N-caffeoyltryptophan) by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2  better than Sirt1. Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 μM) and showed competitive inhibition against the peptide substrate (Ki = 5.3 μM). Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382) in p53, but not (K305). This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II ( Coffee is one of most popular beverages worldwide. SeveralTryptophan, caffeic acid, dichloromethane and other chemicals, were purchased from Sigma Chemical Co. . The Sirt1, 2 and 3 inhibition assays were performed using human Sirt1, 2 and 3 Direct Fluorescent Screening Assay kits .Coffea arabica) from four different coffee brands  to resolve possible variety issues for HPLC analyses. After several preparations of coffee samples, the 5g coffee was finely grinned and extracted for 5min with boiling 100mL water. Then, the samples were centrifuged (7000 rpm) and filtered using Millex syringe filter . The filtered samples were ready for High Performance Liquid Chromatography (HPLC) analysis.Coffee samples were prepared using the equal amounts of roasted coffee beans  for 0–5 min, a linear change from buffer A to buffer B (40% acetonitril) for 5–40 min, and buffer B for 10 min at the flow rate of 1 mL/min. The samples were injected by an auto-sampler into Alliance 2690 HPLC system , and were monitored by CoulArray electrochemical detector with four electrode channels . Using the HPLC method, 50 factions were collected at one min interval and each fraction was monitored using the electrochemical detector. The fractions were individually heat-dried and dissolved in 10% ethanol (0.2 mL).For HPLC fractionation, a 150 mm × 2.1 mm i.d., 4 μm, Nova-Pak C18  was used as the stationary phase to separate coffee samples. The samples were separated using a gradient condition; buffer A , according to the manufacturer’s manual. The kits provide a convenient fluorescence-based method for screening Sirt1/2/3 inhibitors. The assay was performed in a 96-well microplate. First, human recombinant Sirt1, 2 or 3 is incubated with potential inhibitors. Then, the fluorescent substrate and its co-substrate NADThe fraction with the highest activity was selected, heat-dried and dissolved in 10% ethanol. Then, the sample was injected again into HPLC and separated using the HPLC method used above. The peaks were separately isolated and screened again using the Sirt2 inhibition assay. The peak with the highest activity was saved for NMR analysis.1H and 13C spectra were acquired at ambient temperature on the JEOL BCX-400 NMR spectrometer operating 400 MHz for 1H and 100 MHz for 13C.For NMR experiments, the sample was prepared by dissolving javamide-II (20 mg) in d6-DMSO (0.75 mL). The synthesis of javamide-II was performed as previously described . Brieflyi value was determined using Lineweaver-Burk plot. Data points in all figures represent the mean ± SD (n = 3).Kinetic analysis of the Sirt2 inhibition was performed, and Kbinding).Molecular docking study was performed using an algorithm-based docking program ICM-pro . The proposed binding to the active pocket of the Sirt enzymes was determined as the best ranked scoring function, representing the conformational structures with the most favorable free binding energy (ΔG2.Neuroblastoma×glioma (NG108-15) cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum at 37°C in a humidified atmosphere of 5% CO™ Blot Module kit , acetylated-cortactin antibody  and acetylated-lysine, acetylated-histoneH4, acetylated-histoneH4(K56), acetylated-p53, and alpha-tubulin antibodies .For the Western blot, the extracts were prepared using NG108-15 cells treated with various concentrations of javamide-II  for 18 hr. The amounts of protein in the extracts were determined using Bio-Rad protein assay kit  and an equal amount protein was used per samples for the blot. The Western blots for total acetylated lysine and alpha-tubulin were performed using Novex 4–12% Tris-Glycine Mini Gel and XCell II® acetylated-alpha-tubulin Sandwich ELISA kit  as described in the manufacturers' manuals.The quantification of histone H3 acetylation was performed using a histone H3 total acetylation detection kit . The extracts were prepared using NG-108 cells treated with various concentrations of javamide-II  for 24 hr, according to the kit’s manual. The extracts were used immediately or stored at–80°C for later use. The equal amounts of the protein determined by Bio-Rad Protein Assay Kit  were used for the determination of histone H3 acetylation. Similarly, the measurement of p53 and α-tubulin acetylation was also performed using p53 acetyl K382 Human ELISA kit and p53 Acetyl K305 Human ELISA Kit , and PathScanTreatment effects on the parameters measured were compared by analyzing the means for differences using One-way ANOVA with Holm-Sidak method. Differences were considered to be significant when p< 0.05. Data points represent the mean ± SD of three or more samples.In this study, coffee samples were prepared using roasted coffee from four different coffee brands to resolve possible variety issues for the high-performance liquid chromatography (HPLC) analyses. For an optimal HPLC fractionation, a new HPLC method was developed and utilized to fractionate the coffee sample as described in “Materials and Methods”. Several different buffer conditions were tested to improve the resolution of the coffee compounds with a reliable detection, and then a gradient condition was selected for optimizing separation, detection and sample collection as described in "Materials and Methods". The chromatograms of the coffee sample were shown in The fractions were individually heat-dried, and then dissolved in the buffer A for Sirt1/2 inhibition assay as described in "Materials and Methods". As shown in 1H and 13C spectra were acquired at ambient temperature on the JEOL BCX-400 NMR spectrometer operating 400 MHz for 1H and 100 MHz for 13C. Chemical shifts were referenced to DMSO . The NMR data were following: 1H NMR  7.33  15.6 Hz, H-7), 7.20 , 7.08  7.3 Hz, H-16), 6.68  8.2, 1.4 Hz, H-5), 6.41  15.6 Hz, H-8), 7.58  8.2 Hz, H-18), 6.99  7.3 Hz, H-17), 6.81  8.7 Hz, H-4), 7.05 , 7.35  7.8 Hz, H-15), 4.71  6.0, 6.9 Hz, H-10), 2.94  7.3 Hz, H-11), 8.19  5.5 Hz, H-a), 10.79 ; 13C NMR  174.7 , 166.7 , 147.5 , 145.7 , 141.5 , 136.4 , 127.4 , 126.9 , 122.8 , 121.1 , 120.2 , 118.9 , 119.5 , 118.4 , 117.0 , 115.0 , 112.1 , 111.5 , 58.7 , 26.2 . Based on the NMR data, the structure of the HPLC purified compound with Sirt2 inhibiting activity was determined as being (E)-acryloyl)tryptophan (N-caffeoyltryptophan), and the compound was named as javamide-II  in d6-DMSO (0.75 mL). amide-II .To further demonstrate that javamide-II is the compound able to inhibit Sirt1/2, the synthesis of javamide-II was performed as previously described . BrieflyThe specificity of inhibitors is often regarded as a very critical factor in determining which inhibitor may be appropriate in treating disease conditions. In fact, Sirt proteins are found in specific cellular compartment; for instance, Sirt1, 6 and 7 are found in the nucleus, Sirt2, and often Sirt1 are found in the cytoplasm, and Sirt3, 4 and 5 are found in the mitochondria in human cells. Since javamide-II showed strong Sirt2 inhibition activity, the efficacy of the amide was also investigated related to srituin1 (nucleus) and 3 (Mitochondria) was investigated. As shown in 50 = 8.7 μM) stronger than Sirt1 (IC50 = 34 μM) (50 = 4.3 μM) and EX527 [50 = 5.9 μM) performed in our laboratory. Therefore, kinetic experiments were performed to determine the Ki values of javamide-II against Sirt2. As shown in +, and the Ki value was approximately 9.8 μM. Additionally, the amide showed competitive inhibition against the peptide substrate with the Ki value of 5.3 μM (Javamide-II inhibited Sirt2 (IC= 34 μM) , which ind EX527  . As shown in Because javamide-II was identified as a better inhibitor for Sirt2 than Sirt1 and because histone H3 is a well-known substrate for Sirt2 , we inveLike histone H3, α-tubulin is a known substrate for Sirt2. Therefore, we did investigate the potential effects of javamide-II on the acetylation of α-tubulin in NG108-15 cells. However, the α-tubulin acetylation was not increased significantly with the treatment of javamide-II in the NG108-15 cells (Data not shown here).Another famous substrate for Sirt1/2 is p53 which is a potent transcription factor involved in cell cycle arrest and apoptosis via activating numerous genes, and its growth suppressive and pro-apoptotic activity could be utilized in fighting cancer cells . TherefoFor validating the ELISA data, we also performed Western blots using the extracts prepared using the NG108-15 cells treated with javamide-II . As shown in +-binding domain (Sirt1–7) [in vitro inhibition assays performed in this study , although the amide can inhibit Sirt2 to some extent better than Sirt1 in vitro assays. Therefore, there is a great possibility that the amide could be used as a parent/lead molecule which could lead to yielding Sirt inhibitors with great efficacy and selectivity through structural optimization. Currently, in our laboratory, several analogues of javamide-II are being synthesized for investigating their activity and selectivity related to Sirt inhibition, which may help find greater Sirt inhibitors with better selectivity.The Sirt consists of a family of seven proteins with highly conserved NADSirt1–7) , 34–43. Sirt1–7) –44, theySirt1–7) . Due to Sirt1–7) . Our andSirt1–7) . Consequis study . Related


N-methyl-D-aspartate (NMDA) receptor phosphorylation at the NR2B Y1472 subunit, influencing receptor internalization at the synaptic plasma membrane. However, it is unclear whether this phosphorylation is upstream to and/or necessary for ERK1/2 activation, which is known to be crucial for synaptic plasticity and memory consolidation. Here, we tested the hypothesis that tyrosine phosphorylation of NR2B at Y1472 is correlated with ERK1/2 activation by dopamine and necessary for it as well. We find that dopamine receptor D1, but not D2, activates ERK1/2 and leads to NR2BY1472 phosphorylation in the mature hippocampus and cortex. Moreover, our results indicate that NR2B Y1472 phosphorylation is necessary for ERK1/2 activation. Importantly, application of dopamine or the D1 receptor agonist SKF38393 to hippocampal slices from NR2B F1472 mutant mice did not result in ERK1/2 activation, suggesting this site is not only correlated with ERK1/2 activation by dopamine stimulation, but also necessary for it. In addition, NR2B F1472 mice show impairment in learning of attenuation of taste neophobia but not associative taste learning. Our study shows that the dopaminergic and glutamatergic transmission converge on the NMDA receptor itself, at the Y1472 site of the NR2B subunit, and that this convergence is essential for ERK1/2 activation in the mature brain and for processing new sensory information in the cortex.Understanding the heterosynaptic interaction between glutamatergic and neuromodulatory synapses is highly important for revealing brain function in health and disease. For instance, the interaction between dopamine and glutamate neurotransmission is vital for memory and synaptic plasticity consolidation, and it is known to converge on extracellular signal-regulated kinase (ERK)-MAPK signaling in neurons. Previous studies suggest that dopamine induces  N-methyl-D-aspartate (NMDA) receptor and metabotropic dopamine receptors. Interaction between glutamatergic and dopaminergic synapses in the mature hippocampus is important for modulation of long-term potentiation (LTP) and long-term memory in the CA1 region of the hippocampus , and C57BL/6 mice (20–22 g) were procured from Harlan . NR2B F1472 mutant mice lacking the Y1472 phosphorylation site by substituting Tyr-1472 (Y) with Phe-1472 (F) were given to us by Prof. Nakazawa and Prof. Yamamoto . AnimalsN,N,N-trimethylethanaminium chloride); D(-)-APV , dopamine receptor D1 agonist R-(+)-SKF38393 ; D1 antagonist R-(+)-SCH-23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (40 μM); D2 agonist (-)-quinpirole hydrochloride ; and D2 antagonist eticlopride (3-chloro-5-ethyl-N-{[(2S)-1-ethylpyrrolidin-2-yl] methyl}-6-hydroxy-2-methoxybenzamide (60 μM), were purchased from Sigma Aldrich. Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolopyrimidine (10 μM) was purchased from Calbiochem. All compounds were dissolved in artificial cerebro-spinal fluid (ACSF) on the day of the experiment.For pharmacological manipulations: dopamine  was co-applied with the antioxidant ascorbic acid (1 mM); carbachol 2-[(aminocarbonyl)oxy]-2, 5% CO2; ACSF, comprising 124 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 26 mM NaHCO3, 10 mM D-glucose, and 2.4 mM CaCl2), and after approximately 2 min, both hippocampi or insular cortex were dissected out in a plate filled with cold (4°C) ACSF on ice. The hippocampi or insular cortex were then put on a cooled stand of a McIlwain tissue chopper TC752 , cut into 400 μm slices, and then put back into a chamber filled with carboxygenated cold (4°C) ACSF  carboxygenated (95% O°C) ACSF , 2007.n = 1).The slices were kept in six holding chambers designed by Scientific Systems Design Company  heated to 32°C and kept for 3 h before any pharmacological intervention. Each chamber contained four slices. Within each experiment, two chambers were used as a positive control for the quality of the slices. The remaining four chambers were the experiment chambers. 10 min. after the pharmacological manipulation, slices were removed from the pharmacological chamber and snap-frozen on dry ice. After freezing, the slices were homogenized in 200 μl of SDS sample buffer, comprising: 1 ml glycerol, 2 ml of 10% SDS, 1.2 ml of 0.5% Tris-HCl (pH 6.8), 4.8 ml of DDW, and 0.5 ml of β-mercaptoethanol. Each specimen comprised two hippocampal/insular cortex slices combined  and administered intraperitoneally (5 mg/kg) to C57BL/6 and NR2B F1472 mice. Mice were sacrificed 15 min later and tissue was collected. For incidental taste learning experiment, SCH23390 was dissolved in saline (0.9%) and administered intraperitoneally  to C57BL/6 mice 30 min prior to the novel taste exposure.Fifteen minutes following SKF38393 injection  or 20 min following novel taste exposure, animals were decapitated and the hippocampus and insular cortex were collected and frozen in liquid nitrogen and stored in -80°C. The obtained tissues were homogenized in a glass-teflon homogenizer in a lysis buffer containing 10 mM HEPES, pH 7.4, 2 mM EDTA, 2 mM EGTA, 0.5 mM DTT, 1% phosphate inhibitor mixture (Sigma), and 1% protease inhibitor mixture (Sigma). Protein quantification was made with BCA Protein Assay Kit . Appropriate volumes of 2XSDS sample buffer  were added to the homogenates, and samples were boiled for 5 min and stored at -20°C.Samples in SDS sample buffer were subjected to SDS-PAGE (7.5–10%) and Western blot analysis. Lanes were loaded with equal amount of protein. Following transfer to a nitrocellulose membrane , bands were visualized with Ponceau staining (Bio-Rad). Membranes were blocked in 3–5% BSA (depending on the primary antibody) for 1 h at room temperature, before being incubated overnight at 4°C with the primary antibodies: p44/42 MAP kinase (1:1000) and ; Src ; NR2B antibody (1:500), β-actin ; Phospho-(Tyr1472)-NR2B ; and Phospho-(Tyr418)-Src .Following three washing steps with Tris-buffered saline  plus 0.1% Tween 20 (TBST), membranes were incubated for 1 h at room temperature with secondary HRP-linked antibodies: goat-anti-Rabbit (IgG) HRP conjugated; Goat anti-mouse (IgG) HRP conjugated; and rabbit anti-goat (IgG) HRP conjugated . Immunodetection was accomplished with the Enhanced Chemiluminescence EZ ECL kit . Quantification of immunoblots was performed with a CCD camera and Quantity One software (Bio-Rad). Each sample was measured relative to the background. Phosphorylation levels were calculated as the ratio of phosphorylated protein and total amount of protein.CTA acquisition and extinction. Conditioned taste aversion was performed as described previously . Moreover, co-application of the D2 agonist quinpirole with D1 antagonist SCH23390 did not result in ERK1/2 activation in either the hippocampus -APV  inhibited ERK1/2 activation in hippocampal slices . These results indicate that indeed the convergence of the dopaminergic and glutamatergic inputs converge on the NMDA receptor. Following the results above , we investigated whether the convergence of the dopamine and glutamate signals on the NMDA receptor is mediated via the phosphorylation of the NR2B Y1472 residue. Indeed, dopamine application increased NR2B Y1472 phosphorylation compared to control in hippocampal slices . We next examined whether this NR2B Y1472 phosphorylation was NMDA receptor-dependent. Interestingly, blocking the NMDA receptor with 40 μM APV did not affect NR2B Y1472 phosphorylation induced by dopamine , indicating that NR2B Y1472 phosphorylation is NMDA receptor independent.Similarly to p < 0.05, n = 12; Figure 1D). However, application of APV did not inhibit Cch-dependent ERK1/2 . These results suggest segregation between dopamine and acetylcholine effects in the mature brain.As known from previous studies, other neuromodulators such as acetylcholine can increase ERK1/2 activation in various brain areas . In ordep < 0.05, n = 9, insular cortex: 1.28 ± 0.06, p < 0.05, n = 9; Figures p < 0.05 vs. SKF38393, n = 9; insular cortex: 1.04 ± 0.09, p < 0.05 vs. SKF38393; Figures Following our observation that dopamine induces ERK1/2 activation and NR2B Y1472 phosphorylation, we tested whether NR2B Y1472 phosphorylation was also D1 receptor-selective. We treated hippocampal and insular cortex slices with D1 or D2 agonists separately or concomitantly with their antagonists. Immunoblot analysis showed that indeed the D1 but not D2 receptor agonists mediate NR2B Y1472 phosphorylation in both hippocampus and insular cortex  while the SFK inhibitor PP2 (10 μM) blocked phosphorylation on Y418 . Moreover, dopamine increased phosphorylation of ERK1/2  and NR2B Y1472 . Inhibition of the SFKs with PP2 blocked dopamine-dependent tyrosine phosphorylation of NR2B Y1472  and, more importantly, inhibited ERK1/2-increased activation at the same time . In addition, we observed correlations between ERK1/2 activation and phosphorylation of NR2B Y1472 , and between SFK activation and NR2B Y1472 phosphorylation .Src family kinases (SFKs) modulate the NMDA receptor function and are important for different aspects of the receptor properties including for trafficking, mainly through phosphorylation of the NR2B subunit at Y1472 . We therp < 0.001, n = 6; Figure 3A) and increased phosphorylation of ERK1/2  and NR2B Y1472 . Co-application with the inhibitor PP2 blocked the activation of SFKs  and phosphorylation of ERK1/2  and NR2B Y1472 . These data suggest that dopamine induces ERK1/2 activation via NR2B Y1472 phosphorylation by the SFKs in mice as in rats.In order to test the general significance of these results to other species and to use transgenic mice to demonstrate causality, we repeated the experiment described above in C57BL/6 wild-type mice. As we had seen in the rat, dopamine increased activation of the SFKs in the hippocampus of mice . There was a significant effect of the genotype . Moreover, there was a significant interaction between treatment × genotype . Similar results were found with the D1 receptor agonist SKF38393 which activated ERK1/2 in wild-type mice . There was a significant effect of the genotype  and also interaction between treatment × genotype . Following our results above that muscarinic receptor-dependent ERK1/2 activation is NMDA receptor independent , we hypothesized that carbachol will induce ERK1/2 activation in both wild-type and NR2B F1472 hippocampal slices. Indeed, application of Cch induced ERK1/2 activation in the NR2B F1472 mutant mice similar to that in the wild-type ones . These results indicate the pathway connecting specifically dopamine, NR2B Y1472 phosphorylation, and ERK1/2 activation in the mature brain.In light of the above results, we hypothesized that NR2B Y1472 phosphorylation may be upstream and essential for ERK1/2 activation by dopamine. In order to test this hypothesis, we tested hippocampal slices from non-phosphorylatable NR2B F1472 mutant mice and their wild-type littermates . Two-wayin vivo and its impact on cortical-dependent novelty learning NR2B F1472 mutant mice and wild-type littermates were injected with either saline or low concentration of SKF38393 (5 mg/kg) and both hippocampus and insular cortex tissues were collected 15 min post-injection. Western blotting analysis showed that indeed in vivo as in vitro, D1 receptor stimulation induces NR2B Y1472 phosphorylation in wild-type hippocampus  and insular cortex . However, two-way ANOVA followed by post hoc analysis showed that ERK1/2 activation following the D1 agonist treatment was detected in the wild-type mice but not in the NR2B F1472 mice in both brain areas . There was also a significant effect of the genotype  and interaction of treatment × genotype in the hippocampus ; Figure 4A) and in the insular cortex  for the genotype . These results demonstrate the necessary role of NR2B Y1472 phosphorylation in ERK1/2 activation by dopamine in the mature brain.In order to evaluate the physiological effect of the pathway described above in vitro and in vivo pharmacological results, we set to examine whether the dopamine-induced NR2B Y1472 phosphorylation was important for consolidation of novel sensory information. It is known that novel taste learning induces both ERK1/2 and NR2B Y1472 phosphorylation in the gustatory cortex which resides within the insular cortex . There was also significant effect of the genotype  and interaction of treatment × genotype . In addition and as was reported before, novel taste induced NR2B Y1472 phosphorylation in the wild-type but not in the NR2B F1472 mice (data not shown). Moreover, novel taste memory is dopamine D1 receptor dependent as was shown previously . There was significant effect of the testing days, indicating normal attenuation of neophobia . There were no differences in the total drinking volume during the test days  and second test day .The results above suggested that dopamine plays a key role in novel taste memory. We thus hypothesized that impairment of dopamine-dependent ERK1/2 activation in the NR2B F1472 mutant mice would lead to a behavioral deficit in learning of novel taste information. C57BL/6 wild-type and NR2B F1472 mutant mice were subjected to two cortical-dependent learning tasks: attenuation of neophobia of novel taste learning and CTA. NR2B F1472 mutant mice showed lower preference to the novel taste saccharin than the wild-type mice at the first test day  and also a significant effect of the test days . There were no differences in total volume drinking during the test days . The NR2B F1472 mice showed high aversion index at the first test day similar to the wild-type littermates . Repeated-measures ANOVA showed a significant effect of the testing days  indicating normal extinction for both groups. There were no differences in the total drinking volume during the test days  and the insular cortex (shown in vivo). This mechanism was found also in other brain areas. For example, in striatal and pre-frontal cortex neurons, activation of the D1 receptors increases tyrosine phosphorylation of NR2A and NR2B, and produces rapid alteration in the distribution of the NR1, NR2A, and NR2B subunits of the NMDA receptor, with accumulation of these proteins in synaptosomal membranes  but not in associative learning of CTA. Although our results indicate impaired cortical-dependent learning of the NR2B F1472 mice, it was shown in previous studies that these mice are not impaired in hippocampal-dependent learning , suggestIn addition to dopaminergic and glutamatergic signaling, muscarinic transmission also plays an important role in ERK1/2 activation and consolidation of memory, as well as synaptic plasticity. For example, the muscarinic agonist carbachol (Cch) induces prolonged dose-dependent activation of ERK1/2 in the CA1 pyramidal neurons of mice . In addiIn conclusion, we have demonstrated that dopamine converges on the NMDA receptor and leads to phosphorylation of NR2B Y1472 which is both correlated with and necessary for ERK1/2 activation in the mature hippocampus and cortex. Malfunction of dopamine or NMDA receptors causes various types of mental disorders, such as schizophrenia. Our results, taken together with recent findings in schizophrenia patients , may impThe 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.


Recent paleoclimate reconstructions have challenged the traditional view that Northern Hemisphere insolation and associated feedbacks drove synchronous global climate and ice-sheet volume during the last glacial cycle. Here we focus on the response of the Patagonian Ice Sheet, and demonstrate that its maximum expansion culminated at 28,400 ± 500 years before present (28.4 ± 0.5 ka), more than 5,000 years before the minima in 65°N summer insolation and the formally-defined Last Glacial Maximum (LGM) at 21,000 ± 2,000 years before present. To investigate the potential drivers of this early LGM (eLGM), we simulate the effects of orbital changes using a suite of climate models incorporating prescribed and evolving sea-ice anomalies. Our analyses suggest that Antarctic sea-ice expansion at 28.5 ka altered the location and intensity of the Southern Hemisphere storm track, triggering regional cooling over Patagonia of 5°C that extends across the wider mid-southern latitudes. In contrast, at the LGM, continued sea-ice expansion reduced regional temperature and precipitation further, effectively starving the ice sheet and resulting in reduced glacial expansion. Our findings highlight the dominant role that orbital changes can play in driving Southern Hemisphere glacial climate via the sensitivity of mid-latitude regions to changes in Antarctic sea-ice extent. The Last Glacial Maximum (LGM) – traditionally defined from sea level records as having taken place between 23,000 and 19,000 calendar years ago – was apparently driven by insolation changes at 65°N1The timing of glacial advance is complicated in the mid-latitudes of the Southern Hemisphere (SH), where ice fields and glaciers appear to have reached their maximum extent prior to the global LGM, suggesting sensitivity to forcing mechanisms other than Northern Hemisphere insolation3456414C calibrationHere we focus on the timing of maximum ice-sheet expansion in Patagonia, southernmost South America, and across the wider south Pacific region, where paleo-environmental, radiocarbon and relative sea level constraints strongly support a strong eLGM signal4612Here we recast previously published cosmogenic isotope studies from a series of glacial advances of the Patagonian Ice Sheet that spans more than ten degrees of latitude111718192021We apply the production rate derived in New Zealand42 and obliquity, we use a coupled ocean-atmosphere-land-sea-ice climate modelTo investigate the mechanisms that drove this apparent early eLGM, we simulate the effects of orbital changes using a suite of climate models incorporating prescribed and evolving sea-ice anomalies. Previous modeling studies have argued that sea ice plays a critical role in atmospheric circulation change in the Southern Hemisphere, and can significantly alter the location and intensity of the Southern Hemisphere jet stream7We simulate a suite of key time slices: (i) the Last Glacial Maximum, centered on 21 ka, (ii) the obliquity minimum centered on 28.5 ka, (iii) the obliquity maximum centered on 49 ka, and (iv) the pre-industrial climate based on year 1780 parameters as the control simulation . The 50-°S to beyond 60°S and is centered on 53°S, covering a broad longitudinal swathe of the region (120 to 30°W). This temperature reduction is accompanied by a marked increase in wind strength, implying a notable shift in Southern Hemisphere atmospheric circulation between maximum and minimum obliquity. Our simulations suggest that during the austral summer the westerly winds intensify and broaden hemisphere wide, only reducing marginally over New Zealand . Sea surface salinities increase south of Drake Passage, and decrease to the north of this region . This paOur simulations suggest that low obliquity triggered marked intensification of atmospheric circulation in the SH, resulting in a significant increase in the Southern Ocean barotropic streamfunction between 49 ka and 28.5 ka . AlthougThe mechanisms behind the pronounced atmospheric cooling driven by expansion of Antarctic sea ice and orbital parameters can be explored through an independent suite of numerical experiments performed with the atmospheric component of the NCAR CCSM3 model (See methods)2 when compared to 21 ka , allowing us to test for the synchroneity of maximum ice extent in South America as recorded by the outermost moraines at each of the sites in order to compare with potential forcing mechanisms.Here we apply the production rate derived in New Zealand11192021223911171819202111To gain insights into the potential drivers of the eLGM in Patagonia and across the broader Southern Hemisphere we examine the response of the atmosphere, ocean and sea ice to changes in orbital forcing parameters and greenhouse gas concentrations using a fully coupled climate model CSIRO Mk3L. We also examined sensitivity experiments using NCAR CAM3. The climate modeling experiments presented here do not provide a full reconstruction of these past climates; rather they are designed as sensitivity experiments to assess the mechanism of obliquity forcing upon climate as proposed in this paper. The full details of these simulations are outlined in detail in the How to cite this article: Fogwill, C. J. et al. Obliquity Control On Southern Hemisphere Climate During The Last Glacial. Sci. Rep.5, 11673; doi: 10.1038/srep11673 (2015).


To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K This protein is highly expressed in mammalian brain tissue, including hippocampus, cerebral cortex, striatum, and thalamus  ethanesulfonic acid (HEPES). Cells were grown in 100 × 20 mm dishes and transfected when at 85% confluence using Lipofectamine (7.334 μg DNA in 55.1 μl Lipofectamine per dish) . To prepare total lysates for western blot experiments, we transfected cells with either wild type or mutant Kv2.1 DNA  and an appropriate amount of control empty vector, pcDNA3. For immunoprecipitation experiments, total DNA for transfection contained 10% wild type Kv2.1 or 30% mutant Kv2.1 plasmid DNAs (because of the low expression of mutant Kv2.1 proteins in CHO cells) as well as 15% of p38 or Src plasmid DNA plus appropriate amounts of control empty vector, pcDNA3. After 24 h, transfected CHO cells were utilized for electrophysiological recordings or briefly rinsed twice with ice-cold phosphate-buffered saline (PBS) and harvested for protein biochemistry. Cells were lysed in either Cell Extraction Buffer  or NP40 Cell Lysis Buffer (for immunoprecipitation). Both were supplemented with complete protease inhibitor cocktail tablet  and freshly made phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions. Cellular debris in samples were removed by centrifugation at 10,000 × g for 10 min at 4°C. Protein samples were stored at -20°C and concentrations were measured using BCA protein assay kit.Equal amounts (0.7–1 mg) of total protein from cell extracts were pre-cleared by adding 50 μl of resuspended volume of protein A/G plus-agarose  for 1 h at 4°C. After centrifugation, supernatants were mixed with mouse anti-Kv2.1 monoclonal antibody at 4°C for 3 h with gentle agitation before adding 90 μl of resuspended volume of beads overnight. The immune complex of protein-bound beads was washed and used in gel electrophoresis. Either beads or equal amount of total protein samples were incubated with 2× reducing sample buffer and heated at 100°C for 5 min before loading onto 8% gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was run using the Mini Protein 3 System  and separated proteins were transferred onto 0.2 μm nitrocellulose membranes. The membranes were blocked with 1% BSA in PBS containing 0.05% Tween 20 (PBST) at room temperature for 1 h. In most experiments in this study, we took the advantage of Odyssey Infrared Imaging System  to detect two specific antigens of Kv2.1 simultaneously. To do so, two primary antibodies derived, respectively, from mouse and rabbit (mouse anti-Kv2.1 plus rabbit anti-phosphoKv2.1(S800) antibodies or mouse anti-phosphotyrosine plus rabbit anti-Kv2.1 antibodies) were diluted together in PBST and exposed to the blots. Later, after washing (3×) in PBST, membranes were incubated simultaneously with two Li-Cor IRDye-conjugated secondary antibodies raised specifically against mouse or rabbit. Either mouse or rabbit secondary antibody was labeled with IRDye 680LT (for 680 nm) or 800CW (for 780 nm). Different fluorescent signals from specific proteins were scanned and discriminated using two independent infrared detection channels at 685 and 785 nm excitation wavelengths and were quantified by the imaging system. Normalization procedures and other calculations, as well as statistical analyses are detailed in the figure legends.2, 1 CaCl2 x 2H2O, 10 HEPES; pH adjusted to 7.2 with concentrated KOH; 2.2 ATP and 0.33 GTP were added and the osmolarity was adjusted to 280 mOsm with sucrose. The extracellular solution contained (in mM): 115 NaCl, 2.5 KCl, 2.0 MgCl2, 10 HEPES, 10 D-glucose; pH adjusted to 7.2. Measurements were obtained with an Axopatch-1D amplifier and pClamp software , using 2–3 MΩ recording electrodes. Electrodes were pulled from 1.5 mm borosilicate glass  with a model P-97 mechanical pipette puller . Series resistance was partially compensated (80%) in all cases. Currents were filtered at 2 kHz and digitized at 10 kHz with a Digidata 1440A (Molecular Devices). K+ currents were evoked with incremental 10 mV voltage steps to +80 mV from a holding potential of –80 mV. To determine current density values, steady-state current amplitudes were measured 180 msec after the initiation of the +10 mV step and normalized to cell capacitance.Current recordings were performed at room temperature on eGFP-positive co-transfected CHO cells using the whole-cell patch clamp configuration technique as described previously . The intNormalized data are expressed as the mean ± S.E.M. and the level of significance was analyzed by one sample or pair-wise comparisons, or by one-way analysis of variance (ANOVA) with post hoc comparisons, as detailed in each figure legend.To investigate the regulation of the phosphorylation events necessary for Kv2.1-facilitated apoptotic cascades, we employed the Chinese hamster ovary (CHO) cell expression system that we have previously characterized extensively , 27. ThiDuring apoptosis, Kv2.1 is a substrate of p38 and Src kinases; p38 MAPK mediates phosphorylation of Kv2.1 at residue S800 , while STo confirm that p38 MAPK was indeed responsible for Src-stimulated phosphorylation of S800, we pre-treated cells with SB 239063 (5 μM), a selective p38 MAPK inhibitor . We obseNext, we investigated the reverse situation, namely, whether changes in the S800 site would affect Src-mediated phosphorylation of Kv2.1. Although we have not been able to generate successfully a phospho-specific antibody to residue Y124 , it has A recent study showed that a cysteine residue (C73) located in the N-terminus of Kv2.1 is involved in the process of neuronal apoptosis associated with oxidation of the channel. Indeed, mutating C73 to an alanine was sufficient to induce cytoprotection . Further+ current surges in transfected CHO cells. Moreover, mutation of either the Y124 or S800 into non-phosphorylatable residues was sufficient to prevent Kv2.1 current enhancement following oxidant treatment. Moreover, using an alkylatable, extracellular cysteine-containing mutant channel (I379C), we were also able to demonstrate that a serine to alanine mutation at residue S800 prevented the membrane insertion of Kv2.1 following injury [+ current surge. CHO cells expressing either wild type Kv2.1 or either Kv2.1(C73A) or Kv2.1(C710A) were exposed to a 10 minute treatment with the thiol oxidizing agent 2,2’-dithiodipyridine , a treatment that results in a pronounced increase in K+ currents approximately 3 hours post-treatment [+ currents following DTDP exposure  following hypoxia . In addiFyn is a Src family tyrosine kinase that has been shown to physically associate with Kv2.1 protein in mouse Schwann cells . MoreoveIn conclusion, we have demonstrated that Src protein kinase strongly promotes serine phosphorylation of Kv2.1 channel at p38-targeted residue S800 via a process independent of enhanced cellular p38 MAPK activity. Our cysteine mutant experiments also suggest that a single cysteine mutation, either at C73 or C710 of the Kv2.1 channel, significantly alters the state of S800 phosphorylation. As such, the difference in S800 phosphorylation present in these mutants could account, at least in part, for the previously described anti-apoptotic properties of the Kv2.1(C73A) mutant observed in transfected cells following oxidative stress , 30.S1 FigA, CHO cells were co-transfected with 10% plasmid DNA of wild type  expression . The data are expressed as mean ± SEM from three independent experiments; all mutant Kv2.1 protein expression is significantly lower than Kv2.1(WT) protein in transfected CHO cells ; one sample, two-tailed t test).(PDF)Click here for additional data file.


Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact interface between the ribosomal RNA and mRNA. The binding site of amicoumacin A is formed by universally conserved nucleotides of rRNA. In this work, we showed that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We determined the structure of the amicoumacin A complex with yeast ribosomes at a resolution of 3.1  Å. Toxicity measurement demonstrated that human cancer cell lines are more susceptible to the inhibition by this compound as compared to non-cancerous ones. This might be used as a starting point to develop amicoumacin A derivatives with clinical value. Development of small molecule translation inhibitors is needed for progress in antibacterial as well as anticancer therapy1343Thermus thermophilus ribosome as well as biochemical and genetic analysis of bacterial translation inhibition has been reported. It appeared that amicoumacin A binds a conserved site between the E-site mRNA codon and 16S rRNA. The antibiotic contacts only the RNA backbone and nucleobases of rRNA. A number of antibiotics such as pactamycin7In a recent studyThe crystal structure of bacterial ribosome in complex with amicoumacin revealed that antibiotic interacts with universally conserved nucleotides of the small subunit rRNA3in vitro translation and mRNA transfection approaches as well as a toe-printing technique to show that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We also compared human cancerous and non-cancerous cell lines for their susceptibility for protein synthesis inhibition by the antibiotic. The structure of the amicoumacin A complex with yeast ribosomes was determined by X-ray crystallography at resolution up to 3.1 Å. While the overall binding site of amicoumacin A in eukaryotic ribosomes appeared to be the same as in bacterial ones, certain differences in the elements of the binding site may provide a framework for designing selective inhibitors on the basis of the amicoumacin A scaffold.Although the major principles of protein biosynthesis are uniform in all domains of life, the bacterial and eukaryotic translational machineries substantially differ in some particular components, including ribosome constituent elements111214Mammalian mRNAs are known to utilize a wide spectrum of translation initiation pathways14Renilla luciferase (Rluc) or firefly luciferase (Fluc). The former contained the 5′-untranslated region (5′UTR) of the human β-actin mRNA and were m7G-capped. Thus, its translation should reflect a behaviour of a regular cellular mRNA. In contrast, each of the Fluc encoding transcripts harboured one of three well-characterized viral IRESs in their 5′UTR. We used IRES elements that differ in translation initiation mechanisms and have different initiation factor (eIF) requirements. Encephalomyocarditis (EMCV) IRES requires the same set of eIFs as the Actin-Rluc, except for the cap-binding protein eIF4E; it places the 40S ribosome subunit into its internal region, so there is only a limited scanning event across a few nucleotide-long initiation window in this casevia eIF2-independent pathwaysi at all, since it utilizes the most exotic initiation mechanism everWe prepared polyadenylated transcripts that encoded either We transfected the reporter mRNA constructs into cultured HEK293T cells and measured the luciferase activity after 2 hours of expression. Increasing concentrations of Amicoumacin A were added to the culture medium just before the transfection. The short incubation period allowed us to minimize secondary effects that could appear if prolonged incubations with the drug were used. Translation of both scanning- and IRES-dependent mRNAs were inhibited by amicoumacin A in a micromolar concentration range . Almost in vitro translation experiment in the mammalian cell-free system that closely recapitulates in vivo conditionsThe difference we observed could be explained by either an additional inhibitory action of the antibiotic on the scanning process or by some indirect effects of the drug. It is long time known that some protein synthesis inhibitors cause ribotoxic stress response in mammalian cells, including eIF2 phosphorylation and partial inactivation20To dissect this issue unambiguously, we monitored ribosome progression along the capped β-globin mRNA by toe-printing in rabbit reticulocyte lysateAnother eukaryotic system that is widely used for analysis of translation inhibitors is budding yeast. We monitored yeast culture growth in the presence of increasing amicoumacin A concentrations. However, we did not observe substantial growth inhibition up to the highest antibiotic concentration we used . We hypoin vitro experiment in yeast cell-free system (19cI-Fluc) or a 5′ UTR from the natural yeast GCN4 gene (GCN4-Fluc) were used. Both mRNAs initiate translation by canonical scanning mechanism, although the latter one bears four uORFs in their leader and is a subject of a eIF2-P-mediated translational control19cI-Fluc. This result indicates that cell integrity protects yeast ribosomes from amicoumacin A inhibition in vivo, while the ribosomes themselves are highly susceptible to inhibition.To exclude any effect not related to translation, we performed an e system . Two lucSaccharomyces cerevisiae in complex with translation inhibitorsobs − Fcalc) was calculated using the model of vacant 80S yeast ribosome (PDB entry 4V88). The amicoumacin A molecule was located in the E-site of small ribosomal subunit after manual inspection of the peaks of positive electron density . The structure of 80S-amicoumacin A complex was refined with Phenix.refineThe ability of amicoumacin A to inhibit yeast ribosomes prompted us to use this system for structural study. For this, we applied the procedure to determine the structures of 80S ribosome from  density . The atoAmicoumacin A interacts mostly with rRNA residues in helixes 23 and 24 . It is iThermus thermophilus in complex with amicoumacin A reported previouslyWe compared the structure of amicoumacin A bound to the yeast ribosome to the structure of the bacterial ribosome from Thermus thermophilus 70S ribosome, while in the yeast 80S ribosome the terminal leucine of uS11 forms a hydrophobic interaction with the isobutyl group of amicoumacin A. The mutations in the C-terminus of uS11 are known to confer resistance to cryptopleurine and emetine2624in vitro. Interestingly, cryptopleurine and emetine are active only in eukaryotes, while pactamycin is a universal inhibitor.The differences in the binding site of amicoumacin A in bacterial and yeast ribosomes50) roughly matched that of inhibition of reporter mRNA translation . Since this loop has a special importance in the process of start codon selection during translation initiationA conserved loop of the ribosomal protein uS7 is located 3.9 Å from the isocoumarin part of amicoumacin A in the bacterial ribosome and is more than 10 Å away in the yeast 80S structure . Large dAnother intriguing possibility is using the amicoumacin scaffold for designing mRNA-specific translation inhibitors. For example, the HCV IRES domain II binds in close proximity35In summary, we presented evidence for amicoumacin A activity toward the eukaryotic ribosome in both mammalian and yeast systems, we revealed structural details of its interaction with the yeast 80S ribosome, and we showed a relative selectivity of the drug toward human cancer cell lines. Our study could be used for rational drug design aiming to improve amicoumacin A therapeutic potential.Amicoumacin A isolation was described earlierin vitro transcription, pbG was linearized at the EcoRI site. For synthesis of the polyadenylated mRNAs encoding the firefly and Renilla luciferases, a 50T-tailed PCR product was used as a template, as described previously2238The plasmids pbG coded for the rabbit β-globin mRNA2, as indicated. The mixture was incubated for 5 min at 30 °C, then 1 μl of mRNA solution (0.5 pmol/μl) was added followed by incubation for an additional 10 min. After that, 10 μl of RT Mix (including [32P]-labeled primer TCACCACCAACTTCTTCCAC) was added, according toRibosomal complex assembly was performed in the nuclease treated RRL , as described previously2 box and to avoid cooling the plate. Two hours after the transfection, cells were harvested, and luciferase activities were analyzed with the Dual Luciferase Assay kit (Promega). All the transfections were repeated at least three times in different cell passages.HEK293T cells were cultured and transferred into 24-well plates 12–16 h before transfection, as describedMATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) yeast strain was grown in a YPD medium  to exponential phase and inoculated into liquid YPD at OD600 of 0.05. Growth rates (OD600) were measured every 5 min in a 24-well plate in the Infinite 200 PRO microplate reader  at 30 °C with continuous shaking.The BY4741 , 2 u of RiboLock RNase inhibitor (Thermo Scientific), 0.5 mM D-luciferin, 0.25 pmol mRNA, and 1 μl of amicoumacin A solution, as indicated. Translation reactions in the yeast system were performed in a total volume of 15 μl, containing 7.5 μl of the extract, translation buffer 2, 12 mM creatine phosphate, 1 mM ATP, 0.4 mM GTP, 126 mM KOAc, and 50 μM of each amino acid), 3 u of RiboLock RNase inhibitor, 50 μg/ml creatine phosphokinase, 1 mM D-luciferin, 0.12 pmol mRNA, and 1 μl of amicoumacin A solution, as indicated. Translation mixtures were incubated in a white 384-well plate  and covered by a PCR plate seal at 30 °C  or 25 °C (for the yeast one) in the TECAN reader with continuous measurement of the luciferase activity (integration time 3 s). Light intensities at 25 min were taken as luciferase activity values.Translation experiments in the mammalian system were performed in a total volume of 10 μl, which contained 5 μl of the S30 extract, translation buffer S. cerevisiae were purified and crystallized, as previously described80S ribosomes from the yeast http://grade.globalphasing.org) using SMILES strings from the PubChem databasehttp://pymol.org/). Ribosomal proteins were named throughout the manuscript according to the newly established nomenclaturehttp://pdb.org/pdb/home/home.do) under accession code 5I4L.The structure was solved by rigid body refinement of the deposited 80S ribosome structure (PDB 4V88) using Phenix.refine2 for 18 h before treatment. Then we added 15 μl of the drug  and incubated the cells for additional 72 h. Amicoumacin A in final concentrations of 50 nM–100 μM (eight dilutions), in triplicate, was applied. 2 nM–6 μM doxorubicin was used as a positive control. At the end of the incubation we added MTT into the media (up to 0.5 mg/ml), incubated the cells for 2 h, followed by removing the media and addition of 100 μl DMSO. The amount of MTT reduced by cells to its blue formazan derivative was measured spectrophotometrically at 565 nM using a plate reader and normalized to the values for cells treated with the media/DMSO only. IC50 was calculated with “GraphPad Prism 5” software .Cytotoxicity was assessed using the MTT 2,5-diphenyl tetrazolium bromide) assay based on the method described by Ferrari and co-workersHow to cite this article: Prokhorova, I. V. et al. Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome. Sci. Rep. 6, 27720; doi: 10.1038/srep27720 (2016).


Synechocystis PCC 6803. Compared with the wild type, the pfsR deletion mutant displayed stronger tolerance to iron limitation and accumulated significantly more chlorophyll a, carotenoid, and phycocyanin under iron-limiting conditions. The mutant also maintained more photosystem I and photosystem II complexes than the wild type after iron deprivation. In addition, the activities of photosystem I and photosystem II were much higher in pfsR deletion mutant than in wild-type cells under iron-limiting conditions. The transcripts of pfsR were enhanced by iron limitation and inactivation of the gene affected pronouncedly expression of fut genes (encoding a ferric iron transporter), feoB (encoding a ferrous iron transporter), bfr genes (encoding bacterioferritins), ho genes (encoding heme oxygenases), isiA (encoding a chlorophyll-binding protein), and furA (encoding a ferric uptake regulator). The iron quota in pfsR deletion mutant cells was higher than in wild-type cells both before and after exposure to iron limitation. Electrophoretic mobility shift assays showed that PfsR bound to its own promoter and thereby auto-regulated its own expression. These data suggest that PfsR is a critical regulator of iron homeostasis.Iron is an essential cofactor in numerous cellular processes. The iron deficiency in the oceans affects the primary productivity of phytoplankton including cyanobacteria. In this study, we examined the function of PfsR, a TetR family transcriptional regulator, in iron homeostasis of the cyanobacterium  Iron is an essential cofactor in many critical cellular processes, including photosynthesis, respiration, nitrogen fixation, chromophore biosynthesis, and gene regulation. Iron has been considered as a limiting nutrient affecting the marine primary production because iron is poorly soluble in oxygenated seawater 6f complex has five; photosystem I (PSI), which is the largest pool of iron in photosynthetic systems, contains 12 iron atoms; and ferredoxin, the terminal electron acceptor, has two iron atoms The photosynthetic cyanobacteria comprise a large fraction of marine phytoplankton and adapt to the iron-limiting ecosystems, which makes them excellent models for studying iron stress tolerance in relation to photosynthesis. Due to the importance of iron in the photosynthetic electron transport chain, the iron quota in cyanobacteria is much higher than in non-photosynthetic bacteria Synechocystis PCC 6803 (hereafter referred to as Synechocystis 6803) cells Cyanobacteria also exhibit various other physiological and morphological changes in response to iron limitation. For instance, iron deficiency causes a decrease in cellular pigmentation (such as chlorophyll a and phycocyanin) pfsR  deletion mutant, that exhibits enhanced tolerance to iron limitation. PfsR, which is encoded by the open reading frame (ORF) sll1392 in Synechocystis 6803, was discovered in a genetic suppressor screen hli genes, due to the tighter control of free intracellular iron levels Here, we describe a unique cyanobacterial mutant, Synechocystis 6803 enhanced its viability and photosynthetic activity under iron-limiting conditions. Additionally, we demonstrated that PfsR was auto-regulated and modulated iron homeostasis by regulating a set of genes related to iron homeostasis, including fut genes, feoB, bfr genes, ho genes, isiA, and furA.In this study, we investigated the regulatory role of PfsR in iron homeostasis. Inactivation of PfsR in Synechocystis 6803 strains were grown in BG-11 medium at 30°C under 50 µmol photons m−2s−1 continuous white light and bubbling with air. Cell density was determined by measuring the optical density (OD) of the suspension at 730 nm (OD730) with a spectrophotometer . To determine sensitivity to iron, cells grown in BG-11 medium were diluted to OD730 about 0.2 and grown in BG-11 medium that lacked ferric ammonium citrate and contained 10 µM 2,2′-dipyridyl, an iron chelator.The chlorophyll a and total carotenoid in cells were extracted using dimethylformamide (DMF), and their concentrations were determined using the formula −1) = (OD461–0.046×OD664)×4.[Total Carotenoids] (µgml−1) = 12.1×OD664–0.17×OD625.[Chlorophyll a]  at an excitation wavelength of 430 nm with excitation bandwidth of 5 nm and emission bandwidth of 1.5 nm. The chlorophyll concentration was adjusted to 15 µgmlm–Fo)/Fm = Fv/Fm. The effective quantum yield of PSII [Y(II)] was determined as (Fm’–F)/Fm’ = Y(II). The P700 parameter Pm, the determination of which involves far-red preillumination and application of a saturation pulse, represents the maximal change of the P700 signal. The fraction of overall P700 that is oxidized in a given state (P700+) is calculated as P700+ = 1–(Pm’–P)/Pm. To determine the light-intensity dependence of relative electron transport rates (ETR), actinic light intensities were increased stepwise from 0 to 1500 µmol of photons m−2s−1.Chlorophyll fluorescence yield was monitored using a DUAL-PAM-100 P700 & Chlorophyll Fluorescence Measuring System  and DUAL-PAM software. The maximal quantum yield of PSII was determined as  rate was measured in the presence of 1 mM K3FeCN and 0.5 mM 2,6-dichloro-p-benzoquinone. The PSI-mediated electron transport (from DCIP/ascorbic acid via PSI to MV) rate was measured in thylakoid membrane suspensions containing 40 µM methylviologen (MV), 5 mM NH4Cl, 2 mM ascorbic acid, 0.1 mM 2,6-dichlorophenolindophenol (DCPIP), 2 mM NaN3, 40 µM 3--1,1-dimethylurea (DCMU), 40 mM tricine (pH 7.5), and 100 mM sucrose.Cells were grown to the exponential phase and harvested by centrifugation at 500×g for 5 minutes. Whole-chain-, PSII-, and PSI-mediated oxygen evolution activities were measured using a Clark-type electrode at light intensity of 1000 µmol of photon mThylakoid membrane isolation was performed according to the previously reported method http://imagej.nih.gov/ij/).Approximately 20 µg of thylakoid membrane proteins were resolved by SDS-PAGE in 12% polyacrylamide gels with 6 M urea, followed by transferring to nitrocellulose membranes. Immonodetection of proteins was performed using commercial primary antibodies . Bound antibodies were detected using anti-rabbit IgG conjugated to alkaline phosphatase . Protein bands were visualized using a NBT/BCIP reagent kit  and quantified using Image J software  method was used to quantify the relative levels of gene expression. Three biological replicates with three PCR replicates each were performed.Relative transcript levels of various genes were evaluated by quantitative PCR. Briefly, total RNA was isolated from 100 ml Cells were harvested by centrifugation at 500×g for 5 minutes, washed three times with 10 mM EDTA and three times with distilled water, and then dried at 50°C for 48 hours. The dried cells were digested in 70% nitric acid. After all the cells were dissolved, the nitric acid was evaporated and the samples were then reconstituted in 5% nitric acid. Samples were centrifuged at 1100×g for 5 minutes and the supernatant was analyzed for iron content. Atomic absorption was performed in the furnace mode on a Perkin-Elmer Atomic Absorption Spectrometer model AA600 .pfsR ORF was amplified by PCR and cloned into the Sph I and Bgl II sites of the pQE-70 vector . PfsR expression was induced in E. coli cultures with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). The cells were then grown for an additional 8 hours at 20°C. After harvesting the cultures, the cells were broken using a bead beater. His6-tagged PfsR protein was purified under native conditions, using a TALON Spin Column  according to the manufacturer’s instructions. The expression level and purity of the proteins were tested by SDS-PAGE analysis.For overexpression of PfsR protein, the full-length Synechocystis 6803 genomic DNA. The primers used are listed in 2, 12% glycerol]. The reactions were incubated at 25°C for 30 minutes and then subjected to 6% native polyacrylamide gel electrophoresis at 200 V in 0.5×Tris-borate-EDTA (TBE) buffer. The binding of PfsR to promoter regions was detected using the Electrophoretic Mobility Shift Assay (EMSA) Kit , according to the manufacturer’s instructions.The binding of PfsR to the promoter regions of a number of genes was determined by EMSA. DNA fragments for EMSA were amplified by PCR from pfsR deletion on iron stress responses, we examined cell growth of the wild type and the pfsR deletion mutant under conditions with different iron availability. Cells grown in normal BG-11 medium to the exponential phase were washed with iron-free BG-11 medium, and then diluted to an OD730 of 0.2 in iron-free BG-11 medium with supplement of 10 µM iron chelator 2,2′-dipyridyl. Under iron-replete conditions, the growth rates of the wild type and the pfsR deletion mutant were virtually indistinguishable (data not shown). In contrast, the wild type grew poorly and the pfsR deletion mutant grew well under iron-limiting conditions , and of PSI (at 725 nm). After iron deprivation, wild-type cells exhibited a more pronounced decrease in both PSI and PSII fluorescence than did pfsR deletion mutant cells. This decrease correlates with the overall decrease in cellular chlorophyll content in wild-type cells as compared with pfsR deletion mutant cells under iron-limiting conditions. The 77K fluorescence measurements suggest that the pfsR deletion mutant is better able to maintain photosynthetic apparatus/complexes than the wild type under iron-limiting conditions.In order to monitor the changes in the photosynthetic apparatus during iron limitation, we diagnostically investigated PSI and PSII by 77K fluorescence emission spectroscopy using 430 nm light as excitation, which is preferentially absorbed by chlorophyll. As shown in Synechocystis 6803 cells treated by 2,2′-dipyridyl underwent severe iron limitation, their chlorophyll fluorescence emission spectra did not exhibit a distinct shoulder at 685 nm as observed under iron limitation without the iron chelator. This suggests that there exist marked differences in the levels of IsiA and/or the compositions of IsiA complexes between the two different iron-limiting conditions.Interestingly, although pfsR deletion mutant cells that had been shifted to iron-deprived medium for various periods of time were separated on 12% SDS-PAGE gels with 6 M urea and probed using specific antibodies. As shown in pfsR deletion mutant. In addition, IsiA accumulated to a much higher level in the pfsR deletion mutant compared to the wild type under iron-limiting conditions. We also examined the heme cytochrome c550 content in whole cell proteins using heme staining. Similarly, iron limitation caused a more significant loss of heme in the wild type than in the pfsR deletion mutant  and the oxygen uptake activity of the thylakoid membranes (PSI activity) decreased more sharply in the wild type than in the pfsR deletion mutant.We monitored photosynthetic electron transport activity under iron-limiting conditions by measuring oxygen evolution . There wv/Fm) and the effective quantum yield [Y(II)] of PSII (presumably reflecting PSII activity), as well as the maximal P700 changes (Pm) and the proportion of oxidized P700 (P700+) (a representative chosen for the estimation of PSI activity), all markedly decreased in response to iron deprivation in the wild type as compared with the pfsR deletion mutant  levels. From the differences in the initial slopes of light response curves shown in pfsR deletion mutant after iron deprivation. These differences also explain the apparently higher maximal electron transport capacity of the iron-depleted pfsR deletion mutant.The light-intensity dependence of the relative electron transport rate (ETR) is indicative of the relative amount of electrons passing through photosystems during steady-state photosynthesis. The PSII- and PSI-mediated ETR  in both the wild type and the pfsR deletion mutant exhibits higher photosynthetic activity and efficiency than the wild type.Taken together, under iron-limiting conditions, the pfsR after exposure to iron limitation, and the impacts of the pfsR deletion on the expression of a number of genes involved in the iron stress responses were examined by quantitative PCR (pfsR mRNA level increased in the first hour after iron deprivation and reached peak level in three hours (with a 3.3-fold increase), after which, it declined gradually. We noticed that in previous microarray data, iron limitation led to either no change or only slight change (a 1.3-fold increase) in the transcript level of pfsRfutA1, futB, and futC) and ferrous iron transporter encoding gene (feoB) were all found to be up-regulated in the pfsR deletion mutant, even under iron-replete conditions. Moreover, the fut genes were induced immediately and peaked 1 hour after iron deprivation in the pfsR deletion mutant, whereas they only peaked 6 hours after iron deprivation in the wild type. The transcription of bacterioferritins, encoded by bfrA and bfrB, increased to a higher level in the pfsR deletion mutant than in the wild type after 6 hours of iron deprivation. In both the wild type and the pfsR deletion mutant, the expression of ho1 and ho2 genes (encoding heme oxygenases) declined in response to iron limitation. Additionally, the ho1 and ho2 transcript levels of the pfsR deletion mutant decreased more than did those of the wild type. Time course analysis of isiA expression revealed that the induction of isiA by iron limitation was much greater in the pfsR deletion mutant than in the wild type. In addition, the increase in the transcript level of furA (encoding a ferric uptake regulator) in response to iron limitation was more pronounced in the pfsR deletion mutant than in the wild type.In cyanobacteria, transcriptional regulation is one of the most important mechanisms for modulating responses to environmental stresses. To decipher the function of PfsR, a predicted transcriptional regulator, the expression profile of tive PCR . In wildpfsR deletion mutant has increased iron content inside the cells, we measured intracellular iron content of the wild type and the pfsR deletion mutant grown in either iron-replete or iron-limiting conditions using atomic absorption spectroscopy   .Synechocystis 6803 pfsR gene encodes a protein consisting of 203 amino acid residues. Sequence alignment analysis showed that PfsR contains a typical TetR-like N-terminal HTH DNA-binding domain  did not inhibit shifting of the PfsR-bound pfsRp100-2 fragment -type ferric iron transporter that is important in iron acquisition of Synechocystis 6803 fut genes in the pfsR deletion mutant under iron-replete conditions and the more rapid induction of fut genes upon iron deprivation indicate that the pfsR deletion mutant is better able to absorb ferric iron from the environment. The expression of feoB, which encodes a ferrous iron transporter pfsR deletion mutant than in the wild type. This might increase the amount of iron transported into the pfsR deletion mutant.The bfrA and bfrB) in the pfsR deletion mutant likely promotes iron storage when iron is available, and the stored iron is subsequently used for cell growth under iron-limiting conditions. Additionally, it is reasonable to assume that the increased iron storage coincides with an increased iron uptake. The finding that the pfsR deletion mutant contained 15% more intracellular iron under iron-replete conditions and 240% more intracellular iron under iron-limiting conditions than the wild type supports our interpretation well  in the pfsR deletion mutant might reduce not only the demand for the iron-containing ferredoxin, but also the release of detrimental free iron that causes oxidative stress through the Fenton reaction The ferredoxin-dependent heme oxygenase catalyzes the degradation of heme to produce biliverdin IXα with the release of ferrous iron IsiA is critical for the growth of cyanobacteria under iron-limiting conditions and its induction is a hallmark of the transcriptional response to iron stress pfsR deletion mutant than in the wild type after iron deprivation. It may seem paradoxical that the pfsR deletion mutant accumulated more IsiA, as the mutant suffered less iron stress than the wild type when iron was limiting. However, PfsR might be a negative regulator of IsiA, and deletion of pfsR would then lead to an increase in isiA expression, which in turn would enhance the viability of cells under iron-limiting conditions. The regulation of isiA expression is complex, which may involve the transcriptional control by FurA, the posttranscriptional control by the antisense RNA IsrR, and/or a presently unidentified regulatory mechanism isiA promoter directly, the regulation likely requires the participation of other factors yet to be identified.The induction of IsiA was much stronger in the Synechococcus sp. PCC 7492 FurA regulates the expression of numerous genes related to iron stress (such as isiA and isiB) Anabaena sp. PCC 7120 FurA was reported to accumulate under iron-limiting conditions and act as a master regulator of iron metabolism Synechocystis 6803 contains three genes encoding Fur-like proteins , among which, the sll0567-ecoded FurA is most likely the functional homolog of FurA in Synechococcus sp. PCC 7492 and Anabaena sp. PCC 7120 isiAB in Synechocystis 6803 The Fur family proteins are essential in regulating iron homeostasis of cyanobacteria pfsR resulted in a much stronger induction of furA after iron deprivation, implying that PfsR might contribute to the control of furA expression. It has been reported that feoB, bfr, ho, and isiA were all potential target genes of Fur regulation In this study, we showed that inactivation of sll1392 (pfsR), sll1286, and slr0895 (prqR)] in Synechocystis 6803, the PrqR, was found to be an auto-repressor regulating the adaptive responses to the oxidative stress PfsR homologues exist in other cyanobacterial strains. It is likely that these TetR family transcriptional regulators are involved in the adaptation to complex and changing environments. Indeed, one of the three TetR family regulators [encoded by pfsR leads to a relaxed control of certain responses to iron limitation, which may prepare the cells for iron stress before the onset or at the very early stage of iron deficiency.The transcription of PfsR was activated at the early stage of iron limitation, and was then reduced gradually, suggesting an important role of PfsR at the onset of iron-stress responses. PfsR binds to its own promoter, and therefore is auto-regulated. Auto-regulation enables tight homeostatic control of protein expression in response to environmental changes pfsR deletion mutant under the few growth conditions  we tested in the laboratory, however, we cannot rule out the possibility that the mutant carries defects in the natural habitats or under other experimental conditions yet to be tested. Apart from the pfsR deletion mutant, several other single gene inactivation mutants, which displayed improved stress tolerance without deleterious phenotypes, have also been reported; for example, the Arabidopsis oxt1 mutant and the cbf2 mutant exhibit improved tolerance to oxidative stress and freezing stress, respectively We have not been able to observe any obvious defects in the fitness of the pfsR deletion mutant under various stress conditions will provide new insights into the roles of PfsR in regulating stress responses.The observed complexity in the expression of iron stress-associated genes suggests that multiple regulatory mechanisms might be involved in iron stress responses. While the mode of PfsR function and the cross-talks among PfsR and other iron regulators (such as FurA) invite further investigation, the finding that the inactivation of PfsR affects pronouncedly the expression of iron stress-associated genes implies a key regulatory role of PfsR in iron homeostasis. Because various stress responses might share common regulatory mechanisms and regulators pfsR deletion mutant to withstand iron limitation hinges on a mechanism that involves the auto-regulation of PfsR and the altered expression of genes involved in iron homeostasis, including those with roles in iron uptake, storage, and release.The work presented here demonstrates a critical role of PfsR in regulation of iron homeostasis. As depicted in Table S1List of primers used in this study.(DOC)Click here for additional data file.


Although in vitro peptide analysis favoured Ser12 as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser336, but not Ser12, as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser336 may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser100 and Ser326) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3.PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser CDK16) gene, which encodes the protein kinase PCTAIRE-1 , maps to the X chromosome (Xp11.3) whose defects and copy-number variants have been linked to various diseases, including intellectual disability and related disorders ATP was from PerkinElmer. Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson Immuno Research. P81 paper was from Whatman. All cell culture reagents were from Life Technologies. Unless otherwise indicated all other reagents were from Sigma.Peptide substrates for kinase assays were synthesized by GL Biochem. ATP (300 c.p.m./pmol), 1 mM DTT and 50 μM PCTAIRE-tide (PKSPKARKKL) or the indicated concentrations of peptide substrate. Reactions were incubated at 30°C and terminated by spotting onto P81 paper and immersion in 75 mM H3PO4. P81 filters were washed three times for 10 min with H3PO4, rinsed with acetone, air-dried and incorporation of γ-32P determined by Cherenkov counting. Results are expressed in units·mg−1, where 1 unit is defined as the incorporation of 1 nmol of phosphate·min−1.Purified recombinant PCTAIRE-1 or immune complexes isolated from cell/tissue lysates were assayed for phosphotransferase activity in a final assay volume of 50 μl containing 50 mM HEPES, pH 7.5, 0.1 mM EGTA, 10 mM magnesium acetate, 0.1 mM . This result led us to propose that CDK16/PCTAIRE-1 is a novel candidate XLID gene  (http://genetics.emory.edu/egl/emvclass/emvclass.php and ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/variation/166819/). Thus we investigated whether these variants could have an effect on cyclin Y–14-3-3 binding and also on PCTAIRE-1 enzymatic activity. For this purpose, we ectopically expressed both variants of human PCTAIRE-1  and its reference WT (isoform 2) together with WT cyclin Y  is a clinically and genetically heterogeneous disorder with >100 genes identified so far. For some of these XLID genes, only very few families with pathogenic variants have so far been reported, suggesting that mutations in these genes are very rare. In our recent study of 405 families with XLID investigated by X chromosome exome sequencing, we identified a variant in LID gene . An addicyclin Y A and 6B.cyclin Y  and by ocyclin Y B, suggescyclin Y A} and th100 and/or Ser326) and also cyclin Y-like-1 protein  promotes binding to 14-3-3 proteins in which both events are necessary for binding to, and activation of, PCTAIRE-1.Several studies, including our own, have recently established that activation of PCTAIRE-1/CDK16 requires binding of cyclin Y ,6,13,16;12 and Ser336 comprise the preferred consensus sequence for PCTAIRE-1, our initial hypothesis was that PCTAIRE-1 phosphorylates cyclin Y and that this modification influences their interaction. We identified Ser336, but not Ser12, as a PCTAIRE-1-dependent phosphorylation site; however, contrary to our hypothesis, phospho-deficient S336A mutant displayed normal binding to PCTAIRE-1. Although the functional role of Ser336 phosphorylation is still unknown, monitoring the phosphorylation of Ser336 may be a useful read-out to assess activity of PCTAIRE-1 in intact cells/tissues in the absence of well-characterized downstream substrates.Given that residues surrounding Ser100 and Ser326 were indeed phosphorylated in cells. A follow-up study by the same group claimed that they identified several phosphorylation sites on cyclin Y using MS; however, they did not provide data regarding the identity of specific phosphopeptides [100 or Ser326) in the present study contain multiple serine residues  from phosphorylating the Ser100 residue. It is unclear therefore whether Ser100 phosphorylation alone is sufficient for 14-3-3 binding or whether Ser326 phosphorylation also plays a role.To our knowledge, the present study is the first study demonstrating that the interaction between PCTAIRE-1 and cyclin Y requires phosphorylation of cyclin Y that results in 14-3-3 binding. However, it should be noted that a recent study by Li et al.  has repopeptides . Surrounresidues B. The ph119 and Ser153. These sites have been shown to be substrates for protein kinase A (PKA) in vitro [153. Phosphorylation of Ser119 and Ser153 creates 14-3-3 consensus binding motifs, whose binding activity has been confirmed by overexpression and mutagenesis study [In cultured cell lines, PCTAIRE-1 is phosphorylated at several residues, including SerAs part of systematic X-chromosome exome re-sequencing of index patients from 405 families with XLID, a unique CDK16/PCTAIRE-1 variant was identified in a multigenerational family . In addiIn summary, our work has provided a key mechanistic insight into the regulation/activation of PCTAIRE-1, which is mediated through binding of phosphorylated cyclin Y in complex with 14-3-3 protein. CDK16/PCTAIRE-1 has emerged as a potential key player for normal brain development and function, but further studies are required to identify downstream molecules/pathways that link PCTAIRE-1 and biological/physiological processes in brain.


Aphanizomenon flos-aquae after ice formation. A reduction in light resulting from snow coverage exerted a pronounced physiological stress on the cyanobacteria. Consequently, these were rapidly colonized, leading to a sudden proliferation of attached and subsequently of free-living heterotrophic bacteria. Total bacterial protein production reached 201 µg C L−1 d−1, ca. five times higher than spring-peak values that year. Fluorescence in situ hybridization and denaturing gradient gel electrophoresis at high temporal resolution showed pronounced changes in bacterial community structure coinciding with changes in the physiology of the cyanobacteria. Pyrosequencing of 16S rRNA genes revealed that during breakdown of the cyanobacterial population, the diversity of attached and free-living bacterial communities were reduced to a few dominant families. Some of these were not detectable during the early stages of the cyanobacterial bloom indicating that only specific, well adapted bacterial communities can colonize senescent cyanobacteria. Our study suggests that in winter, unlike commonly postulated, carbon rather than temperature is the limiting factor for bacterial growth. Frequent phytoplankton blooms in ice-covered systems highlight the need for year-round studies of aquatic ecosystems including the winter season to correctly understand element and energy cycling through aquatic food webs, particularly the microbial loop. On a global scale, such knowledge is required to determine climate change induced alterations in carbon budgets in polar and temperate aquatic systems.In winter 2009/10, a sudden under-ice bloom of heterotrophic bacteria occurred in the seasonally ice-covered, temperate, deep, oligotrophic Lake Stechlin (Germany). Extraordinarily high bacterial abundance and biomass were fueled by the breakdown of a massive bloom of  Aphanizomenon spp. blooms) bacterial numbers increased during the cyanobacterial bloom to 1010 –1011 cells L−1. At that time the bacterial protein production was between 25 and 350 µg C d−1, respectively On a global scale ice covers, either seasonally or permanently, about 8% of saline and freshwater bodies (not including the Southern hemisphere) It is believed that as a result of low temperatures and reduced nutrient remineralization due to low protozoan grazing, microorganisms under ice are killed, harmed or exist in a dormant state Powerful molecular tools enable systematic explorations of the diverse and largely uncultivable bacterioplankton communities 2 emissions Although lakes cover a little more than 3% of the Earth surface as compared to 71% by oceans and seas, they have an equal contribution to COAphanizomenon flos-aquae in March 2010. Based on this phenomenon during which OM became suddenly available at high concentrations in the cold season, we hypothesize that once winter conditions have reached a steady state, bacterial activity and community composition are mainly regulated by OM availability and subsequent protozoan grazing, and only to a lesser extent by low water temperatures as has been traditionally suggested.In this study we followed a sudden and massive under-ice proliferation of heterotrophic bacteria in oligotrophic Lake Stechlin following the breakdown of an under-ice bloom of the low light-adapted cyanobacterium Field permit was granted to the Leibniz institute by the stechlin natural park authorities on a permanent basis.2 and a maximum depth of 69.5 m, this relatively small, lake is one of the deepest lakes of the Northeastern German Lowlands Lake Stechlin  is a seasonally ice-covered meso-oligotrophic freshwater lake formed after the last glacial period in northern Europe (Weichselian). With an area of 4.23 kmth December 2009 and 21st April 2010 from the epilimnion (integrated from 0–10 m depth) and the hypolimnion (distinct depth of 20 m) (14C] - leucine incorporation in triplicates at in situ incubation for 1 h as described in Rösel & Grossart in situ hybridization (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) and 454 tag pyrosequencing.Our main focus was the ice-covered period and the microbial community residing beneath the ice. Samples were collected between 10of 20 m) . Temperawww.technobiology.ch).For CARD-FISH, water samples were fixed for 24 h at 4°C with formaldehyde . Subsequently, aliquots were separated to determine particle-attached (PA) and free-living (FL) bacteria by sequential filtration through polycarbonate filters  as previously described in Rösel et al. Samples for DNA extraction were size-fractionated immediately after sampling and stored at −80°C for further processing. DNA from 20 epilimnic and 20 hypolimnic samples (10 PA and 10 FL per strata)  was extrwww.bacterio.net) and the bacterial code of nomenclature; therefore, differences in nomenclature may occur when compared to uncurated databases. Diversity and community structure analyses were performed based on >132,000 bacterial sequences. A detailed summary of the analysis process of the pyrosequencing data of each sample, including the total number of reads and length distribution, as well as the results of quality management, dereplication and clustering, can be found in the supplement  remained clear until mid-February, but thereafter the ice sheet was covered by >50 cm of snow. Thermal stratification under the ice was inverted as compared to the summer stratification. In the upper 5 m the water was <1°C while between 5 and 20 m the temperature increased to 3.1°C . Below 2−1 in surface and bottom water, respectively) throughout the ice-coverage period with a background of 75% saturation  and December (A. flos-aquae), respectively A. flos-aquae in Lake Stechlin forms heterocysts in summer, no heterocysts were visible in any of the analyzed filaments collected in winter (n>200 filaments), consistent with data on available combined nitrogen  comprises the late bloom of A. flos-aquae. Phase II (February 23rd-March 16th) depicts the breakdown of the cyanobacterial bloom and the rapid rise of heterotrophic bacteria. Differences between Phases I and II are evident from micrographs of DAPI and FISH stained cyanobacterial filaments collected from the epilimnion  , BPP of PA and FL bacteria, increased abruptly from Phase I to Phase II (5 to 183 and 8 to 58 µg C Lctively) . This inlls mL−1 . At the In Situ Hybridization (CARD-FISH) and 454 tag pyrosequencing results , 16% (0–33%), 7% (0–19%) and 3% (0–28%), respectively) were associated with A. flos-aquae cells (Betaproteobacteria and Bacteroidetes (54% (4–88%) and 31% (4–63%), respectively). These were also the only two bacterial groups associated with A. flos-aquae at that time. Alphaproteobacteria and Actinobacteria  dominated the FL fraction in Phase I, but were replaced in Phase II by a dominance of Bacteroidetes and Betaproteobacteria  (Flavobacteriaceae (21%), Comamonadaceae (27%) and Oxalobacteraceae (48%). While the first two families dominated the PA fraction in the epilimnion during Phase I, Oxalobacteraceae contributed to <0.5% of the BCC  as evident from Denaturing Gradient Gel Electrophoresis (DGGE), Catalyzed Reporter Deposition Fluorescence  results ; Fig. S3ae cells . In Phasctively) ; 5. Compctively) ; 4, but ctively)  & S4. In the BCC . During  the BCC .6 cells mL−1 throughout the entire period. Accordingly, BPP was much lower than in the epilimnion  during the whole period and only slightly increased at the end of Phase II (Our results indicate that the hypolimnion (20 m) was affected by the events in the epilimnion only towards the end of Phase II. Total bacterial numbers (FL) in the hypolimnion (20 m) were <0.5×10Phase II . This, tA. flos-aquaeIn temperate lakes, persistent cyanobacterial blooms frequently occur in winter under the ice e.g.  Table S. HoweverA. flos-aquae were abundant after the mixing in early winter, including nitrogen as indicated by the lack of heterocysts and phosphorus by storage in form of poly-P granules. The high growth potential of the winter bacterioplankton - irrespective of temperature - became evident when A. flos-aquae became light limited A. flos-aquae filaments, leading to an extremely high bacterial abundance and BPP in Phase II. The degradation of cyanobacterial filaments resulted in the release of the protoplasmic content into the surrounding water leaving empty “ghost cells”  bacterial colonization of senescent cyanobacteria resulted in detachment of bacteria and release of substrates into the surrounding water supporting the proliferation of these communities; b) a small fraction of colonized A. flos-aquae aggregates sank into the hypolimnion. The latter process was reflected by significant changes in BCC of hypolimnic PA bacteria greatly reducing microbial diversity in this fraction.High availability of OM during bacterial colonization and lysis of Flavobacteriaceae family, known to degrade high-molecular-weight OM A. flos-aquae cells. Nevertheless, while the marine event harbored numerous genera from the Flavobacteriaceae, Flavobacterium was the sole genus detected under the ice in Lake Stechlin  in parallel to a rather stable BPP. Nevertheless, during the rapid transition between Phase I and II of the bloom, while temperature remained constant, we observed a major change in BCC accompanied by a large increase in activity. The initially low abundant a solely . These pUnder-ice blooms of phytoplankton are not sporadic events since, during the last decades, they have been regularly described from limnic environments and more recently also from marine systems . Using L2 emissions from freshwater have been estimated to 0.75 Pg y−1−1) than marine ones (0.1 Pg y−1)  Current climate change leads to a reduced intensity of ice coverage and hence to dramatic environmental changes with so far unknown ecological consequences, e.g., for biogeochemical processes such as greenhouse gas fluxes across the water-atmosphere boundary. Limnic systems are known to be super-saturated with greenhouse gases Figure S1Oxygen concentration (A) and PH (B) profiles measured during winter 2010 in Lake Stechlin.(TIF)Click here for additional data file.Figure S22. Median and average values are shown by full and dashed lines, respectively.Colonization density of particle-attached bacteria and abundance of free living cells. Since particles vary in size, the cell number  per particle (a cyanobacteria segment) was normalized according to the particle's area to 100 µm(TIFF)Click here for additional data file.Figure S3DGGE analysis of the bacterial diversity of the two phases of the bloom separated into particle-attached (PA) and free-living (FL) bacterial communities of epilimnion (EL) and hypolimnion (HL).(TIF)Click here for additional data file.Figure S4Sequence frequencies of major particle-attached and free-living bacterial genera in the epilimnion and hypolimnion of Lake Stechlin during Phase I (A) and Phase II (B) of the under-ice bloom. Genera and clades of not yet cultured bacteria  are detailed only within families making up over 2% of the total sequences in at least one sample. Numbers in the gray fields refer to all the families that made less than 2% of the total sequences each.(TIFF)Click here for additional data file.Table S1List of samples analyzed for Bacterial Protein Production (BPP), Denaturing Gradient Gel Electrophoresis (DGGE), Tag Pyrosequencing (454) and Fluorescence in situ hybridization (FISH).(DOCX)Click here for additional data file.Table S2Oligonucleotide probes used in this study.(DOCX)Click here for additional data file.Table S3Sequence quality and diversity data for each sample.(DOCX)Click here for additional data file.Table S4List of bacterial tax paths.(PDF)Click here for additional data file.Table S5Under-ice phytoplankton blooms in limnic and marine systems.(DOC)Click here for additional data file.