Mytilus edulis, or on the spines of the Antarctic sea urchin Sterechinus neumayeri) slowed ice formation an average of 25% compared to a grid feature . The geometric dimensions of the features have only a small (∼6%) effect on ice formation. Surface texture affects ice formation, but does not explain by itself the large variation in ice formation and species-specific ice resistance observed in other work. This suggests future examination of other factors, such as material elastic properties and surface coatings, and their interaction with surface pattern.Growth of ice on surfaces poses a challenge for both organisms and for devices that come into contact with liquids below the freezing point. Resistance of some organisms to ice formation and growth, either in subtidal environments , or in environments with moisture and cold air  begs examination of how this is accomplished. Several factors may be important in promoting or mitigating ice formation. As a start, here we examine the effect of surface texture alone. We tested four candidate surfaces, inspired by hard-shelled marine invertebrates and constructed using a three-dimensional printing process. We examined sub-polar marine organisms to develop sample textures and screened them for ice formation and accretion in submerged conditions using previous methods for comparison to data for Antarctic organisms. The sub-polar organisms tested were all found to form ice readily. We also screened artificial 3-D printed samples using the same previous methods, and developed a new test to examine ice formation from surface droplets as might be encountered in environments with moist, cold air. Despite limitations inherent to our techniques, it appears surface texture plays only a small role in delaying the onset of ice formation: a stripe feature (corresponding to patterning found on valves of blue mussels,  Ice nucleation remains a serious issue for many industrial applications where spray, contact with, or submersion in liquids in sub-freezing point environments is inevitable. Piping systems, ship hulls, airplane wings, and road surfaces are all situations where the accumulation of ice can compromise safety or strength, reduce friction available for traction, impose additional weight, reduce stability, or alter aerodynamic or hydraulic performance. Though methods of actively removing ice after its formation , while others are resistant . Accretion of ice can interfere with physiological function and the resulting positive buoyancy can cause removal; as a result, different suceptibility to ice formation affects the community makeup and local ecology  in contrast with the others, as well as the difference between spongy or rubbery organisms and those with hard surfaces like the urchins and sea stars. In both plants and animals, previous work has shown that ice formation can adversely affect physiology and function or present severe ecomechanical  challeng ecology . Denny eThere are clearly many variables that can affect the formation and adhesion of ice. Biological systems may make use of many mechanisms of ice inhibition concurrently and may exploit any or all of the following : shape aSaxidomas nuttalli, Crassostrea gigas, Pisaster ochraceus, and Mytilus edulis, respectively), with all other mechanical properties  held constant. To examine sensitivity, a range of surface parameters (feature spacing and height), bounding the range seen in organisms, was tested. Tests included both the ice formation test of We hypothesized that surface texture provides protection from ice formation, i.e., for purposes of manufacturability, an attractive engineering solution is one using mechanical patterning alone. To address if this is possible, we tested four candidate artificial surface textures  inspired by benthic invertebrates , blue mussels (Mytilus edulis), and Pacific oysters (Crassostrea gigas), and one echinoderm, ochre sea star (Pisaster ochraceus)  and from material leftover from undergraduate biology teaching labs, including three bivalve mollusks, butterclams (hraceus) . The matBiological samples (<2 cm maximum dimension) were placed in open, unstirred 250 ml polypropylene tripour beakers and filled with 150 ml of 32 psu artificial seawater . Samples were placed so that the outer side was facing up. Both sample and seawater were initially at 4 °C. Multiple beakers were placed on a fiberglass tray which was then placed in a −20 °C walk-in freezer, away from overhead fans and partially shielded by surrounding cardboard to the side and above. Beakers were observed for ice formation on the sample within the first 10 min. As in The presence or absence of visible ice growth originating on the sample provides a gross measure of propensity to form anchor ice , subjectSaxidomas, Crassostrea, Pisaster, and Mytilus  respectively. The cone texture is also similar to the Antarctic sea star Odontaster, while the striped texture, consisting of square-edged ridges, is similar to patterning observed on the spines of the Antarctic urchin Sterechinus; both are known to be resistant to ice formation  process to create engineered samples mimicking the biological samples. Samples measured 0.03 m × 0.03 m × 0.005 m overall and included patterning of four textures: grid, valley, cone, and stripes . The texormation . The val to swim , and theSaxidomas; approximately 1 mm–3 mm for the groove-like/valley texture on Crassostrea; 0.5 mm–4 mm for the cone texture of Pisaster; and 0.5 mm for the stripe texture on Mytilus. These measurements were used in designing the textures: for initial screening, a height of 0.5 mm was used, with pattern spacing set at 0.5 mm for grid, valley, and stripes and 1.5 mm for cone.The observed textures  ranged fWe designed the textures using two solid-modeling programs: Solidworks  and Blender . Solid models were used to prepare stereolithography (STL) files that were printed in ABS plastic using a ProJet 3000 3D printer . Newly printed samples were cleaned of support material by gentle heating and use of an 80 °C sonicating warm-oil bath, then washed thoroughly with detergent before testing.As with the biological samples, we tested the four sample plates using the ice formation test of −1 (example video available for download). Samples were observed over 30 min or until all of the samples froze. Samples were classified as frozen once the droplet turned from a slight red tint to opaque white . Plates were dried and stored in 4 °C walk-in freezer overnight to provide isothermal starting conditions. Plates were placed randomly in rows on a plastic tray for testing. Five 0.1 ml droplets of 32 psu artificial seawater, also at 4 °C, were placed on the four corners of the plate and the center –2C. For ue white . The resue white . For theSaxidomas nuttalli, Crassostrea gigas, Pisaster ochraceus, and Mytilus edulis) initiated ice formation during the ice formation test , however, increases in normalized freeze time obtained by varying pattern spacing or height are small relative to the noise in the measurement. An example time-lapse movie of a droplet test is provided as Within both the best-performing texture (stripes) and the worst-performing texture (grid), the pattern spacing appeared to alter the normalized freeze time for droplets up to 28%, though the slowest freeze times were only delayed about 6% relative to the untextured control . Given ain situ field manipulation using portable refrigeration coils or supercooled brine injection  all initiated ice formation , they are not particularly noteworthy as polar species. On the other hand, hard shelled bivalves of closely-related species can be major components of Arctic communities (Odontaster and Sterechinus) are hugely important in the Antarctic . For future work, it may be worthwhile to more systematically probe closely-related pairs with polar versus sub-polar distribution.It is perhaps not a surprise that the four species tested  appears contrary to expectations from surface chemistry alone. In the echinoderms, the presence of a viscoelastic epithelial covering, potentially with actively moving cilia or mucus secretions, may be key.Surface chemistry may have effects on ice resistance; hydrophilic materials reduce resistance to ice adhesion  in air, While texture does have demonstrable effects on freezing time, the effects are small  and 5. MMycale); and elastic and viscoelastic properties ; nanoscale pattern, multi-scale shape and behavior . In an engineering system, these would increase cost; however, perhaps a subset of these may provide synergistic effects resulting in performance improvement in excess of the cost increase. For example, the combination of rigid and viscoelastic materials in a micro-scale composite artificial shark skin was effective in drag reduction ; surfactants, mucus and slime (as in eduction ; similar10.7717/peerj.588/supp-1Movie S1Time lapse video of droplet test, taken at 1 frame/s and played back at 30 frame/s.Click here for additional data file.10.7717/peerj.588/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.588/supp-3Supplemental Information 3This provides the data and R code used to generate Click here for additional data file.


In addition to the disruption of neural function below spinal cord injuries (SCI), there also can be changes in neuronal properties above and below the lesion site. The relevance of these changes is generally unclear, but they must be understood if we are to provide rational interventions. Pharmacological approaches to improving locomotor function have been studied extensively, but it is still unclear what constitutes an optimal approach. Here, we have used the lamprey to compare the modulatory effects of 5-HT and lesion-induced changes in cellular and synaptic properties in unlesioned and lesioned animals. While analyses typically focus on the sub-lesion spinal cord, we have also examined effects above the lesion to see if there are changes here that could potentially contribute to the functional recovery. Cellular and synaptic properties differed in unlesioned and lesioned spinal cords and above and below the lesion site. The cellular and synaptic modulatory effects of 5-HT also differed in lesioned and unlesioned animals, again in region-specific ways above and below the lesion site. A role for 5-HT in promoting recovery was suggested by the potential for improvement in locomotor activity when 5-HT was applied to poorly recovered animals, and by the consistent failure of animals to recover when they were incubated in PCPA to deplete 5-HT. However, PCPA did not affect swimming in animals that had already recovered, suggesting a difference in 5-HT effects after lesioning. These results show changes in 5-HT modulation and cellular and synaptic properties after recovery from a spinal cord transection. Importantly, effects are not confined to the sub-lesion spinal cord but also occur above the lesion site. This suggests that the changes may not simply reflect compensatory responses to the loss of descending inputs, but reflect the need for co-ordinated changes above and below the lesion site. The changes in modulatory effects should be considered in pharmacological approaches to functional recovery, as assumptions based on effects in the unlesioned spinal cord may not be justified. Spinal cord injury is associated with a loss of sensory and motor function below a lesion site due to damage to ascending and descending tracts and local circuitry, as well as disturbances of autonomic function. There is currently no effective intervention to overcome the effects of SPI  between 100–130 mm were obtained from commercial suppliers . All experiments were performed under license and conformed to the requirements of the UK Home Office Animals (Scientific Procedures) Act 1986. For spinal cord transections animals were anesthetized by immersion in MS-222  and a ~5 mm dorsal incision was made approximately 1 cm below the last gill to expose the spinal cord. The spinal cord was transected with iridectomy scissors and the incision site sealed with tissue glue (Vetbond). Following transection animals were kept at 20°C for 8–10 weeks : at this time most animals had recovered locomotor function  recordings were made from unlesioned animals and animals 8–10 weeks after lesioning. EMG recordings were made by inserting bipolar Teflon insulated electrodes (0.075 mm diameter) into the muscle under MS-222 anesthesia. The electrodes were inserted approximately 0.5 cm rostral and caudal to the lesion site. After recovery from anesthesia muscle activity was recorded as the animals swam in a plastic aquarium (29 × 23.5 × 5.5 cm). Swimming activity was initiated by lightly pinching the tail or head of the animal using serrated forceps: stimulation was given 30 s after the end of the previous swimming episode. A qualitative assessment of the functional recovery was made from video recordings based on the scale derived by  to the aquarium water: plateau effects on the episode length and coefficient of variation , aspects where significant effects of 5-HT were seen, occurred at a concentration of 200–400 μM -glucose; 2.0 mM HEPES; 0.5 mM L-glutamine, bubbled with O2 and adjusted to pH 7.4 with 1 M NaOH). The spinal cord was isolated from the notochord and pinned to a Sylgard lined chamber and superfused with lamprey Ringer at 10°C. Intracellular recordings were made from cells above and below the lesion site using an Axoclamp 2B amplifier (Molecular Devices). Motor neurons  and unidentified cells were examined in unlesioned animals and in lesioned animals above and below the lesion site. Given the relative size and number of different types of neurons, unidentified cells are likely to be motor neurons: as there were no obvious differences in motor neurons and unidentified cells they were grouped for analysis . Recordings were not made from different classes of interneurons as this analysis is not trivial even in this “simpler” system  in oxygenated lamprey Ringer at 4°C  spontaneous PSPs were instead measured over a 40 s period in each cell using the event detection feature in Clampfit.The properties of specific presynaptic inputs were examined by making paired intracellular recordings from postsynaptic motor neurons and Müller reticulospinal axons. These inputs were also used to examine the modulatory effects of 5-HT, but for this analysis cord stimulation-evoked responses were also used when reticulospinal axons that connected to the recorded postsynaptic cell could not be found. Cord-stimulation responses were evoked by stimulating the medial column that runs between the cell body layer (the region where locomotor network interneurons and motor neurons are located) and the midline of the spinal cord extracellularly using a glass suction electrode for 1 ms at 0.1 Hz for 50 s. The stimulation strength was adjusted to evoke what appeared to be a unitary EPSP, determined by the short rise time, longer decay time, and absence of multiple peaks . This was done in control and in the presence of 5-HT: the overlaid PSPs were averaged and the peak amplitude was measured. Putative monosynaptic reticulospinal-evoked inputs were examined by intracellular stimulation of reticulospinal axons in the medial column above the lesion site several segments rostral to the postsynaptic cell at 20 Hz for 1 s, followed by low frequency recovery test pulses . The amplitude of the initial EPSP and of EPSPs over the spike train was measured from the baseline preceding each EPSP to the peak of the EPSP. The trains were evoked at 20 s intervals. As there is no activity-dependent plasticity of the input at this frequency, the initial EPSP in the spike trains provided a measure of single low-frequency-evoked EPSPs. Putative monosynaptic connections were determined by the presence of reliable EPSPs that occurred with a consistent short latency  in response to presynaptic stimulation at 20 Hz  could fail the criteria for monosynapticity even though the connections were monosynaptic). Inputs were considered to be putatively polysynaptic if the inputs were unreliable  in the resting potential  or below the lesion site . With 10 μM 5-HT , but in the population as a whole there was still a mean hyperpolarization of −0.54 ± 0.39 mV, while above the lesion site it depolarized the membrane potential in 9 of 24 cells, which in this case gave a mean depolarization of 0.19 ± 0.76 mV . The RMP effect thus changed from a highly consistent hyperpolarization in unlesioned animals to a more variable effect where there was a mix of depolarization and hyperpolarization.In cells from unlesioned animals, 1 μM 5-HT non-significantly hyperpolarised the RMP , but it did significantly reduce the sAHP amplitude in each case (unlesioned (n = 11), above lesion (n = 10), lesioned below (n = 25), the reduction not differing significantly in the different conditions .There was no significant effects of 1 μM 5-HT on the input resistance, I-V relationship, excitability, or sAHP amplitude in unlesioned animals or lesioned animals above or below the lesion site (data not shown). For 10 μM 5-HT there was again no significant effect on the excitability or I-V relationship in cells from unlesioned animals or lesioned animals above or below the lesion site , and in lesioned animals above (n = 19) and below the lesion site (n = 20), the magnitude of the EPSP reduction not differing significantly between conditions . Similarly, 10 μM 5-HT also significantly reduced the amplitude of cord stimulation-evoked EPSPs in unlesioned animals  or below (n = 26) the lesion site  and in lesioned animals above the lesion site (n = 3): it is generally harder to find connections in lesioned animals, especially below the lesion site, which may be a reflection of the sparseness of regenerated inputs . In unlesioned animals and lesioned animals below the lesion site when all connections were averaged the EPSP depressed across the spike train , but not above (n = 15), the lesion site compared to unlesioned animals  was significantly increased in cells above and below the lesion site compared to cells from unlesioned animals Figures ; Table 1n = 1 of 17 in unlesioned animals, n = 3 of 6 lesioned animals, p < 0.05, Chi square). While the properties of spontaneous PSPs above the lesion site did not differ to unlesioned animals, there was additional evidence for an increase in excitatory connectivity: putative polysynaptic EPSPs were more common above the lesion site than in unlesioned animals . The IPSP value for spinal cord injured animals was significantly greater below than those in both the unlesioned and above lesion values, while the EPSP value was significantly greater above than in unlesioned animals  was calculated for each cell there was a significant reduction below the lesion site compared to unlesioned and above lesion cases, presumably reflecting the increase in inhibition  significant effects of 5-HT on the CV and episode length occurred at concentrations of between 200–300 μM , but control values varied and in animals with shorter cycle periods (<400 ms) the cycle period was increased , the swimming episode length was significantly reduced by 5-HT at higher concentrations , but the CV was again significantly reduced , 5-HT could improve swimming in poorly recovered animals (swimming score of 2). In these animals activity was absent below the lesion site but was evoked by 5-HT, and the activity above the lesion site became more regular . The failure of PCPA incubated animals to recover was not due to the acute effect of PCPA as animals were tested after being removed from PCPA for at least 2 weeks. However, in the five animals that recovered in the absence of PCPA, incubation in PCPA for 5 days 10 weeks after lesioning resulted in no disruption of locomotor activity  and lower levels of the swimming score .We have previously found significant differences in larval animals separated into those that recovered well or poorly  or poorly (n = 10), but above the lesion site excitability was significantly reduced with current steps from 1–2.5 nA in poorly recovered animals  than those that recovered well . Excitability did not differ to that in unlesioned animals for either those that recovered well  compared to those that recovered well  compared to those that recovered well (n = 17). The EPSPAmplitude × EPSPNumber was significantly greater above the lesion site in animals that recovered well than in those that recovered poorly  that gave a mean change of 0.31 ± 0.74 mV (n = 12), but in cells from poorly recovered there was a hyperpolarization in every cell that was significantly different to cells from animals that recovered well  effects, above the lesion site the only significant difference was that the sAHP was only significantly reduced in animals that recovered well Figure . Below tWhile several of the effects shown here are preliminary and require further analysis, the results show that there are changes in cellular and synaptic properties in the isolated lesioned compared to the isolated unlesioned spinal cord; that these changes differ above and below the lesion site; and that the modulatory effects of 5-HT are altered by lesioning, with effects again differing above and below the lesion site.There were several changes in functional properties in lesioned animals. Spontaneous synaptic and cord activity was increased above and below the lesion site compared to unlesioned animals. The reticulospinal axon-evoked EPSP half-width and the slow synaptic depolarization during trains of action potentials were also greater above and below the lesion site compared to unlesioned animals. However, the activity-dependent plasticity of reticulospinal inputs differed above and below the lesion site: depression occurred below that matched that seen in unlesioned animals, but above the lesion site the input facilitated which will make these connections functionally stronger. Facilitation is typically associated with a lower release probability and smaller initial EPSP amplitude , changes in EIN excitability , but they were not blocked by specific 5-HT2 antagonists: 5-HT3 and 5-HT4 agonists and antagonists were without effect . Exogenous and endogenous 5-HT slows the frequency of network activity in unlesioned animals  sAHP following an action potential or via activation of NMDA channels in the crossing (CC) inhibitory interneurons, which delays switching of activity between the two sides and thus increases the cycle period (e.g., Matsushima and Grillner, A major issue is to place the differences in 5-HT effects in lesioned animals into a functional context. While this lack of understanding is an obvious weakness, similar uncertainty exists over the effects of 5-HT in unlesioned animals despite several studies that examine these effects. 5-HT has numerous cellular and synaptic effects in unlesioned animals: it reduces somatosensory and reticulospinal-evoked EPSP amplitudes (Buchanan and Grillner, Pharmacological, electrical stimulation, and training effects modulate distinct functions in experimental and clinical situations (Courtine et al., 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.


Their occurrence in the plant kingdom is mainly confined to plant families of Meliaceae and Rutaceae. Owing to their wide range of pharmacological and therapeutic properties, this study was aimed at investigating the potential nitric oxide (NO) and acetylcholinesterase (AChE) inhibitory activity and the cytotoxicity of three limonoids: trichilia lactone D5  and (3) had lower cytotoxicity compared to puromycin and doxorubicin used as reference cytotoxic compounds. Compound (1) (LC50 of 23.55 μg/mL) had good antiproliferative activity against RAW 264.7 cancer cells. At the lowest concentration tested (0.5 µg/mL), compound (2) and (3) released the lowest amount of nitric oxide . The three limonoids had anti-AChE activity with IC50 values ranged of 19.13 μg/mL for (1), 34.15 μg/mL for (2) and 45.66 μg/mL for (3), compared to galantamine (IC50 of 8.22 μg/mL) used as positive control.Results indicated that the three limonoids had low cytotoxicity towards Vero cells with LCThe limonoid compounds studied in this work inhibited nitric oxide production in LPS-stimulated macrophages and had anti-AChE activity. Trichilia lactone D5 had potential antiproliferative activity against RAW 264.7 cancer cells. The limonoids had low cytotoxicity towards Vero cells lines. This study provided further examples of the importance of limonoids compounds as potential AChE inhibitors and anti-inflammatory agents targeting the inhibition of NO production. Nitric oxide (NO) is an important pro-inflammatory mediator involved in a wide variety of physiological and pathophysiological events; however overproduction of NO by inducible nitric oxide synthase (iNOS) results in severe inflammation . An assoHarrisonia sp. of Simaroubaceae -2,5-diphenyltetrazolium (MTT) assay as previously described . The selThe RAW 264.7 macrophages cells were seeded in 96 well-microtitre plates and were activated by incubation in medium containing 1 µg/mL LPS alone (control) or lipopolysaccharide with different concentrations of the samples dissolved in DMSO. Quercetin served as a positive control NO inhibitor for the reduction of NO production .Nitric oxide released from macrophages was determined by measuring the nitrite concentration in culture supernatant using the Griess reagent. After 24 h incubation, 100 µL of supernatant from each well of cell culture plates was transferred into 96-well microtitre plates and an equal volume of Griess reagent was added. The absorbance of the resultant solutions was determined on a BioTek Synergy microplate reader after 10 min at 550 nm. The concentrations of nitrite were derived from regression analysis using serial dilutions of sodium nitrite as a standard. Percentage inhibition was calculated based on the ability of compounds to inhibit nitric oxide formation by cells compared with the control (cells in media without compounds), which was considered as 0 % inhibition.To determine whether the observed nitric oxide inhibition was not due to cytotoxic effects, MTT assay was also performed on the macrophage cells as previously described .2·6H2O), 50 µL of Buffer B  and 25 µL of samples . Then, AChE (0.2 U/mL) was added to the wells and the absorbance was determined spectrophotometrically (BioTek Synergy microplate reader) at 405 nm. Distilled water was used as negative control. The percentage of inhibition was calculated as follow: max is the reaction’s rate of the control without inhibitor. The IC50 values of samples leading to 50 % inhibition were calculated by plotting the percentage of inhibition against the concentrations.Inhibition of acetylcholinesterase activity was determined using Ellman’s colorimetric method  with sliAll results are presented as means of triplicate experiments. All experiments were conducted in triplicate and values expressed as mean ± standard deviation. Statistical analysis was performed with GraphPad InStat Software and results were compared using the Student-Newman Keul test at 5, 1 or 0.1 % significance level.


We examined the natural variability of carbon and nitrogen isotopes in the symbiotic sea anemone Anemonia viridis to investigate dietary shifts (autotrophy/heterotrophy) along a natural pCO2 gradient at the island of Vulcano, Italy. δ13C values for both algal symbionts (Symbiodinium) and host tissue of A. viridis became significantly lighter with increasing seawater pCO2. Together with a decrease in the difference between δ13C values of both fractions at the higher pCO2 sites, these results indicate there is a greater net autotrophic input to the A. viridis carbon budget under high pCO2 conditions. δ15N values and C/N ratios did not change in Symbiodinium and host tissue along the pCO2 gradient. Additional physiological parameters revealed anemone protein and Symbiodinium chlorophyll a remained unaltered among sites. Symbiodinium density was similar among sites yet their mitotic index increased in anemones under elevated pCO2. Overall, our findings show that A. viridis is characterized by a higher autotrophic/heterotrophic ratio as pCO2 increases. The unique trophic flexibility of this species may give it a competitive advantage and enable its potential acclimation and ecological success in the future under increased ocean acidification.Contemporary cnidarian-algae symbioses are challenged by increasing CO Such a location has been reported in the Levante Bay of Vulcano Island  in the Mediterranean Sea where many studies have examined physiological adaptations of biota to OA, including seagrass811pCO2 gradient of several hundred meters moving away from the venting source. The submarine gas emissions in Levante Bay are characterized by high CO2 content volume (>90%) and variable low H2S (ranging 0.8 to 2.5% volume)Increasing carbon dioxide CO emissionSymbiodinium) and from a variety of external food sourcesSymbiodinium utilize bicarbonate (HCO3−), rather than CO2(aq), as the primary source for photosynthesisAnemonia viridis Forskål (Cnidaria: Anthozoa), the temperate Mediterranean species chosen for this study, occurs naturally at high densities throughout Levante Bay and harbors the dinoflagellate Symbiodinium muscatinei LaJeunesse and Trench (Dinomastigota: Dinophyceae)pCO2 gradient. Other reports on the response of A. viridis near CO2 vents discovered changes in their associated microbial communitiesA large body of research has focused on the potential impact of OA on reef organisms, particularly scleractinian corals. However, non-calcifying cnidarians such as sea anemones have received less attentiononate HCO−, ratherA. viridis using isotopic compositions, particularly carbon source shifts in the anemone metabolism, in response to high pCO2/low pH conditions in situ. We measured how the natural variability of carbon and nitrogen isotopes in Symbiodinium and host tissues of A. viridis varies along a natural pCO2 gradient. This was compared with other key physiological parameters  which were used in the present and in previous studies13C and δ15N signatures of an organism are related to those of its diet20212223versus heterotrophically derived food to the anemone energetic budget (autotrophic/heterotrophic ratio) with increasing seawater pCO2. This may facilitate better understanding of the environmental fate of cnidarians in a high CO2 world.The purpose of this paper is to investigate dietary changes of −2), consistent with previous findingspCO2 site  = 1.438, P = 0.248]  = 0.583, P = 0.562] and cell chlorophyll a concentration  between sites (−1 wet wt ± SE) between sites was 37.65 ± 1.12. Symbiodinium density (cells mg protein−1 ± SE) between sites averaged to 1.06 ± 0.07 × 106 and mean chlorophyll a content (pg cell−1 ± SE) was 4.57 ± 0.27. The number of dividing Symbiodinium cells (MI) was progressively greater in anemones inhabiting the higher pCO2 sites , increasing from 3.69 ± 0.76% at the control site to 7.12 ± 1.44% and 9.8 ± 0.54% at the intermediate and high pCO2 sites, respectively , with an average of 0.98 ± 0.01‰  and Symbiodinium (δ13CS) decreased under high pCO2 conditions  = 42.901, P = 0.000003], with a decrease from −16.66 ± 0.2‰ at the control site to −17.62 ± 0.19‰ and −19.12 ± 0.16‰ at the intermediate and high pCO2 sites, respectively. δ13CS also differed significantly between all sampling sites , decreasing from −15.1 ± 0.28‰ at the control site to −16.65 ± 0.37‰ and −18.21 ± 0.24‰ at the intermediate and high pCO2 sites, respectively. The difference in δ13C between the anemone tissue (δ13CT) and Symbiodinium (δ13CS) at each site was calculated as δ13CS- δ13CT to evaluate changes in autotrophic/heterotrophic ratios. δ13CT was considerably lighter than δ13CS at all sampling sites with δ13CS- δ13CT reduced with increasing pCO2  .δnditions . One-waying pCO2 . In ambi15N values of anemone tissue (δ15NT)  and Symbiodinium (δ15NS)  with increasing pCO2 (15NT was lowest at the control site (4.32 ± 0.12‰) and increased to 4.55 ± 0.16‰ and 4.6 ± 0.18‰ at the intermediate and high pCO2 sites, respectively. δ15NS averaged to 1.34 ± 0.36 at the control site and increased to 1.41 ± 0.42 and 1.82 ± 0.41 at the intermediate and high pCO2 sites, respectively. δ15NS was substantially lighter compared to δ15NT at all sampling sites, with an average difference of 2.5 ± 0.23‰  = 0.301, P = 0.745 for anemone tissue; F  = 0.069, P = 0.934 for Symbiodinium)  . The C/NA. viridis collected at all pCO2 sites lacked any apparent signs of stress  is the most important trace element for algal growth13The substantial increase in dividing algal cells under elevated pCO2 MI;  is in acpCO2 site as compared to the control, CO2(aq) increased near the venting source  increased from less than 1% at the control site to 4% at the high pCO2 site  . This is typical of cnidarian-algae symbioses, in which Symbiodinium may contribute up to 95% of their photosynthetically-produced carbon to the hostpCO2 site the heterotrophic input to δ13CT reduced to about 2.5% , leading to a greater autotrophic input. This observation is also supported by the difference in δ13C values between host tissue and Symbiodinium, which reflects the relative contribution of heterotrophy and photosynthesis to fixed carbon20Symbiodinium stable carbon isotopic values are usually within 2‰ of each other203913CS- δ13CT with increasing pCO2 from 1.56 ± 0.21‰ at the control site to 0.96 ± 0.31‰ and 0.9 ± 0.17‰ at the intermediate pCO2 and high pCO2 sites, respectively (δ ± 0.2‰) , suggestectively . This fupCO2 near the vent promotes carbon isotope fractionation by Symbiodinium during photosynthesis, leading to lighter δ13CS values. δ13CS showed a substantial decrease from −15.1 ± 0.28‰ at the control site to −16.65 ± 0.37‰ and −18.21 ± 0.24‰ at the intermediate and high pCO2 sites, respectively (13C is depleted in marine photosynthetic organisms under elevated pCO2414243Symbiodinium carbon requirements (~85%) are met via energy-demanding carbon-concentrating mechanisms (CCMs), whilst the remainder diffuses passively from seawater to the Symbiodinium cellspCO2 is elevated, CO2(aq) can replace HCO3− as the main carbon source for photosynthesis while energy-consuming CCMs become less important43Symbiodinium13CpCO2 in the proximity of the vent diffuse to the Rubisco, which favors 12C for carbon fixation and ultimately results in a lightning trend of δ13CS values. Krief et al. (2010) reported the same trend in two species of scleractinian corals after experimental exposure to high pCO2 in a controlled pCO2 system. While Krief et al. (2010) kept corals under elevated pCO2 for a period of 14 months, our in situ study at the CO2 vent site lends insight into a long-term exposure scenarioOur results suggest that elevated ectively . Many st15NT and δ15NS values did not change along the pCO2 gradient, suggesting that the anemones' function and performance reside within normal bounds close to the vent after long-term exposure to acidification conditions  demonstrated the high capacity of A. viridis to regulate against decreases in internal and external pH, thereby maintaining normal cellular metabolism and physiologyA. viridis to acidification conditions, as physiological data , part of the Aeolian Island chain, NE Sicily  in May 2pCO2) was ~300 m away from the CO2 vents; Site 3 (high pCO2) was in the proximity of the CO2 vents (~260 m). Sampling at the primary vent site ], selecting the constants of Mehrbach et al. (1973)2 s−1  and temperature were measured at all sites several times a day for 4 days using a pH meter . Water samples for total alkalinity (TA) analysis were collected from each site, cooled and stored in the dark until analysis. TA was quantified with a Metrohm 862 compact titrosampler73)2 s−1 55.A. viridis, a dominant benthic organism in Levante Bay, was prevalent throughout the study area. Sixteen anemones were collected randomly from each site at a depth of 1–2 m and immediately frozen until further analyses. To minimize any confounding responses due to age and/or size all samples were of similar size Symbiodinium density, chlorophyll a concentration and mitotic index) at the sampling site. Samples were weighed  and homogenized in 0.2 μm sterile filtered seawater (FSW) with an electric homogenizer . The homogenate and all anemones were immediately frozen and then transported on dry ice to the Interuniversity Institute for Marine Sciences (IUI), Israel, where they were stored at −80 °C pending analyses.13CDIC) and oxygen isotopic analysis (δ18Oseawater). Triplicate samples for δ13CDIC analysis were immediately poisoned upon collection with 60 μl saturated solution of mercuric chloride and stored in 60 ml brown bottles at room temperature until analysis. Triplicate samples for δ18Oseawater analysis were collected in 50 ml test tubes (Stardest) and stored at room temperature until analysis.Seawater samples were collected from the four sites for carbon isotopes of dissolved inorganic carbon  was further processed and analyzed for measurements of physiological parameters. Total protein analysis was performed by removing 100 μl of the tissue homogenate and sonication on ice with a Branson Sonifier B12  for 20 s. Quantification was done after Bradford (1976) using the Quick Start Bradford Protein Assay Kit and Quick Start Bovine Serum Albumin Standard Set Symbiodinium were used for chlorophyll a extraction in acetone (100%) at 4°C in the dark for 24 hours. Concentrations of chlorophyll a were measured using spectrophotometry  and calculated using standard equationsSymbiodinium cell. Symbiodinium densities were quantified from 4 replicate counts using a Neubauer haemocytometer and normalized to protein concentration. Mitotic index (MI) was measured as an indicator of Symbiodinium growth and was calculated as a percentage of doublets with a complete cleavage furrow observed per 1000 cells (n = 8 per sampling station)For measurement of algal characteristics, 2 ml of homogenate of each sample (n = 16) were centrifuged  and re-suspended four times in FSW. Re-suspended Symbiodinium was done by the following protocol. The homogenate was centrifuged for 5 min at 5000 rpm  to separate the algae (pellet) and the host tissue (supernatant). Visual inspections revealed no crossover of material between these components, but both were washed carefully.Sub-samples of 250 mg were excised from the tentacles of each anemone (n = 5 per site) and placed in sterile 15 ml falcon tubes (Stardest). After adding 1 ml 0.2 μm filtered seawater (FSW), an electric homogenizer  was used to homogenize the tissue extract for 2 min. Separation of anemone tissue and Symbiodinium pellet was then re-suspended in 1 ml FSW, homogenized, and centrifuged for 5 min at 5000 rpm . The procedure was repeated twice in order to remove remaining tissue. All samples were washed with double-distilled water (DDW) to remove any remaining salts. Both the host tissue and Symbiodinium samples were dried with a lyophilizer  for 24 h for further isotopic analysis.The host supernatant was homogenized and centrifuged for 10 min at 13,500 rpm , resulting in pelleted host material for analysis. The The isotopic measurements were made at the stable isotopes laboratory in the Department of Earth and Planetary Sciences, the Weizmann Institute of Science, Israel. The oxygen, carbon and nitrogen isotope measurements are reported in the conventional δ-notation.13C and δ15N of 240–270 μg of dried tissue and algae were analyzed using an elemental analyzer (CE 1110) interfaced to the MAT 252 mass spectrometer. Long term precision of working standards for δ13C is 0.05‰ and for δ15N is 0.1‰ relative to V-PDB and Air respectively (±1σ SD). The carbon to nitrogen ratios (C/N) of anemone tissue and Symbiodinium were calculated from simultaneous %C and %N.δ18Oseawater was analyzed by equilibrating 0.5 ml of samples with a mixture of 0.5% CO2 in He at 25 °C for 24 h. The samples were analyzed on a Gas Bench II connected in-line to a Finigan MAT 252 mass spectrometer. The results are reported relative to VSMOW with 0.08‰ (±1σ SD) long-term precision of the laboratory working standards.δ13CDIC analysis, 1 ml sea water was injected into vials, flushed with He gas, acidified with 0.15 ml orthophosphoric acid (H3PO4) and left to react for 24 h at 25 °C. The samples were analyzed on a Gas Bench II and Finigan MAT 252. The results are reported relative to VPDB with 0.08‰ long-term precision (±1σ SD) of the NaHCO3 laboratory standard.For δAll data was checked for normality using the Kolmogorov-Smirnov test and for homogeneity of variance using Cochran's test. In cases in which homogeneity of variance was achieved, we used one-way ANOVA and a multiple comparison test (Tukey). If homogeneity of variance or normality was not achieved, we used a non-parametric Kruskal-Wallis ANOVA and post-hoc Mann-Whitney U-tests for separation of significant factors. Differences between factors were considered significant for a P value < 0.05. Unless otherwise specified, mean values are presented ± standard error of mean (SEM). All data were analyzed using SPSS version 20 .R.H., E.M.B. and M.F. conceived the overall project. R.H. and E.M.B. conducted the field and laboratory work and analysed data. R.Y. and A.S. carried out stable isotope analyses. All authors reviewed and edited the manuscript.


Aberrant intramuscular triacylglycerol (TAG) storage in human skeletal muscle is closely related to insulin insensitivity. Excessive lipid storage can induce insulin resistance of skeletal muscle, and under severe conditions, lead to type 2 diabetes. The balance of interconversion between diacylglycerol and TAG greatly influences lipid storage and utilization. Diacylglycerol O-acyltransferase 1 (DGAT1) plays a key role in this process, but its activation and phosphorylation requires further dissection. In this study, 12 putative conserved phosphorylation sites of DGAT1 were identified by examining amino acid conservation of DGAT1 in different species. Another 12 putative phosphorylation sites were also found based on bioinformatics predictions and previous reports. Meanwhile, several phosphorylation sites of DGAT1 were verified by phosphorylation mass spectrometry analysis of purified DGAT1 from mouse myoblast C2C12 cells. Using single point mutations, a regulatory role of 3 putative phosphorylation sites was dissected. Finally, using truncation mutations, a potential domain of DGAT1 that was involved in the regulation of enzymatic activity was revealed. This study provides useful information for further understanding DGAT1 activity regulation. Triacylglycerol (TAG) is the main energy storage compound in mammals. Due to their hydrophobicity, TAG has an energy density six times greater than hydrated glycogen  to provide substrates for mitochondrial β-oxidation and the production of adenosine triphosphate (ATP).However, ectopic storage of TAG is closely related to human diseases, such as obesity, diabetes, and steatohepatitis. Insulin-regulated glucose metabolism is mainly accomplished by skeletal muscle in humans  and acetyl-CoA charged fatty acids are most likely involved in this process. Dgat1 encodes a protein of ~500 amino acids with molecular weight ~55 kDa. DGAT1 contains a large hydrophobic domain, localizes in the endoplasmic reticulum (ER), can form homo-tetramers through N-terminal interactions  to higher animals   Fig. . Among tan) Fig. , and T15an) Fig. , S17 (Huan) Fig. , S20 (Haan) Fig. , S40 (Tran) Fig. , and S67an) Fig.  were preTo confirm the phosphorylation of potential sites predicated by bioinformatic methods and to identify novel phosphorylation sites, we analyzed the enriched DGAT1 by phosphorylation mass spectrometry. We overexpressed Myc-His tagged mouse DGAT1 in C2C12, enriched the protein by nickel-affinity chromatography, and confirmed with immunoblots. The DGAT1 was eluted with 500 mmol/L imidazole with no detectable cytosolic protein contamination. We further concentrated DGAT1 with ultra-filtration  or glutamate (E) to mimic dephosphorylation or phosphorylation, respectively. These mutations showed different phenotypes when overexpressed in C2C12 cells (data not shown). However, only mutations of S83, S86, and S89 could apparently regulate DGAT1 enzymatic activity  in the N-terminus could have a great influence on the activity of DGAT1 in C2C12 cells. When each of these three serines was mutated to glutamate to mimic phosphorylation, there was an apparent increase in DGAT1 activity, suggested that phosphorylation at each of these three sites is likely to potentiate DGAT1 activity. However, we failed to detect phosphorylation at any of these three sites by phosphorylation mass spectrometry. The biochemically enriched DGAT1 might be at basal level when lacking upstream stimulating.Truncation of the N-terminus of DGAT1 increased enzymatic activity, suggesting that N-terminus possibly acted as a negative regulator, and the mechanisms underlying this phenomenon require further study. It is possible that the N-terminus is required to permit the formation of homo-tetramers, which may suppress enzymatic activity /myc-his A plasmid were from Invitrogen. Sodium palmitate and the TAG mixed lipid standard were from Sigma-Aldrich. Tritium labeled palmitic acid was from PerkinElmer. Myc antibody was from CST. Antibody recognized GAPDH was from Millipore. The goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were from ZSGB-BIO.2. For plasmid transfection, C2C12 cells were trypsinized, mixed with the indicated plasmids, and nucleoporated by a standard program according to instructions from Amaxa. The transfected cells were then seeded to 12-well plates and treated cells with FFAs as described previously  were maintained in DMEM (Macgene Biotech.) supplemented with 10% FBS (Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Macgene Biotech.) at 37 °C, 5% COCloning and mutagenesis primers are listed in Table 3H-palmitic acid. The medium was cooled to 37 °C, and cells were treated for 12 h. After FFAs treatment, cells were washed three times with PBS and then lysed with 1% Triton X-100 (in PBS) at room temperature for 20 min. Cell lysates were further processed with sonication and the protein concentration determined by BCA assay (Thermo). Lipids were extracted from the cell lysates by adding 1 mL lipid extraction buffer . The mixture was vortexed and then centrifuged at 20,000 g for 10 min at 4 °C. The bottom lipid phase was transferred to fresh tubes for further analysis.After C2C12 cells were transfected with the indicated plasmids and cultured for 24 h, the cells were treated with FFAs. Complete medium was pre-warmed to 60 °C and sodium palmitate was added to a final concentration of 500 µmol/L with 5 µCi/mL v/v/v). The lipids were visualized by iodine staining. The TAG and DAG fractions, identified relative to standards, were scraped into tubes. The lipids were quantified by liquid scintillation.The extracted lipids (30 µL) were spotted onto TLC plates (Yantai Jiangyou silicone company) and developed with neutral lipids developing buffer  and eluted with a step gradient of imidazole at 4 °C. The eluate was concentrated by ultra-filtration . The chromatography was followed by immunoblots. The concentrated DGAT1 proteins were separated and visualized by SDS-PAGE and Colloidal blue staining, and were excised for phosphorylation mass spectrometry analysis.DGAT1-Myc-His A overexpressed C2C12 cells were harvested in lysis buffer with 5 mmol/L β-glycerophosphoric acid, 10 mmol/L NaF, 1 mmol/L NaMS/MS spectra were searched by the Sequest algorithm against a database containing the mouse DGAT1 protein database and its reversed complement. Search parameters included a static modification of 57.02146 Da (carboxyamidomethylation) on Cys; dynamic modifications of 79.96633 Da (phosphorylation) on Ser, Thr, and Tyr; and 15.99491 Da (oxidation) on Met. Results were first filtered to contain only fully tryptic peptides, and then other cutoffs were established to achieve maximum sensitivity levels at 0.1% FP results using decoy matches as a guide. Sample-specific filters for the solution charge state and mass accuracy were used. A 3 Da mass tolerance window was applied separately to each analysis. After filtering by tryptic state, solution charge, and mass accuracy, only minimal filtering with Sequest scoring (XCorr and dCn) values at the level of the entire dataset was then required to achieve less than 0.1% FP rate.http://www.cbs.dtu.dk/services/NetPhosK/) and PhosphoSitePlus , and the parameters were set default.Cross-species sequence conservation was analyzed by ClustalX2 (Larkin et al. All experiment results were analyzed by GraphPad Prism 6.0 using one-way ANOVA.


Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD ‘code’ for co-transcriptional histone modifications.Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast  In contrast, the Spt5 CTD repeat array is variable, between taxa and within the Spt5 CTD array of a given species. The consensus repeated motif in human Spt5 is TP(M/L)YGS(R/Q) (Schizosaccharomyces pombe Spt5 contains a relatively homogeneous CTD consisting of 19 tandem copies of consensus TPAWNSGSK. The consensus repeat in budding yeast Spt5 (referred to as the CTR) is highly divergent (SAWGGQ) (in vivo (The RNA polymerase II (RNAPII) elongation complex coordinates transcription with key co-transcriptional events such as mRNA processing and histone post-translational modification. Association of processing factors and regulatory partners with the RNAPII elongation complex generally depends upon Rpb1, the RNAPII large subunit, and/or Spt5. Rpb1 and Spt5 have distinctive C-terminal domains (CTDs) comprising multiple repeats of a short motif; their CTDs interact with multiple regulators and are critical for assembly of a functional elongation complex . Whereas, methylated H3K79 and dimethyl forms of both H3K4 and H3K36 are all found broadly distributed across coding regions. These modifications have roles in nucleosome dynamics, mRNA processing and DNA repair –16. How Underlying the program of co-transcriptional histone modifications is an equally complex pattern of phosphorylation on the Rpb1 and Spt5 CTDs, which regulates their interactions with elongation factors. All five of the potential phosphoacceptor sites within the Rpb1 CTD motif are phosphorylated at various stages of the transcription cycle . Most phin vitro  and treated with the indicated concentration of 3-MB-PP1 for 3 h. Mycophenolic acid  was dissolved in DMSO and added to solid YES media at a concentration of 25 μg/ml.Liquid cultures were grown at 30°C using standard YES media . 3-MB-PP1  was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 50 mM. For inhibition of analogue sensitive alleles, cells were grown to early log phase  was used as a negative control.ChIP was carried out as previously described . The SptS. pombe in which a truncated but fully functional CTD  was replaced with CTD variants harboring single amino acid substitutions in all or some of the repeats , set1Δ  H3K4me; ), set2Δ H3K36me; ) and setH4K20me; )11 strain in which 11 of 14 Rpb1 CTD repeats were S5A, or a strain in which all 14 repeats were S5A in the context of an rpb1-CTD-S5A-MCE1 translational fusion. However, there was no effect on these modifications in the (S5)7(S5A)7 strain in which the CTD contains seven Ser5 heptads. Apparently, there is a threshold level of Ser5 content that can sustain H3K4me and H3K36me3 modifications. By contrast, H2Bub1 was only reduced in the rpb1-CTD-S5A-MCE1 strain, suggesting that this modification has a less stringent requirement for Rpb1 Ser5 content.We found that S5A mutation dramatically reduced the levels of all the co-transcriptional histone modifications tested without affecting H4K20me, affirming the general importance of this site for coupling histone modifiers to elongating RNAPII Figure . H3K4me1S2A mutant strain spt5-T1A mutation decreased H3K4me3 levels when combined with rpb1-S2A and led to an additive decrease in H3K4me3 in combination with rpb1-S7A, consistent with independent functions for the two CTDs in regulating H3K4me3 , sensitivity to which can often indicate a transcription elongation defect .It is possible that the observed effects of n defect . Whereas+brl2 gene, encoding a ubiquitin ligase for histone H2B. We observed decreased levels of Spt5 T1-P in the brl2Δ strain, in accord with the previously described positive feedback loop linking Spt5 T1-P and H2Bub1 (Since H2Bub1 and H3K4me depend on Spt5 CTD Thr1 (presumably via its phosphorylation), we tested whether the impact of Rpb1 Ser5 mutations on these modifications could reflect an effect of Rpb1 Ser5 on Spt5 T1-P. Immunoblotting using antibodies that recognize total Spt5 or Spt5 T1-P showed no effect of the Rpb1 S5A mutations on levels of Spt5 T1-P in extracts. As a control for these experiments we included extracts derived from a strain harboring a deletion of the d H2Bub1  assays demonstrated that association of Spt5 with the s Figure . A contrS. pombe Cdk12 orthologue) and Cdk9. Inhibition of Lsk1as via a 3-h treatment with 20 μM of the ATP analogue 3-MB-PP1 rendered the Rpb1 S2-P mark undetectable, as shown previously . We conclude that Lsk1 positively regulates H3K36me3 by phosphorylating Rpb1-Ser2.To determine whether the effects for Rpb1 CTD Ser2 and Ser5 mutations on histone modification were attributable to the lack of phosphorylation, we inhibited Rpb1 CTD kinases implicated in phosphorylation of these sites. We employed strains harboring engineered ‘analogue sensitive’ (as) kinase variants that are inhibited by bulky ATP analogues. We focused first on kinases shown to target Rpb1 Ser2: Lsk1 . Le. LeS. pon Figure . A knockas inhibition caused partial loss of H3K36me3 and Rpb1 S2-P .,41.lsk1Δ pathway .as caused dramatic reduction in the levels of H2Bub1 and H3K4me3  due to a reduction in ATP binding affinity. Thus, the fact that increased in H3K4me was apparent in the aslsk1 allele into rpb1-CTD-14 or rpb1-S2A strains. In the rpb1-CTD-14 background, aslsk1 caused a ∼50% increase in H3K4me3 and ∼30% increase in H3K4me2, as assessed by immunoblotting   ,58. Globgure S3) . This inas and Cdk9as decreased the levels of H3K36me3 more strongly than inhibition of either kinase individually . These data suggest that the requirement of Rpb1 Ser5 for co-transcriptional H3K36me3 in S. pombe involves a mechanism that is independent of Mcs6/Cdk7, the major Rpb1 Ser5 kinase in this organism.Combined inhibition of Mcs6y Figure . Howevery Figure ). Thus, 1 Figure , but Cdks Figure . H3K36meY1F, T4A and S7A mutations may have subtle effects on levels of Rpb1-S2P and/or Rpb1-S5P that contribute to their effects on histone modifications , inactivation of which has been shown to disrupt the entire TFIIH complex at gene promoters , raising the possibility that the S5A mutation exerts its effects by perturbing more than one mark (as inhibition in the rpb1-CTD-S5-MCE1 background with minimal effects on H3K36me and H2Bub1. It is also possible that the effects of the S5A mutation on co-transcriptional histone modifications are an indirect consequence of chronic S5-P depletion, or are due to effects of S5A that are unrelated to Ser5 phosphorylation. Either of these possibilities would imply that H3K36me is in fact independent of Rpb1 S5-P per se, an idea that would be difficult to reconcile with the biochemical evidence linking the H3K36 methyltransferase Set2 to Rpb1 S5-P (in vivo, experiments in vitro show that its preferred site on Rpb1 CTD-derived peptide substrates is in fact serine 5 (How do we reconcile the requirement of Rpb1 Ser5 for H3K36me with the relative insensitivity of this mark to loss of Mcs6/Cdk7 activity? Phosphorylation of both Ser5 and Ser7 was eliminated and Ser2 phosphorylation was reduced slightly in the one mark . Howeverpb1 S5-P ,25. We tpb1 S5-P . Second,serine 5 . Finallyserine 5 . Our dat


Amaranthus retroflexus pollen is very common in tropical and subtropical countries. In this study we aimed to produce a recombinant allergenic Ole e 1-like protein from the pollen of this weed. To predict cross-reactivity of this allergen (Ama r 1) with other members of the Ole e 1-like protein family, the nucleotide sequence homology of the Ama r 1 was investigated. The expression of Ama r 1 in Escherichia coli was performed by using a pET-21b(+) vector. The IgE-binding potential of recombinant Ama r 1 (rAma r 1) was evaluated by immunodetection and inhibition assays using 26 patients' sera sensitised to A. retroflexus pollen. The coding sequence of the Ama r 1 cDNA indicated an open reading frame of 507 bp encoding for 168 amino acid residues which belonged to the Ole e 1-like protein family. Of the 26 serum samples, 10 (38.46%) had significant specific IgE levels for rAma r 1. Immunodetection and inhibition assays revealed that the purified rAma r 1 might be the same as that in the crude extract. Ama r 1, the second allergen from the A. retroflexus pollen, was identified as a member of the family of Ole e 1-like protein. Sensitisation to Amaranthus retroflexus (redroot pigweed), a well-known species of the Amaranthaceae family which is found throughout the world, is an important trigger of respiratory allergies in different regions with temperate and dry climates, such as Saudi Arabia, Kuwait, India, Iran, the western United States, Australia, and the Mediterranean area  at 4°C for overnight. After blocking with a solution composed of 150 μL of phosphate buffered saline (PBS) and 2% bovine serum albumin (BSA) solution for 1 hour at 37°C, the plates were incubated with 100 μL of patients' sera for 3 hours at room temperature. Each well was then incubated for 2 hours at room temperature with a 1 : 500 dilution of biotinylated goat anti-human IgE antibody  in 1% PBS. The wells were then washed five times with PBS containing 0.05% Tween-20 (T-PBS) to eliminate unbound anti-IgE. Thereafter, 100 μL of horseradish peroxidase- (HRP-) conjugated streptavidin  was placed in each well, and each well was then incubated for 1 hour at room temperature. After five washes with T-PBS, each well received 100 μL of tetramethylbenzidine substrate solution  that was placed in each well, and the plate was incubated at room temperature for 20 min before the reaction was stopped by addition of 100 μL of 2 M HCl. Subsequently, the absorbance in each well was measured at 450 nm using an ELISA plate reader. All results were expressed as optical density (OD) units. An OD value four times higher than the average values of three determinations of a pooled sera from negative controls  was considered to be positive.Total serum IgE levels were evaluated in serum samples in duplicate using a commercially available ELISA kit according to the manufacturer's instructions . To evaluate the levels of specific IgE to A. retroflexus pollen by using the Chomczynski and Sacchi method [™ First Strand cDNA Synthesis Kit  according to the manufacturer's instructions. The primers used for cDNA amplification were designed according to the known nucleotide sequence for reported allergens from the Ole e 1-like protein family that have a high degree of amino acid sequence identity [™ PCR Cloning Kit , according to the manufacturer's instructions. E. coli TOP10 cells  were transformed with the ligation products using the manufacturer's protocol. A recombinant plasmid was selected by white/blue screening and then purified from the gel using a plasmid extraction kit . DNA sequence analysis was performed using the dideoxy method at the Bioneer Inc. .Total RNA was extracted from 100 mg ofi method . The firidentity , 21–23. http://www.ncbi.nlm.nih.gov/) under accession number KR870437.The obtained sequence was submitted to the GenBank database of the National Center for Biotechnology Information ( Not I and Xho I restriction enzyme sites were introduced at the 5′- and 3′-ends of the Ama r 1 cDNA by the polymerase chain reaction (PCR) method using two specific primers as follows: the sense primer 5′-TCCgcggccgcATGGGGAAGTGTCAAGCTGT-3′ (Not I restriction site is in lowercase) and the antisense primer 5′-CCctcgagTTAATTAGCTTTAACATCATAAAGATCC-3′ (Xho I restriction site is in lowercase). After PCR amplification, the resulting product was digested with Not I and Xho I restriction enzymes according to the manufacturer's protocol . The purified digested PCR product was ligated into the digested pET-21b(+) plasmid with the same enzymes. Correct constructs were transformed into competent E. coli BL21 (DE3) cells .To create a pET-21b(+) expression plasmid , restriction sites ofμg/mL of ampicillin and incubated at 37°C. Expression of the recombinant protein was induced by adding isopropyl β-d-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM [A clone of recombinant plasmid pET-21b(+)/Ama r 1 was inoculated into 2 mL of lysogeny broth (LB) medium containing 100 f 0.5 mM . AfterwaμL/well of the purified rAma r 1 at a concentration of 2 μg/mL in coating buffer  overnight at 4°C. The results were expressed in OD units. Based on the mean value of two normal sera, OD450 greater than three times the median values of negatives controls was considered to be positive.In order to assess the serum IgE levels to the purified rAma r 1, an indirect ELISA was developed as described above, except that the wells of the ELISA microplate were coated with 100  A. retroflexus , which was preincubated for overnight at 4°C with either 1000, 100, 10, 1, 0.1, or 0.01 μg of rAma r 1 as an inhibitor or with BSA as a negative control. The inhibition percentage was calculated using the following relationship: ELISA inhibition was performed as described above, except for a pooled serum (1 : 2 vol/vol) from patients allergic to A. retroflexus pollen extract and E. coli lysate and purified rAma r 1 were analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% acrylamide separation gels and under reducing conditions according to the method of Laemmli [ A. retroflexus pollen were electrotransferred to polyvinylidene difluoride (PVDF) membranes , as described elsewhere [ A. retroflexus allergy or with control sera (1 : 5 dilutions) for 3 hours. Biotinylated goat anti-human IgE  (1 : 1000 vol/vol in PBS) was added to the blotted membrane strips and incubated for 2 hours at room temperature. The unbound antibodies were removed from blots by washing with T-PBS and incubated at 1 : 10,000 vol/vol in T-PBS-HRP-linked streptavidin  for 1 hour at room temperature. After several washes with T-PBS, strips were incubated using the SuperSignal West Pico Chemiluminescent Substrate Kit  for 5 minutes, and proteins were then visualised by chemiluminescence using the ChemiDoc XRS+ System .Proteins from Laemmli . The mollsewhere . In brieμL of pooled serum (1 : 5 vol/vol) was incubated with natural A. retroflexus pollen extract , rAma r 1 , or BSA (as a negative control) overnight at 4°C with shaking. Preincubated sera were used to assess the reactivity of a PVDF membrane blotted with natural A. retroflexus pollen extract and rAma r 1.To study cross-inhibition among natural and recombinant Ama r 1, a mixture of 100  A. retroflexus pollen extract. All of these patients had significantly elevated specific IgE levels to the extract of A. retroflexus pollen . The mean OD450 for specific IgE in patients reactive to rAma r 1 was 0.95 ± 0.16   .The sequence analysis of Ama r 1 indicated an open reading frame of 507 bp coding for 168 amino acid residues with a predicted molecular mass of 18.379 kDa and a calculated isoelectric point (pI) of 4.70. We compared the deduced amino acid sequence of Ama r 1 with other allergenic plant-derived Ole e 1-like proteins in the protein database , and we  A. retroflexus pollen extract with molecular weights ranging from approximately 10 to 85 kDa  pLysS as a fusion protein with a His6-tag in the C-terminus. rAma r 1 was present in a soluble form in the supernatant, where it was further purified by Ni2+ affinity chromatography to yield purified protein. The purified rAma r 1 was quantified by using Bradford's protein assay, which showed that approximately 17 mg of recombinant protein had been purified from 1 L of the bacterial expression medium. SDS-PAGE revealed that the apparent molecular weight of the fusion protein was about 19 kDa  Allergen Nomenclature Subcommittee .A pET-21b (+)/Ama r 1 clone was constructed and confirmed by digestion witht 19 kDa . The all A. retroflexus pollen were further tested for IgE reactivity to rAma r 1 by immunoblotting assays. The results showed that the recombinant form of Ama r 1 was reactive with 10 individuals' sera. These results were consistent with those obtained by specific IgE ELISA  had significant specific IgE levels to rAma r 1 . Serum sgE ELISA . A. retroflexus pollen extract. The ELISA inhibition results revealed a dose-dependent inhibition of the IgE directed towards rAma r 1 in patients' sera positive to A. retroflexus. Preincubation of pooled sera with 1 mg/mL of rAma r 1 and A. retroflexus pollen extract showed significant inhibition  of IgE binding to rAma r 1 in microplate wells , and 17–20 kDa (glycosylated and nonglycosylated) in Plantago lanceolata (Pla l 1) [The open reading frame of Ama r 1 contained a sequence encoding an 18.37 kDa protein related to the molecular specifications of a known plant Ole e 1-like protein family , 14, 21.Pla l 1) , 22, 25. A. retroflexus pollen extract using pooled sera from the patients also revealed an IgE-binding protein band with an estimated molecular weight of 19 kDa .Immunoblotting assays off 19 kDa . The IgEThe results of immunoblotting assays for natural Ama r 1 with a molecular weight of 19 kDa were consistent with those obtained for rAma r 1. A nearly complete inhibition of IgE-binding to natural Ama r 1 was also obtained after preincubation of pooled sera with purified rAma r 1. It seems that rAma r 1 is composed of IgE epitopes similar to those of its natural counterpart. A. retroflexus pollen components and other allergenic members of the Amaranthaceae family  and some unrelated allergenic plants such as Acacia farnesiana and Prosopis juliflora has been described previously [ C. album (Che a 1), C. sativus (Cro s 1), and S. kali  . Identification of the Ama r 1 sequence will warrant further studies on the basis of in vitro assays to investigate the molecular basis of cross-reactivity among these important pollen allergens.Cross-reactivity betweeneviously , 7, 17.  A. retroflexus pollen, Ama r 1, with a detectably specific IgE in 38.4% of patients allergic to A. retroflexus pollen. Ama r 1 was identified as a member of the Ole e 1-like protein family. In addition, the results demonstrate that rAma r 1 expressed in E. coli has immunoreactivity similar to that of the natural form of the allergen. Analysis of the amino acid sequences of Ama r 1 and several allergenic members of the Ole e 1-like protein family from other plants also indicated that cross-reactivity between plants belongs to unrelated families, which may be predicted by the degree of amino acid sequence identity of potential conformational epitopes.In conclusion, in this study, we investigated a new allergen from A. retroflexus pollen and the abundance of it in different countries throughout the world, identification and production of the recombinant forms of common allergens of this pollen may lead to the exploration of new guidelines for diagnostic, therapeutic, and preventive purposes.Concerning the more prevalent of sensitisation to


Protein phosphorylation is a modification that offers a dynamic and reversible mechanism to regulate the majority of cellular processes. Numerous diseases are associated with aberrant regulation of phosphorylation-induced switches. Phosphorylation is emerging as a mechanism to modulate ubiquitination by regulating key enzymes in this pathway. The molecular mechanisms underpinning how phosphorylation regulates ubiquitinating enzymes, however, are elusive. Here, we show the high conservation of a functional site in E2 ubiquitin-conjugating enzymes. In catalytically active E2s, this site contains aspartate or a phosphorylatable serine and we refer to it as the conserved E2 serine/aspartate (CES/D) site. Molecular simulations of substrate-bound and -unbound forms of wild type, mutant and phosphorylated E2s, provide atomistic insight into the role of the CES/D residue for optimal E2 activity. Both the size and charge of the side group at the site play a central role in aligning the substrate lysine toward E2 catalytic cysteine to control ubiquitination efficiency. The CES/D site contributes to the fingerprint of the E2 superfamily. We propose that E2 enzymes can be divided into constitutively active or regulated families. E2s characterized by an aspartate at the CES/D site signify constitutively active E2s, whereas those containing a serine can be regulated by phosphorylation. In thUbiquitin Conjugation (UBC) domain and contains the catalytic cysteine, which forms a thioester bond with Ub4546495051525455E2 Ub-conjugating enzymes are critical in this cascade, as they regulate both the topology of the poly-Ub chains and the processivity of the polyubiquitination reaction454645464243Ubiquitin-like (Ubl) attachment to substrates57His-Pro-Asn (HPN) motif, Tyr87 and Asp127 play important roles in lowering the substrate lysine pKa to allow its nucleophilic attack of the Ube2I ~ SUMO thioester bond. Several studies have demonstrated the importance of these residues for the catalytic function of different E2s, especially the HPN motif5960616263Saccharomyces cerevisiae Rad6 and its human ortholog hHR6A (Ube2A), the site homologues to Ube2I Asp127 is a serine .Although several high-resolution three-dimensional (3D) structures of E2 and E3 enzymes have yielded many insights into the mechanisms of Ub or  Ser120, , whose pIn this study we investigate this site in the whole E2 superfamily by phylogenetic analysis and more than 60 atomistic MD simulations, which are a powerful approach to investigate the role of phosphorylation in atomic detail91215161718206566676869707129Noncanonical Ubiquitin Conjugating Enzymes (NCUBE) as they lack the conserved HPN motif and have structural reorganizations in the region surrounding the catalytic cleft7475−1 in In the E2 superfamily, the site, which corresponds to hHR6A Ser120 (CES/D), generally contains an acidic residue (mostly Asp) or a ‘phosphorylatable’ serine residue . ConversInterestingly, the conserved site is almost invariably accompanied by a proximal C-terminal proline . TherefoIn summary, we propose that a negatively charged residue at this site, which can be either constitutive or modulated by phosphorylation, is important for optimal E2 catalytic activity. We here defined an additional conserved site for the E2 superfamily, that together with HPN, the catalytic cysteine and the invariant tryptophanThe conserved patterns observed in our phylogenetic analysis prompted us to compare the 3D structures and dynamics of representative E2 enzymes, which feature either an aspartate or a serine at the CES/D site, by multi-replicate atomistic MD simulations in explicit solvent . In part29To compare the dynamic properties and the structural effects induced by different amino acids or phosphorylation at the CES/D site, we carried out a principal component analysis (PCA) of a Cα concatenated trajectory of all the MD simulations798081We thus quantified the similarities in protein dynamics of the different variants using the first 20 PCs, which account for more than 80% of the total variance of the system. We calculated the root mean square inner product (RMSIP) as a measure of overlap. In particular, we carried out a boxplot analysis  comparinIn summary, a PCA analysis of our MD trajectories shows that mutations at the CES/D site that abolish or impair (S120E and S120T) the Ub-conjugation activity induce perturbations in E2 native dynamics, whereas the other variants do not feature any remarkable effects.To further unveil the effects induced upon phosphorylation or mutation in the active site, we calculated the solvent accessible surface (SAS) of the catalytic cysteine in our simulations. The phospho-hHR6A and -Rad6 variants present a higher average solvent accessibility of the cysteine than the non-phosphorylated or S120D counterparts (hHR6A and Rad6) . The solMutations of the conserved CES/D site of hHR6A, Rad6 or Ube2I do not remarkably affect the Ub/Ubl-charging step of the E2, i.e., the step in which the E1 enzyme transfers the Ub/Ubl to the cognate E2292934We first monitored the distances between the substrate lysine and either CES/D side chain or the catalytic cysteine. We observed that the catalytically active or partially active variants  allow for interactions between the residue at the CES/D site and the substrate lysine at distances below 0.48 nm, with the phospho-variant exhibiting the minimal distance of ~0.38 nm. The interaction is not observed when alanine or threonine occupies the CES/D (distances higher than 0.7 nm). A similar trend is observed when we calculated the distances between the side chains of the substrate lysine and the E2 catalytic cysteine. The distances between the catalytic cysteine and target lysine residues in our simulated E2-RanGAP1 complex, correlate with an exponential behaviour to the experimentally observed Ub-conjugation activity of hHR6A on its substrate histone H2Ap127S simulations  thus cooperate for the optimal coordination of the substrate lysine within the E2 catalytic pocket . Furtherhttp://tree.bio.ed.ac.uk/software/figtree) was used for the tree illustration.We obtained the unrooted phylogenetic tree using the PHYLIP packageSupplementary Fig. S148S120A, hHR6AS120T, hHR6AS120E, hHR6AS120D, hHR6ApS120, Rad6S120A, Rad6S120D, Rad6pS120 is provided in 29D127S-RanGAP1, Ube2ID127A-RanGAP1, Ube2ID127E-RanGAP1, Ube2ID127T-RanGap1 and Ube2IpS127-RanGAP1).A summary of the starting structures, number of replicates and simulation length used for simulations of wild-type hHR6A, hHR6B, Rad6, Ube2I and Ubc1, along with hHR6AMD simulations were performed with Gromacs 4.59412We discarded the first ten ns of MD simulations from analysis, since they are required to reach stable main-chain root mean square values in all the MD replicates . For eacPCA of MD trajectories reveals large amplitude concerted motions in the MD ensemble through the eigenvectors of the mass-weighted covariance matrix of the atomic positional fluctuations98The cosine content of the principal components (eigenvectors) of the covariance matrix of atomic positional fluctuations is an index that can range between 0 (no cosine) and 1 (perfect cosine). It has been demonstrated that insufficient sampling lead to high cosine content values in MD simulations, representative of random motionsHow to cite this article: Valimberti, I. et al. E2 superfamily of ubiquitin-conjugating enzymes: constitutively active or activated through phosphorylation in the catalytic cleft. Sci. Rep.5, 14849; doi: 10.1038/srep14849 (2015).


Rangifer tarandus caribou from the Rivière-aux-Feuilles herd in northern Québec (Canada) with GPS telemetry collars and studied their space use. We measured contemporary  and historical  variations in freshwater-ice availability and evaluated the concurrent responses of caribou to these changes.Freshwater lakes and rivers of the Northern Hemisphere have been freezing increasingly later and thawing increasingly earlier during the last century. With reduced temporal periods during which ice conditions are favourable for locomotion, freshwater bodies could become impediments to the inter-patch movements, dispersion, or migration of terrestrial animals that use ice-covered lakes and rivers to move across their range. Studying the fine-scale responses of individuals to broad-scale changes in ice availability and phenology would help to understand how animals react to ongoing climate change, and contribute to the conservation and management of endangered species living in northern environments. Between 2007 and 2014, we equipped 96 migratory caribou Ice had a positive influence on caribou movement rates and directionality, and caribou selected ice and avoided water when moving across or in the vicinity of large water bodies. When ice was unavailable, caribou rarely swam across (6 % of crossings) and frequently circumvented water bodies for several km. Although ice phenology did not change significantly during our study, climate projections indicated that ice availability could decrease considerably before the end of the century, generating a ~28 % increase in distance travelled by caribou during the early spring and fall migrations.We demonstrated that ice availability influenced the movements of a migratory arctic ungulate. Warmer air temperatures in the Arctic will undoubtedly modify the phenology of ice forming on freshwater lakes and rivers. If migratory caribou are unable to adjust the timing of their migrations, they could be forced to circumvent unfrozen water bodies more frequently and over broader areas, which may increase the distance, time, and energy they use to reach wintering areas. The long-term conservation of wide-ranging species will ultimately depend on our ability to identify the fine-scale behavioural reactions of individuals to broad-scale changes in climate and land use.The online version of this article (doi:10.1186/s40462-016-0079-4) contains supplementary material, which is available to authorized users. Climate is changing rapidly , and theUrsus maritimus  reveals that caribou in Northern Québec are initiating their spring and fall migrations earlier. This response may allow caribou to avoid a potential mismatch between the timing of migration and freshwater ice availability. Assuming caribou are unable to adjust the timing of their migrations further , delaysCaribou in Northern Québec seemed to favour ice substrates during migrations and winter displacements. Their movements in water were slower and more sinuous than their movements on ice. The relatively long “pauses” caribou often made on lake shores could have allowed them to assess the quality  of the ice before crossing . CaribouNeovison vison) spend 2.7 times more energy swimming compared to running [Caribou swim at the water surface using quadrupedal paddling, which is amongst the least efficient types of swim in vertebrates . Few com running . To cove running . It is pR.t. platyrhynchus) increased their displacement distances following rain-on-snow events, which are predicted to occur more frequently during warm winters. In Northern Québec, we revealed a link between caribou movements and inter-annual variations in ice availability. Caribou avoided water and selected ice in years with long and short ice-availability periods, respectively, suggesting that caribou were able to respond to ice availability and, indirectly, prevailing climatic conditions. Ice availability could thus be one of many drivers explaining the distribution of caribou following climate change. Predicting future migration routes of caribou, however, remains challenging [The movements of large herbivores are influenced by a suite of environmental and anthropogenic factors, including the type of substrate , topograllenging .Antilocapra americana) and mule deer (Odocoileus hemionus) of the Greater Yellowstone Ecosystem  to migrate through relatively narrow bottlenecks surrounded by topographic and anthropogenic features. Although Northern Québec is much less disturbed than Wyoming [Other landscape features, notably anthropogenic disturbances, also have the potential to disrupt ungulate migrations . Berger  Wyoming , 26, the Wyoming . Hydroel Wyoming . Along tThe dataset supporting the conclusions of this article is available in the Dryad Digital Repository (DOI:10.5061/dryad.4k275).