 8c. 242526272210Be surface exposure ages we have constrained the Holocene glacial history of the QL – the southernmost part of the GrIS. The locations of the threshold lakes that have been analysed from various places around the QL, in conjunction to the published data, provide a unique opportunity to assess both the spatial and temporal resolution of the ice marginal changes. At the central part of the QL the ice margin was retracted from the initial deglaciation, ~9.3–9.0 cal. ka BP until the LIA ~0.5–0.2 cal. ka BP. At the margins of the QL and adjacent to the ice lobe this long period of retracted ice margin position was interrupted by a Neoglacial readvance to the present ice extent between ~4.4 and 1.9 cal. ka BP. In all sectors of the QL, the ice margin re-advanced during the LIA between ~0.5 and 0.2 cal. ka BP. These results clearly show that multiple threshold lakes may be needed to obtain a comprehensive history of ice marginal fluctuations of a larger ice lobe and that a single threshold lake record may not be sufficient to infer ice marginal fluctuations for a large part of the ice sheet. However, in general, the results show that the southern margin of the GrIS responded to the recorded Holocene climate forcings and also emphasises the role of local topography on ice marginal changes.In summary, using six new threshold lake records combined with two existing threshold lake records, radiocarbon ages of reworked organic remains in LIA moraines, and published We analysed sediment cores from six threshold lakes located adjacent to the present ice margin distal to the QL in South Greenland to obtain a better understanding of its Holocene ice marginal fluctuations. We chose these threshold lakes because they only receive meltwater and clastic sediments (silt and clay) during periods of close-to-present ice extent, whereas non-glacial organic rich sediments (gyttja) are deposited during intervals with restricted ice extent in the catchment1113141618193 sample at 550 °C for 4 hoursThe lakes were identified using satellite and aerial photographs. In the field, an echo sounder with a built-in GPS was used to measure the lake depth and create a bathymetric map. The lakes were cored using a custom built piston corer capable of obtaining 4 m of sediments at a lake depth up to 50 m. One exception is the Kangerdluatsiaup tasia (lake) that was cored using the Uwitec coring equipment. After retrieval, the cores were kept in an upright position and drained before being packed and shipped back to the laboratory where they were stored at 2 °C. In the laboratory the cores were split in half, cleaned and a lithological description was made. An ITRAX core scanner was used to take high-resolution pictures and measure micro-XRF and magnetic susceptibility (MS) at every 2 mm and 0.5 mm, respectively. The XRF scans were made at Aarhus University core scanning facility with a molybdenum tube set at 30 kV and 30 mA with a dwell time of 30 seconds. Prior to analysis the sediment surfaces were carefully flattened and subsequently covered with thin (4 μm) ultralene film. A step size of 2 mm was selected to capture possible elemental variations in even small laminations. To illustrate the changes from gyttja to clastic sediments Ti, K or Si are often used as a measure of clastic input1114C ages therefore often deviate substantially from the true age of sediments. Further, as the origin of bulk carbon will depend on hydrology, catchment processes and morphology the bulk 14C age offset will most often deviate between different lakes3814C bulk age offset can be estimated using paired bulk and terrestrial macrofossil samples in order to correct unpaired bulk 14C ages14C Centre at Aarhus University (AAR), Lund University (LU) and Belfast University (UBA). The age models are based on radiocarbon ages of 40 macrofossils and 18 bulk sediment samples that have been converted into calendar years using Oxcal 4.2 and calibrated to calendar years using IntCal1340Samples for radiocarbon dating were sub-sampled with the primary objective to date clastic layers (meltwater) within the cores. We preferably sampled and identified terrestrial macrofossils in 1-cm intervals. When terrestrial macrofossils were absent, we used moss or bulk samples (humic acid) for radiocarbon dating. Using bulk sample to estimate the age of sediments can be problematic because bulk samples integrate carbon of both allochthonous and autochthonous origins. Consequently eroded soil carbon may for example in some cases constitute a major fraction of the bulk carbon. Hence bulk How to cite this article: Larsen, N. K. et al. Holocene ice marginal fluctuations of the Qassimiut lobe in South Greenland. Sci. Rep.6, 22362; doi: 10.1038/srep22362 (2016).