Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response.Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in  Arabidopsis thaliana, we found AtPLC2 as the enzyme that is decisive in phosphoinositide metabolism by analytical lipidomics of the gene knockout study. Functional characterization of AtPLC2 knockout plant enabled us to find two novel roles of phosphoinositides: requirement in seedling growth and ER stress tolerance. Because economically important environmental stresses such as salinity and temperature upshift cause ER stress, our findings may open up a new avenue in addressing ER stress responses via phosphoinositide signaling.Plant growth requires continuous signal transduction in response to ever-changing environment. Phosphoinositides represent primary lipid-derived signals that are involved in various plant responses to surrounding environment. However, enzymes that determine the complex phosphoinositide molecular profiles remained elusive due to the existence of a number of candidate genes for each step. Here, in  GUS staining was observed in trichomes, particularly at the base, in developing true leaves of ProPLC2:PLC2-GUS transgenic plants . In agreement with GUS staining, strong signals appeared at the tip of main roots  media showed growth retardation both in shoots and roots (plc2-1 mutant were fully complemented in two independent lines harboring a genomic sequence of AtPLC2 (ProPLC2:PLC2) in the plc2-1 mutant (plc2-1 ProPLC2:PLC2 #1 and #3) in root length, fresh weight and dry weight (plc2-1 mutant was introgressed into Columbia-0 (Col) ecotype by backcrossing six times. In consistent with that in the Ws background, the plc2-1 (Col) mutant showed growth retardation both in shoots and roots (AtPLC2 in the plc2-1 (Col) [plc2-1 (Col) ProPLC2:PLC2] as shown in plc2-1 mutant is due to knocking out of AtPLC2.The seedlings of nd roots . The ovend roots . The frend roots . These gy weight . To exclnd roots . Moreoveplc2-1 mutant against the tunicamycin (TM)–induced ER stress. TM inhibits the protein N-glycosylation pathway in the ER and thus induces the ER stress. Treatment of TM produced growth defects in the wild-type seedlings: smaller rosette leaves and shorter roots  ProPLC2:PLC2 transgenic plant: reductions of root length and fresh weight upon the ER stress were 26.7% and 19.9%, respectively (binding protein 3 (BiP3), calnexin (CNX) and calreticulin (CRT) . After incubation in the dark at 37°C for 30 min, staining was stopped by replacing the solution with 70% ethanol. For colored tissues, pigments were removed by immersing the tissue in ethanol:acetic acid = 6:1 (in vol).Harvested fresh tissues other than roots were immediately immersed in ice-cold 90% (v/v) acetone for 15 min, then with GUS staining solution [10 mM EDTA, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.1% (w/v) Triton X-100, 0.5 mg/ml X-Gluc  analysis, the plants were grown on MS media for 7 days. Then, the seedlings were transferred to MS media containing 5 μg/ml tunicamycin, and were harvested at the times indicated as described . DimethyProPLC2:PLC2-Venus transgenic line in 12-day-old seedlings was observed under a microscope  equipped with LCI Plan-Neofluar 63x/1.3 NA immersion, Plan-Apochromat 20x/0.8 NA, and Plan-Apochromat 10x/0.45 NA. For plasma membrane staining, seedlings were immersed in 10 μg/ml of FM 4–64  for 10 min and subjected to confocal microscopy observation. For nucleus staining, samples were immersed in 10 μg/ml of DAPI  for 10 min and subjected to confocal microscopy observation. Images were captured by use of LSM 510 v3.2  with filters for Venus , FM 4–64 , DAPI , and chlorophyII autofluorescence .Venus expression in the AtPLC6 using plant RNeasy mini kit  according to the manufacture’s instruction. RNase-free DNase  was used during the on-column digestion to remove genomic DNA contamination. For AtPLC6, total RNA isolated from the flowers of 5-week-old plants were used because AtPLC6 highly expresses during flower development [Actin (ACT) was used as a control. The primers used are listed in Total RNA was isolated from 2-week-old seedlings except elopment . The cDNACT used as an internal control [Total RNA was isolated from 7-day-old seedlings using TRI reagent  including DNase treatment according to the manufacture’s instruction, and cDNA was synthesized by the SuperScriptIII First-Strand Synthesis System . qRT-PCR was performed with the 7500 Real Time PCR System . The comparative threshold cycle (CT) method was used to determine the relative amount of gene expression, with the expression of  control . The meaS1 Table(XLS)Click here for additional data file.


Purpose. To investigate the preventive effect of resveratrol (RES) on the formation of portal vein system thrombosis (PVST) in a rat fibrosis model. Methods. A total of 64 male SD rats, weighing 200–300 g, were divided into five groups: Sham operation, Splenectomy I, Splenectomy II, RES, and low molecular weight heparin (LMWH), with the former two groups as nonfibrosis controls. Blood samples were subjected to biochemical assays. Platelet apoptosis was measured by flow cytometry. All rats were euthanized for PVST detection one week after operation. Results. No PVST occurred in nonfibrosis controls. Compared to Splenectomy II, the incidences of PVST in RES and LMWH groups were significantly decreased (both p < 0.05). Two rats in LMWH group died before euthanasia due to intra-abdominal hemorrhage. In RES group, significant decreases in platelet aggregation, platelet radical oxygen species (ROS) production, and increase in platelet nitric oxide (NO) synthesis and platelet apoptosis were observed when compared with Splenectomy II , while in LMWH group only significant decrease in platelet aggregation was observed. Conclusion. Prophylactic application of RES could safely reduce the incidence of PVST after splenectomy in cirrhotic rat. Regulation of platelet function and induction of platelet apoptosis might be the underlying mechanisms. Hepatic cirrhosis was most commonly caused by viral hepatitis and alcohol abuse  with hepPortal vein system thrombosis (PVST) referred to thrombosis in the portal vein, splenic, and superior mesenteric veins or intrahepatic portal vein branches . It was Resveratrol , a natural polyphenol, was first isolated from the roots of white hellebore in 1940  and is nIn light of these, the present study aimed to investigate the preventive effect of RES on the formation of PVST in rats after splenectomy in the context of liver cirrhosis. The findings will provide some clues that RES can be a potential antithrombotic agent for reducing PVST formation after splenectomy for cirrhotic patients.4, Sinopharm Chemical Reagent Co. Ltd. Shanghai, China; 0.6 mL/kg of body weight) in olive oil, twice a week for six weeks, and was confirmed by liver biopsy when animals were euthanized. All animals were allowed free access to water and standard laboratory chow, except for an overnight fast before undergoing surgery. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals . All procedures were reviewed and approved by the Ethics Committee, Xi'an Jiaotong University Health Science Center.Male Sprague-Dawley (SD) rats weighing between 200 and 300 g 9 to 10 weeks old were purchased from the Laboratory Animal Center of Xi'an Jiaotong University Health Science Center . Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride . Dimethyl sulfoxide (DMSO) and RPMI-1640 were purchased from Wuhan Boster Biological Technology, Ltd. . The RES was dissolved and sterilized in DMSO and then diluted in RPMI-1640 to 5 mg/mL.α = 0.05 and β = 0.20), when the forecasting incidences of PVST in nonfibrosis controls, fibrotic rats without anticoagulation, and fibrotic rats with anticoagulation were 1% [We estimated that a total of 64 rats would be needed to detect a difference between groups, with a one-tailed chi-square test  at a count of 1 × 108/mL.5 mL of PRP was added with equal volume of the washing buffer  and centrifuged at 800 ×g for 15 min. The pellet was resuspended in washing buffer and washed twice. Then the platelets were suspended in Krebs solution  as agonist to 0.5 mL platelet suspension and was determined using a computerized dual channel Chronolog Aggregometer  at 37°C as previously described [Platelet aggregation was initiated by adding 10 escribed . At the 8/mL) were loaded with 5 μM of 2′,7′-dichlorofluorescein diacetate  for 15 min at 37°C. Then the platelet samples were centrifuged at 800 ×g for 10 min and resuspended in 500 μL of Krebs solution. The ROS-generated fluorescence was read using a Becton Dickinson flow cytometer  equipped with a 488 nm wavelength argon laser, 510–540 nm band pass filter, as described [For the measurement of the generation of ROS by activated platelets, washed platelets in Krebs solution  as reported previously .For measuring platelet apoptosis, an Annexin-V Apoptosis Detection Kit FITC  was used according to manufacturer's instructions.t-test or chi-square test using SPSS 11.5 statistical software . A p value <0.05 was considered statistically significant.Continuous data were presented as mean ± SD and categorical data were presented as frequencies. Statistical differences were calculated by Student's p = 0.037 and p = 0.039, resp.).No PVST occurred in Sham group and Splenectomy group I. For the other three groups of rats with liver fibrosis, the incidence of PVST 1 week after operation was 43.75% (7/16) in Splenectomy group II, 6.25% (1/16) in RES group, and 7.14% (1/14) in LMWH group . Both pretreatment with RES (50 mg/d per nasogastric tube for 10 days) and a short term postoperative administration of Enoxaparin  showed a significant decrease in the incidence of PVST than in Splenectomy group II .p < 0.001). Both pretreatment with RES (50 mg/d per nasogastric tube for 10 days) and a short term postoperative administration of Enoxaparin  caused a significant reduction in collagen-induced platelet aggregation 1 week after splenectomy, compared with Splenectomy group II .Data on the effects of RES and Enoxaparin on platelet aggregation induced by collagen are presented in p < 0.001). However, this increase in ROS formation was significantly alleviated by in vivo pretreatment with RES (p < 0.001), but not by postoperative administration of Enoxaparin . However, this decrease in NO release was significantly alleviated by in vivo pretreatment with RES (p < 0.001) but not by postoperative administration of Enoxaparin , suggesting more platelet apoptosis occurring in rats pretreated with RES.As seen in Although the pathogenesis of PVST following splenectomy is still controversial, it is generally agreed that it is related to blood hypercoagulability , hemodynFor cirrhotic patients, preoperative platelet count was generally lower than normal due to concurrent hypersplenism. Under such condition, removal of spleen would lead to a platelet rebound phenomenon and the soaring count and augmented aggregation competence of platelet after operation would contribute to the hypercoagulable state, which, in combination with other underlying causes, may synergistically result in the formation of PVST.Currently, the prophylaxis of PVST after splenectomy in liver cirrhosis remains controversial. The main concern is that prophylactic anticoagulation might induce relevant complications, mainly related to bleeding. So far, the management of PVST after splenectomy is mainly based on individual experience . Severalp < 0.05 and p < 0.001, resp.). However, two rats in LMWH group died before euthanasia due to intra-abdominal hemorrhage, suggesting that the anticoagulants under such conditions should be used more carefully. We speculated that a possible reason might be the increased release of tissue plasminogen activator and consequent fibrinolysis induced by LMWH.The activation of thrombin is a crucial step in the formation of thrombosis. LMWH could suppress the activation of thrombin via suppressing coagulation active factor X. Moreover, LMWH could reduce platelet aggregation induced by thrombin and inhibit platelet aggregation in response to adenosine diphosphate (ADP) . In the 2+ influx into thrombin-stimulated platelets [κB/Rel-dependent transcription in both endothelial cells and monocytes [One of the most studied actions of RES was its antiplatelet aggregation property. RES exerted this function through multiple mechanisms. First, RES could inhibit both type I collagen mRNA expression and platelet adhesion to the collagen, which was the initiation of platelet activation ; second,latelets ; third, onocytes .Besides, RES was shown to reduce oxidative stress by different mechanisms including activation of antioxidant enzymes, inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, and chelation of metal catalysts , thus reOf late, there were reports of plant-derived molecules, such as RES, causing platelet apoptosis , 42. FurA previous study had reported that RES exerted protective effect on rats subjected to PVST via its antioxidant and antiaggregant properties . The autIn the present study, we investigate the preventive effect of RES on the formation of PVST after splenectomy in a rat fibrosis model. The reported interval between splenectomy and PVST development varied and in our previous study the interval was 4 days . Taking Taken together, our present study has provided evidence for the first time that RES exerts its preventive effect on PVST formation after splenectomy, through simultaneously inhibiting aggregation, decreasing ROS, and increasing NO production, as well as stimulating apoptosis effects on platelets, in a rat fibrosis model. RES, as a natural substance found in multiple plants, could be a promising candidate in the development of an effective antithrombotic therapy for the prevention of PVST after splenectomy for cirrhotic patients.


Vulpes lagopus) during a single winter in the High Canadian Arctic, we investigated the detection range of arctic foxes by detecting hotspots of fox activity on the sea ice.The scale at which animals perceive their environment is a strong fitness determinant, yet few empirical estimates of animal detection ranges exist, especially in mammalian predators. Using daily Argos satellite tracking of 26 adult arctic foxes  contains supplementary material, which is available to authorized users. The scale at which animals perceive their environment determines their ability to locate resources and avoid predators , 2, and Corvus corax) and bald eagles  [Scavengers should be excellent study models to analyze detection range of animals, for they need to locate carrion, a spatially and temporally aggregated resource pulse that can be readily identified by observers. Yet, whereas many studies have focused on the organization of scavenger guilds , 17–20, ephalus) , 17, whiephalus) , can covephalus)  and alsoephalus) , 11. Mamephalus) , 23.Vulpes lagopus) are facultative scavengers that can feed on marine carrion found on the sea ice during winter [Arctic foxes  and collared lemmings (Dicrostonyx groendilandicus), but also on greater snow geese (Chen caerulescens atlantica) [Ursus maritimus) [Phoca hispida) pups [We worked in the south plain of Bylot Island , which is part of Sirmilik National Park, Nunavut, Canada. The 600 kmlantica) . Arctic lantica) . Land–falantica) . Arctic ritimus) . They cada) pups  when theda) pups .As part of an ongoing study on arctic fox ecology, 6 to 26 adults were collared annually with Argos Platform Terminal Transmitters from 2007 to 2010 . Collars weighed 2.5–4.4 % of individuals’ body mass. We captured adults between May and August using padded leghold traps . If necessary, we anaesthetized animals through injection of medetomidine (0.05 mL/kg) and ketamine (0.03 mL/kg). We used atipemazole (0.05 mL/kg) as an antidote. All capture and handling of animals was approved by the appropriate authority and ethical committee . Field research was approved by the Joint Park Management Committee of Sirmilik National Park of Canada (permit # SNP–2009–2218).n = 26) since the beginning of the study. In addition, foxes foraged primarily on land during the winter 2010–2011, making relatively few excursions on the sea ice . We set speed values from GPS data collected from the same population . After the removal of a location, the filter recalculated and evaluated again the speed between the new successive locations. We mapped locations using ArcMap 9.3 . The mean (± SD) number of locations per day per collar was 5.3 ± 2.1. We used locations from 25 October 2010 to 1 June 2011, starting from when the sea ice was completely formed around Bylot up to the beginning of cub rearing. Seven foxes dispersed during winter . First, we kept only positions with a location class of LC 3, 2 and 1, respectively corresponding to errors < 250 m, between 250 and 500 m, and between 500 and 1500 m . We thenmove [2m) parameter that reflects changes in the movement behavior of the animal along the trajectory [w locations along the path, and compares fit of models that use either one or two estimates of σ2m for the window. Models using two estimates of σ2m split the window in two parts at all possible breakpoints. The model with the lowest Bayesian Information Criterion (BIC) value is chosen. The sliding window produces several estimates of σ2m for each segment, which are then averaged for the segment. The dBBMM requires a user–defined margin of at least 3 locations on each end of the window in which no breakpoint can be estimated [We used the dynamic Brownian bridge movement model (dBBMM) implemented in the R package move  to estimajectory . Like thajectory . To detestimated . The chostimated . Followistimated , the comRangifer tarandus) or muskox (Ovibos moschatus) carcasses, we did not expect scavenging on land since there are no large herbivores in the study area. To facilitate visualization of the UD of the sea ice, we thus substracted ad hoc all land cell probabilities and re–scaled the resulting UD so that it summed to 1. One fox using repeatedly the same area sometimes led to high cell values in the population–level UD. In addition, pair mates foraging on the sea ice close to their home ranges also yielded high cell values along the coast , with fox and hotspot identities included as random grouping variables. As a fox closer to the coast may be more aware of events occurring on the sea ice, we also assessed if the likelihood that a fox joined a hotspot depended on the distance of its home range center to the coastline. We compared each model with a null model using a likelihood–ratio test. We similarly assessed the relationship between the day of arrival of a fox at a hotspot and the length of its stay in this hotspot using a linear mixed effect model with foxes present at the hotspot as sampling units  and hotspot identity included as a random grouping variable. We could not add fox identity as a second random factor in this analysis because of the low sample size. This variable was however not significant  when tested as a single random effect using a likelihood–ratio test and restricted maximum likelihood (REML) models [nlme [lme4 [We used a mixed logistic regression to assess whether the probability that a fox joined a hotspot depended on its distance from it. Foxes that had not dispersed from Bylot Island were used as sampling units  models . Finallyls [nlme  and lme4me [lme4  librarieThe 26 tracked foxes  yielded 47,634 locations which were reduced, after filtering, to 23,779 locations of LC 3, 2 or l . Foxes arriving late at a hotspot stayed fewer days than those arriving early. On average, a fox decreased the length of its stay at a hotspot by 1 day for every 4 days passing since the beginning of the hotspot . However, the distance a fox traveled to a hotspot did not influence the length of its stay .Thirty–five movements to the sea ice by 13 individuals formed five spatio–temporal hotspots on the land–fast ice of Navy Board Inlet Fig. . Four toFrom October to mid–March , the 13 foxes that were detected at least once at a hotspot remained inland 83.7 ± 9.1 % (range: 66.7–95.6 %) of the days they were tracked. These foxes were located at hotspots 35.2 ± 24.1 % of the days they used the sea ice. However, for nearly half of these foxes (6 out of 13), 44.8 to 75 % of their days spent on the sea ice were at a hotspot , with some foxes present at identified hotspots during a relatively high proportion of the days they spent on the sea ice. In addition to the use of the sea ice by foxes being relatively low and often associated with visits to hotspots, four individuals left their inland home range for the first time of the winter to go to a hotspot. These results may indicate that foxes did not forage on the sea ice routinely during that winter, but rather used this habitat opportunistically, when the availability of carrion was detected, possibly from the home range. On these occasions, foxes traveled long distances to reach sea ice hotspots, often more than 10 km and up to 40 km. Altogether, the above suggests that foxes may be attracted to carrion from a relatively long distance. Note that although some fox trips on the sea ice appeared unrelated to any hotspot, not all foxes were collared in the area, and these trips could thus have led to undetected hotspots.2) of hotspots may be explained by a combination of 1– polar bears leaving clusters of carcasses rather than single carcasses, 2– foxes moving in the vicinity of carcasses between meals, and 3– Argos location error.The dark and cold conditions prevailing during the study prevented us from sampling food sources on the sea ice, yet we can assume that food of marine origin was present. In addition, since all hotspots were detected from December–February, before seal pups were born, this food must have been carrion. Whereas all species of arctic whales leave the area before winter , ringed An alternative, non–mutually exclusive hypothesis may explain the specific case of Hotspot–3 Fig. . A few dThe concurrent visit of the same areas by several individuals may indicate that they are guided by the same cues. Mammalian scavengers rely mostly on olfaction to find carcasses , 20, thuCanis familiaris) tracking seal lair or excavation [Canis latrans) from 12.2 km and 20.5 km, respectively [. [Diomedea exulans) is capable of olfactory detection from over 20 km.Among other arctic mammals, polar bears are notorious for their excellent sense of smell , 52. Thecavation , 57 or wcavation  reportedectively , but it ively [.  who founVulpes vulpes) track wolf trails in the snow to find their kills [Ursus americanus) [a priori knowledge of food location. All of these hypotheses regarding long–range detection need testing and the arctic fox study system could offer productive avenues for experimental research, especially in late winter when light and temperature constraints are released in the Arctic. In particular, the experimental use of seal carcasses, coupled with Argos telemetry and camera traps [Other foraging tactics could be involved, such as the following of cues left by polar bears in the same way coyotes, ravens and red foxes  , coarse–ricanus)  or interricanus) . Until tra traps , could yra traps .Because of the patchiness and unpredictability of marine resources, foraging on the sea ice is usually considered to be more risky for foxes than foraging on land , 67. OurIn general, the lack of information on resource detection abilities could lead to erroneous conclusions in animal movement research. For example, a straight–line movement of an animal towards a resource can result either from goal–oriented navigation based on a cognitive map or from a discovery made using long–range detection. Distinguishing between these alternatives requires measuring detection distances for specific resources . Our resOur results, based on the most extensive set of satellite tracking data obtained to date on territorial arctic foxes, provide the first indication that this species may have a large food detection range that extends far beyond the boundaries of the territory. This study presents estimates of detection range useful for interpreting and modeling movements of this mobile predator. The ability to detect a food source from a long distance may shape foxes’ decisions to stay inland or move onto the sea ice during winter. As mobile links, foraging arctic foxes contribute to the transfer of resources from the marine to the terrestrial ecosystem , 31. A l


The aim of this study was to design a new minimally invasive percutaneous lag screw guide apparatus and to verify its adjuvant treatment of acetabular anterior column fracture on pelvis specimens.This guide apparatus was self-developed based on the principles of “two points form a line” and “Rectangle”. Using C-arm fluoroscopy, this guide apparatus was used to conduct minimally invasive percutaneous lag screw internal fixation of acetabular anterior column fractures. Ten hollow lag screws were placed into 5 pelvis specimens.Result showed no sign of any screws puncturing the cortex or entering into the hip joint on radiological assessment. The cross-section reconstructed vertical distance to the screw, on the cross-section acetabular notch and the cross-section of the screw where the distance of between the screw and the iliopectineal line’s arc roof was at its shortest, indicate that at all points  under the line with an inclination of 1 (namely T = R-r) the screw is within the cortex and does not puncture the acetabula anterior column or enter into the hip joint.We may conclude that this self-developed guide apparatus solves the screw precision problem during the treatment of acetabular anterior column fractures through a minimally invasive percutaneous lag screw. Pelvic fractures are mainly caused by high-energy injuries and are usually followed by multiple injuries. The traditional open reduction and internal fixation (ORIF) treatment will aggravate the injury; therefore, Roberts  claimed Minimally invasive percutaneous lag screw is a stable and reliable technology that has slight injury . For traFor the traditional minimally invasive percutaneous lag screw technique, the position of entry point is always determined first, followed by the direction. The puncture angle has strong subjectivity, because there is no supporting point in the acetabular and no correct direction when the guiding needle drilled during the operation. During the drilling, X-ray fluoroscopy is repeatedly used to determine the direction and to obtain satisfactory results. In addition, the need for readjustment of the needle depends on the operator’s subjective feeling, which obviously prolongs the time of the operation and increases the X-ray exposure time for the physician and the patient. Carmack  showed tBased on the principle of “two points form a line”, the position and direction can be determined by the screw’s entry and exit points. To self-develop minimally invasive percutaneous lag screw guides the apparatus has to ensure that the guiding needle or screw gets through the entry and exit point and enter the anterior column cortex, to reduce the complication of screw puncturing out, simplify the operation and reduce the radiation during the operation.This in vitro study was approved by the Ethics Committee of Chinese PLA General Hospital. Due to the fact that the cadaver pelvises used in this study were provided by Anatomy Lab of Chinese Aerospace General Hospital, no informed consent was needed. For this study we used 5 pelvises, self-made pelvis upper and lower pads, a C-arm X-ray machine (SIEMENS ARCADIS ORBIC), minimally invasive percutaneous lag screw guiding apparatus, and protractor.The principle of “Two Points Form a Line” helps determine the position of the two points, the entry and exit points. Based on the structure of the rectangle, which shows that the length of the corresponding two sides are equal, the minimally invasive percutaneous lag screw guide apparatus is developed and is expected to solve the difficult problem that the hollow screw cannot be placed successful in the acetabula anterior column Fig. .Fig. 1ThThis apparatus has two important parts, including two locators A1 and A2), two side connection bars (B1 and B2) and one square connection bar (C), which ensure that the entire apparatus form a rectangle and fulfill the principle of “Two Points Form a Line”.  as the transverse axis and T as the vertical y-axis to describe the distribution. When points  were under the line with an inclination of 1 (namely T = r-r), the screw was suggested to be within the cortex and not puncturing the acetabula anterior column or the hip joint.After the minimally invasive percutaneous lag screw fixation operation on acetabular anterior column fractures were performed, 10 hollow lag screws were placed into five pelvises using the minimally invasive percutaneous lag screw guide apparatus. All the screws were placed in one trail.Visually, we observed the five pelvises and no hollow lag screw could be seen puncturing the cortex or entering the hip joint.When the operation was completed, the five pelvises were examined under the C-arm to obtain views from the front and back, as well as inlet and outlet views of the pelvis. The results are presented in Fig. After the experiment, CT scan and 3-D reconstructions were obtained for the five pelvises to confirm the position of the screws further see Fig. .Fig. 6aIn Fig. Acetabula anterior column fractures are caused by high-energy injury. The traditional method is to treat with open reduction and internal fixation. However, this approach has disadvantages such as significant tissue injury, blood lost and slower recovery. The wound is easily infected and may result in injury to blood vessels and nerves and heterotopic ossification –14. TherHowever, due to the high technical requirement, it depends to a large extent on the operator’s subjective feeling and lacks objective evidences. The guiding needle will definitely deviate from the correct position and puncture the cortex and enter into the pelvis cavity or the hip joint causing injury to blood vessels and nerves. Therefore, its clinical application is limited , 16. TheBy introducing the principle of “two points form a line”, the guide apparatus dramatically simplifies and improves the precision of the screw placement. The task group shaped the device to a rectangle. During the operation, a screw can be accurately placed under the guidance of the guiding needle, whose direction has been determined by two points on the four sides of the rectangle as the entry and exit points. In other words, as long as the two points on the guide apparatus are determined, precise screw placement can be achieved.Another obvious advantage of the guide apparatus is that when the guide apparatus is used to place anterior column hollow screws, there is no need to shoot X-ray images, such as the traditional front, inlet and outlet views and to repeatedly obtain images to confirm the position and direction of the guiding needle during the operation. The guiding apparatus does not need repeated fluoroscopy instead the obturator oblique and the iliac oblique views are needed to determine the position of the two points. The group’s experiment reduced the times of radioscopy and shortened the operation time by using the minimally invasive percutaneous lag screw guide apparatus.The guide apparatus needs further verification with follow up cadaver experiments and clinical application.In conclusion, the proposed minimally invasive pecutaneous lag screw guide apparatus should be used after miniinvasive or closely anatomical reduction of fractures, which include manual reduction, traction and locations with several K-wires under intro-operative fluoroscopy,and based on comprehensive knowledge of the pelvic anatomy and fractures. According to the principle of “two points form a line” and the shape of the Rectangle, the self-developed minimally invasive percutaneous lag screw guide apparatus solves the screw precision problem for the treatment of acetabular anterior column fracture with minimally invasive percutaneous lag screw and is a feasible and effective apparatus for its treatment.


Establishing the trajectory of thinning of the West Antarctic ice sheet (WAIS) since the last glacial maximum (LGM) is important for addressing questions concerning ice sheet (in)stability and changes in global sea level. Here we present detailed geomorphological and cosmogenic nuclide data from the southern Ellsworth Mountains in the heart of the Weddell Sea embayment that suggest the ice sheet, nourished by increased snowfall until the early Holocene, was close to its LGM thickness at 10 ka. A pulse of rapid thinning caused the ice elevation to fall ∼400 m to the present level at 6.5–3.5 ka, and could have contributed 1.4–2 m to global sea-level rise. These results imply that the Weddell Sea sector of the WAIS contributed little to late-glacial pulses in sea-level rise but was involved in mid-Holocene rises. The stepped decline is argued to reflect marine downdraw triggered by grounding line retreat into Hercules Inlet. The precise contribution of the West Antarctic Ice Sheet (WAIS) melt to global sea-level rise remains enigmatic. Here, the authors generate an ice sheet thinning history for the Weddell Sea embayment and propose that this sector of the WAIS contributed to mid-Holocene, rather than late-glacial sea-level rise. The West Antarctic ice sheet (WAIS) has undergone a complicated process of thinning and retreat since the last glacial maximum (LGM) with different parts of the ice sheet behaving in contrasting ways1234689715161713An alternative view based on the latest high-resolution dating of the WAIS Divide ice core is that the central ice dome was lower than present during the LGM and thickened in response to increased snowfall caused by late-glacial climatic warming20The Patriot, Independence and Marble Hills are situated in the southern Heritage Range, Ellsworth Mountains in the heart of the inner Weddell Sea embayment . The mou116710Be, 26Al and 36Cl exposure ages indicate the ice sheet was close (∼20 m below) to its LGM maximum position in the early Holocene at 10 ka; this suggests deglacial thinning began ∼5 ka later than previously thoughtHere we present new evidence from the southern Heritage Range that allows us to constrain both the deglacial history of the ice sheet and its relationship to changing patterns of ice flow. Our evidence derives from detailed multi-isotope cosmogenic nuclide analyses tied closely to the geomorphology. We find geomorphological evidence indicating an easterly ice flow direction at the LGM that contrasts with the more northerly ice flow direction at present. Cosmogenic 10Be and 36Cl ages of 17–43 ka from large boulders on the blue-ice moraine ridge itself are consistent with the moraine reflecting ice flow at the LGM  or higher in the Marble, Patriot and Independence Hills, respectively. When considering all sites together, the modelling suggests a lower and linear thinning rate of 8.8±0.2 cm a−1, initiated at about 8.5–9 ka. Using the same statistical approach but including all deglacial exposure ages (<15 ka) suggests the onset of thinning occurred no earlier than 9–10.5 ka –14.3 ka67Landforms were mapped in the field using differential GPS and satellite imagery. This formed the basis for detailed work on sediment morphology, lithology, weathering and cosmogenic nuclide analysis. Selected areas, such as complex debris accumulations were mapped with a terrestrial laser scanner as well as high-resolution vertical aerial photographs, taken from an unmanned aerial vehicle3526Al and 10Be data set here. In addition, we also include in our data set 11 samples previously reported from the same bedrock spursThe cosmogenic nuclide data are presented in 10Be and 26Al, equivalent to 0–0.8 ka and in two cases 1.5 ka of pre-exposure. In view of the similar lithologies, and thus origin, of quartz-rich erratics at higher elevations, it is reasonable to argue that they too were deposited with pre-exposure of a similar magnitude. On the Independence Hills medial moraine ridge, we collected four large, broad-based limestone and sandstone boulder samples to determine the age of this feature.The sampling strategy for cosmogenic nuclides was designed to reduce the chance of nuclide inheritance, and exclude the possibility of nuclide loss through postglacial erosion. We targeted sub-glacially derived clasts with striated surfaces and sub-angular to sub-rounded shapes. We sampled the freshest-appearing, quartz-bearing, brick-sized clasts resting on flat bedrock to minimize problems of post-depositional movement and self-shielding. It is crucial to be convinced that the exposure ages reflect the time since deposition of a freshly exposed clast rather than a signal inherited from the past. To test whether clasts emerging on the glacier surface in blue-ice areas have no inherited cosmogenic nuclides, we analysed seven clasts on the present ice margin. All had relatively low amounts of cosmogenic −1 would be to increase LGM ages by ∼4% and Holocene ages by 2%. The high end of most Antarctic sandstone erosion rates is ∼1 mm ka−1; applying this rate would increase LGM ages by 2% and Holocene ages by 1%. Therefore, we argue the results are insensitive to our assumption of zero erosion.The presence of striations on most sampled rock surfaces validates our assumption of negligible rock surface erosion. However, it is worth exploring the sensitivity of our results to this assumption. The effect of incorporating even an exceptionally high Antarctic erosion rate of 2.5 mm kaIn this study, we favour the youngest exposure age at each altitude to best represent the elevation of the ice surface at the time . This iset al.10Be exposure ages <10 ka on each massif. For each iteration, the age for each point is randomly chosen from within the uncertainty bounds on that sample and then a linear regression is fit to those points, removing any results with negative slopes (negligible: <0.001%). Uncertainties are calculated statistically so that 68% of the resulting slope values fall within the given range. The results suggest average thinning rates of 20.9±2.7 cm a−1 at Marble Hills, 6.7±0.4 cm a−1 at Patriot Hills, and 5.1±0.2 cm a−1 at Independence Hills (1σ), but these rates would be higher following the youngest exposure age approach. When considering all sites together, the modelling suggests a lower and linear thinning rate of 8.8±0.2 cm a−1, but initiated a little earlier at about 8.5–9 ka. The exercise demonstrates that our conclusion of a mid-Holocene pulse of thinning, which was complete by 3.5 ka, is not sensitive to our interpretation based on the youngest exposure. In 10Be exposure ages <15 ka, and then with three clear outliers (3σ) removed to obtain a linear regression through all deglacial exposure ages. The model suggests the onset of initial deglaciation was at 9–10.5 ka, with a lower average thinning rate of 8.1±0.2 cm a−1. The onset of thinning implied is similar to that inferred from the youngest exposure age at 10Be/9Be and 26Al/27Al ratios were measured in 20–30 g of quartz at the Scottish Universities Environmental Research Centre (SUERC) Accelerator Mass Spectrometry (AMS) Laboratory in East Kilbride, UK. Measurements are normalized to the NIST SRM-4325 Be standard material with an assumed10Be/9Be of 2.79 × 10−11, and the Purdue Z92-0222 Al standard material with a nominal 26Al/27Al of 4.11 × 10−11, which agrees with the Al standard material of Nishiizumi10Be-AMS is insensitive to 10B interference26Al detection are well characterizedn=6) were spiked with 250 μg 9Be carrier  and 1.5 mg 27Al carrier . Samples were spiked with 250 μg 9Be carrier and up to 1.5 mg 27Al carrier . Blanks range from 3.3–9.3 × 10−15 [10Be/9Be] ; and 1.6–7.5 × 10−15 [26Al/27Al] . Concentrations in 27Al measurement (ICP-OES) uncertainty.Whole rock samples were crushed and sieved to obtain the 250–710 μm fraction. Be and Al were selectively extracted from the quartz component of the whole-rock sample at the University of Edinburgh's Cosmogenic Nuclide Laboratory following established methods37et al.35Cl carrier  was added to the samples before dissolution. The sample was dissolved using trace analysis quality nitric acid and Cl was precipitated as AgCl. The AgCl was purified, pressed and then measured at the SUERC AMS Laboratory. The sample was blank-corrected individually for 36Cl and total Cl concentrations (∼2% each) in the process blanks for that batch of samples. Full sample chemistry and measurement information can be found in Cl was extracted from three carbonate samples at the University of Edinburgh following a similar procedure to those outlined in Marrero 10Be half-life of 1.387 Ma  per a−1 for 36Cl–Ca and 759±180 neutrons per (g air) per a−1 for 36Cl–Pf(0). The sample had very low Cl (15.6 p.p.m.), so the choice of Pf(0) does not change the age. The use of Lal/Stone51−3 for quartz-bearing erratics and 2.5 g cm−3 for limestone; the attenuation length used is 153±10 g cm−2. No corrections are made for rock surface erosion or snow cover and thus exposure ages are minimal. Finally, we make no attempt to account for production-rate variations caused by elevation changes associated with glacial isostatic adjustment of the massif through time. Any glacial isostatic uplift would cause the exposure ages to be too young. However, we note that glacial isostatic adjustment in this area is lowFor exposure age calculations we used default settings in Version 2.0 of the CRONUScalc programme Ma refs , 46, an  Ma ref. , and a 3The authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Hein, A. S. et al. Mid-Holocene pulse of thinning in the Weddell Sea sector of the West Antarctic ice sheet. Nat. Commun. 7:12511 doi: 10.1038/ncomms12511 (2016).Supplementary Figures 1-4 and Supplementary ReferencesCosmogenic 10Be and 26Al data from the southern Heritage Range, Antarctica.Cosmogenic 36Cl data from the Independence Hills.Cosmogenic 26Al, 10Be and 36Cl exposure ages from the southern Heritage Range, Antarctica.


Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation. The differentiation of skeletal muscle cells involves the exit of mononucleated myoblasts from the cell cycle, changes in activity of an array of signaling pathways, altered gene expression, and cell fusion to form multinucleated myotubes.mTOR is contained within 2 distinct complexes: mTORC1 and mTORC2.Protein synthesis is carried out in 3 stages , with the initiation stage of translation generally accepted as a major site of regulation of gene expression in mammalian cells.The activity of the eIF4F complex is regulated its assembly, phosphorylation of components and the inherent structural properties of the recruited mRNA.Previously, we have shown that myogenic differentiation is associated with increased rates of translation, biphasic activation of p70S6K, and the phosphorylation of both eIF4E and 4E-BP1.13 and KU0063794,We and others have previously shown that rapamycinFigure S1 shows that in differentiated cell extracts, this phosphatase was effective within 10 minutes, removing modification on Ser 65 more efficiently than Thr70 (lane 3 vs. lanes 2 and 1); Thr37/46 remained resistant to dephosphorylation (lanes 4–6) under these assay conditions. Similar results were seen with extracts from cells allowed to differentiate in the presence of RAD001 (lanes 8–13). Western blotting with phospho-specific antisera strongly suggests that even in the presence of RAD001, the phosphorylation of 4E-BP1 was maintained during differentiation. To address this more directly, we have used extracts prepared from cells incubated in the absence or presence of RAD001 or KU0063794 for 24 or 48 hours and subjected them to lambda phosphatase treatment. The work described above shows that the PI3-kinase inhibitor, LY294002, could partially inhibit phosphorylation of 4E-BP1 in C2C12 cells, similar to that seen with RAD001. Published work has suggested that insulin-induced phosphorylation of 4E-BP1 on Ser111 occurs in an Ataxia-Telangiectasia Mutated kinase (ATM)-dependent manner.Figs. S2A and B). Consistent with published data,Figures S2A and B show that, relative to untreated cells, mRNA and protein for both raptor and rictor were depleted by >95% within 48 hours of transfection. In subsequent experiments, cells were incubated for 96 hours to allow for 48 hours in differentiation medium before extracts were prepared. SDS-PAGE analysis of extracts showed that depletion of raptor provoked an increase in the phosphorylation of Akt Ser473 . After transfection, cells were harvested at different times and the level of raptor and rictor mRNA and protein depletion analyzed using qRT-PCR and Western blotting, respectively , but rather increased Akt Thr308 phosphorylation, probably reflecting loss of inhibition of feedback signaling to IRS1 from p70S6K.Fig. S3A, lane 9 vs lane 6) led to decreased AktThr308 and p70S6K phosphorylation, and partial inhibition of ERK phosphorylation. However, depletion of raptor seemed to protect 4E-BP1 phosphorylation from the effects of serum starvation. On the addition of serum there was an increase in ERK and total 4E-BP1 phosphorylation; consistent with published findings,Fig. S3A, lane 12 vs. lane 9). To determine whether ERK activity was responsible for 4E-BP1 phosphorylation here, we repeated experiments in the absence or presence of PD184352, a characterized inhibitor of ERK.Figure S3C, inhibition of ERK did not prevent phosphorylation of 4E-BP1 in refed cells, either in the presence or absence of raptor. Ablation of rictor (lane 10 vs lane 6) led to increased p70S6K activity possibly reflecting increased expression of raptor.Considering that the mTORC1 complex is known to control 4E-BP1 phosphorylation during the upregulation of growth in a number of cell systems, we were somewhat surprised to find that extensive depletion of raptor had no effect on the KU0063794-sensitive phosphorylation of 4E-BP1 in C2C12 cells. Therefore, we used the same siRNAs to ablate raptor expression in murine NIH3T3 cells to see whether effects were cell-specific. The differentiation of skeletal muscle cells involves their exit from the cells cycle, changes in activity of an array of signaling pathways, altered gene expression, and cell fusion to form multinucleated myotubes.in vitro .In the present study we show that myogenic differentiation promotes the hyperphosphorylation of 4E-BP1 and activation of p70S6K in C2C12 cells. Similar findings with 4E-BP1 have been reported for human megakaryocyte cells undergoing differentiation.The nature of the RAD001-resistant kinase responsible for 4E-BP1 phosphorylation here is unknown. However, one potential candidate is the Ataxia-Telangiectasia Mutated kinase, ATM.As KU0063794 has the ability to inhibit mTORC1, mTORC2, phosphorylation of 4E-BP1, p70S6K, rpS6, protein synthesis and differentiation, we explored mTORC signaling further using siRNA-mediated ablation of raptor or rictor to specifically target these signaling nodes.S2 show that depletion of raptor was effective in these cells, it allowed cell fusion but delayed differentiation (data not shown). Relative to untreated cells, >95% depletion of raptor provoked an increase in the phosphorylation of Akt Ser473  and lysed by vortexing following the addition of 0.5% (v/v) Igepal and 0.5% (v/v) deoxycholate. Cell debris was removed by centrifugation in a microfuge for 5 min at 4°C2.Following treatment, cells were isolated in a cooled centrifuge and washed briefly with 0.5 ml ice-cold PBS containing 2 mM benzamidine. Pellets were resuspended in 100 µl ice-cold Buffer A (20 mM MOPS(KOH), pH 7.2, 20 mM sodium fluoride, 1 µM microcystin, 75 mM KCl, 2 mM MgClSamples containing equal amounts of protein were resolved by PAGE (SDS-PAGE) and processed for Western blotting as described previously.2. Incubations were carried out for the times indicated, in the absence or presence of 50 mM NaF; aliquots containing 10 µg extract protein were then removed into SDS-PAGE sample buffer and the phosphorylation of 4E-BP1 visualized by Western blotting.Aliquots of total cell extract (90 µg) were incubated with 200 units of lambda phosphatase (NEB) using the manufacturer’s conditions and buffers, in a final volume of 100 µl, in the presence of MnCl4) plated in 6 well plates were grown to confluency and transfected with 9.4 nM Ambion Silencer pro-sequence siRNAs (Life Technologies) specific to either murine rictor, raptor or both together (or scrambled control) using Lipofectamine mRNAi Max. After 24 hours, the medium was changed to DMEM in the presence of 2% (v/v) horse serum, 10 µg/ml insulin and 10 µg/ml transferrin and cells incubated for the times indicated in the figure legends.C2C12 cells (5 × 105) were plated in 6 well plates, grown for 24 hours and then transfected using Lipofectamine mRNAi Max with 9.4 nM Ambion Silencer pro-sequence siRNAs and cells were incubated for 48 hours before harvesting.Cells (1 × 10−ΔΔct).RNA was extracted from cell extracts using an RNA easy mini-kit  as per manufacturer’s instructions. RNA concentration was then quantified using a Nanodrop and 1µg of RNA was used for cDNA synthesis using the Promega ImpromII kit. The SYBR real-time PCR system (Kapa Biosystems) was used to quantify transcript abundance for genes of interest and 18S rRNA was used as a control. Template equivalent to 5 ng of RNA in cDNA library per reaction was added to each 20 μl reaction with a final primer concentration of 200 nM per reaction. Crossing thresholds were determined using MxPro software (Agilent), and fold-difference in RNA quantity was calculated using the relative quantification method (2


E. coli hydrogenase 1 (Hyd-1) is adsorbed on a high surface-area carbon electrode and subjected to the same electrochemical control and efficient supply of substrate as in protein film electrochemistry during spectral acquisition. The spectra reveal that the active site state known as Ni-L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non-turnover conditions. The observation that Ni-L is present at all potentials during turnover under H2 suggests that the final steps in the catalytic cycle of H2 oxidation by Hyd-1 involve sequential proton and electron transfer via Ni-L. A broadly applicable IR spectroscopic technique is presented for addressing electrode-adsorbed redox enzymes under fast catalytic turnover.A novel in situ IR spectroscopic approach is demonstrated for the characterization of hydrogenase during catalytic turnover.  Hydrogenases, widespread in the microbial world, are finely tuned for these reactions, and use active sites based on iron, or nickel and iron. The fundamental active-site structure for the nickel iron (NiFe) hydrogenases is well-established. The Ni is coordinated by two terminal cysteines, and a further two bridging cysteines which link to Fe. The Fe is additionally coordinated by two CN− and one CO. Fast transport of electrons between physiological donors or acceptors and the active site is achieved via a chain of iron sulfur clusters. Recent work has shown that the potential of the proximal cluster (nearest to the active site) is important in tuning the properties of the active site.[2-tolerant NiFe hydrogenases, including Escherichia coli hydrogenase 1 (Hyd-1) have an unusual proximal cluster structure,[Oxidation and production of Hive site. Certain tructure,− ligands give rise to vibrational bands (νCO and νCN) in the mid-IR, the frequency of which is highly sensitive to changes in electron density at the active site.[2 reacts with the EPR-silent Ni-SI state and is cleaved heterolytically[2 remains unclear, although long-range proton transfer channels have been identified.[IFeII state of the active site. Sequential proton- and electron-transfer steps via a NiI intermediate are well-established for small molecule H2-production catalysts based on Ni.[IR spectroscopy and spectroelectrochemistry has been particularly useful in evaluating active site redox states in NiFe hydrogenases because the intrinsic CO and CNive site. Despite ive site., 4, 5 Inlytically to form lytically The initentified. In Schementified. Concerteentified. and in tentified. In theseed on Ni.IFeII redox level has been identified for most NiFe hydrogenases following photo-illumination of Ni-C at cryogenic temperatures.[CO and νCN bands. It has been suggested by Neese and co-workers that the additional electron density resides in a metal–metal bond rather than being localized on Ni, with a nearby thiolate base suggested as the proton acceptor.[I-containing bimetallic model for Ni-L which is reduced by H2 to give a species containing a bridging hydride.[An active site state at the Nieratures. This staacceptor. Protonatacceptor. The specacceptor. Interest hydride.−1),[2-saturated static solutions are likely to suffer from substrate depletion and limited electron delivery.[Ralstonia eutropha in the presence and absence of H2,[E. coli.[2-saturated electrolyte through a custom-built spectroelectrochemical ATR-IR cell provides efficient mass transport, shown by the similar waveshape of the catalytic voltammogram recorded in this cell , even studelivery. The apprdelivery., 21 A tedelivery. notably ce of H2, and comp[E. coli. Herein w[E. coli. Flow of l Figure  with thal Figure . The higCO region recorded at selected electrode potentials, under 1 bar Ar or H2, on a single sample of adsorbed Hyd-1, together with the corresponding current–time trace. The adsorbed enzyme was first activated at low potential under H2. At the lowest potential applied, −0.594 V, there is no detectable electrocatalytic current under either Ar or H2, consistent with the poor ability of Hyd-1 to reduce protons, even at pH 6  provides effective mass transport of H2, and that the enzyme is adsorbed robustly on the carbon. At flow rates greater than 50 mL min−1, increasing the flow rate led to no further increase in the electrocatalytic current. At the most positive potential applied (+0.356 V) the current under H2 decays over time, consistent with the well-established slow anaerobic, oxidative inactivation of NiFe hydrogenases . At −0.594 V, a potential at which there is no detectable catalytic turnover, there are no obvious differences to the spectrum under Ar. However at more positive potentials where Hyd-1 engages in electrocatalysis under a H2 atmosphere, there are significant differences in the distribution of states. Under H2 Ni-R persists as the majority species at all applied potentials, together with a significant proportion of Ni-C and Ni-L, which are now observed even at +0.356 V where they are absent under an inert atmosphere. Under H2, Ni-SI is observed in a lower concentration at −0.074 V and persists up to the highest potential, and the onset potential for formation of Ni-B is more positive.Figure 2 data can be explained by reference to a catalytic cycle involving the Ni-SI, Ni-R, Ni-C, and Ni-L states (Scheme 2 is consistent with fast attack on Ni-SI by substrate: the H2 concentration is well above the Michaelis Menten constant for Hyd-1 (9 μm).[2 at the highest potential, outside the potential window where they are observed under non-turnover conditions . At the hDesulfovibrio vulgaris Miyazaki F hydrogenase has been reported by Tai et al.,[2 oxidation at room temperature in the dark provides strong evidence that it is the intermediate in conversion between Ni-C and Ni-SI in the catalytic cycle of Hyd-1.Recent EPR measurements at cryogenic temperatures have shown the presence of Ni-L in Hyd-1 without external illumination, and Ni-Li et al., who suggi et al., Furtherm2 oxidation by a NiFe hydrogenase. The high surface area carbon electrode used in this work provides facile, robust adsorption of the enzyme without the need for specific surface modification. This new approach adds structural insight to the information available from electrochemistry alone and will be widely applicable to redox proteins involved in small molecule binding and activation.In summary, this study builds upon the direct electrochemical control provided by the technique of PFE which has been important in addressing the activity and inhibition of redox enzymes via variations in current under a range of solution conditions. We have demonstrated a method for coupling PFE, which provides no direct structural information, with IR spectroscopy at a carbon electrode to provide insight into the active site states present during catalytic H


One disadvantage of cement-retained crowns is the lack of predictable irretrievability. This problem can be overcome through designing a screw access hole in the metal substructure of cement-retained restoration and using porcelain stain to define this area.This study aimed to evaluate the influence of existence of screw access hole on porcelain fracture resistance of metal-ceramic implant-supported crowns. Thirty six standardized metal-ceramic crowns were fabricated and divided into 3 groups (n=12); group 1 conventional cement-retained metal-ceramic crowns as control group, group 2 cement-retained MC crowns in which porcelain stain was used to define the location of screw access channel, and group 3 cement-retained metal-ceramic crowns in the metal substructure of which a hole and ledge was designed in the location of screw access channel. The specimens were cemented  to their dedicated abutments. A hole was made in the location of screw access channel in group 2 and 3 and filled with photo-polymerized composite resin . All specimens were thermocycled and loaded in universal testing machine at crosshead speed of 2mm/min until fracture. Mean values of load at fracture were calculated in each group and compared with One-way ANOVA (α=0.05).p> 0.05) Mean value of the load required to fracture the restorations was 1947±487 N in group 1, 1927±539 N in group 2, and 2170±738 N in group 3. No statistically significant difference was found between the fracture resistance values of the three groups ( Presence of screw access channel in cement-retained implant restorations does not compromise fracture resistance. Dental implants have been approved for their durable predictable success in treatment of both completely-2 and paRetrievability is the main advantage of screw-retained prosthesis,-16 becauFurthermore, natural occlusal morphology might be interfered due to the presence of a screw access opening, which miAmong the advantages of cement-retained restorations are the lower costs of fabrication, facilitated procedure of implant restoratives, and a better passive fit, as well as preventing the interference of screw access opening with the esthetic or the occlusion of the restoration., 25 MeanCement-retained restorations have been advised for treating partial edentulous patients with implant and theyLiteratures have suggested several methods to provide retrievability of cement-retained implant restorations; using provisional cement is one of them, although the retention may be damaged. Placing In the present study, a special feature was designed in the metal framework to support the remaining porcelain after the screw access channel was created. There is little data in the literature regarding the fracture resistance of cemented prostheses with screw access channel. The current investigation aimed to evaluate the influence of access hole in the occlusal surface of cement-retained implant restoration with and without specially designed feature on the fracture resistance of the restoration. The null hypothesis was that creating screw access on cement-retained crowns to permit retrievability would not compromise the strength of restoration. A 5×6.5 implant analog  was connected to a straight abutment. Then it was duplicated as 36 brass dies by using a lathe . Then, five millimeters of each die was embedded vertically in an acrylic resin block with the aid of a dental surveyor .Subsequently 36 metal ceramic crowns were fabricated with 3 designs; group 1 consisted of 12 conventional metal-ceramic crowns with cement-retained restorations as control group a, group Porcelain application to all specimens was standardized by using a silicone index. The crowns were then cemented on their dedicated master dies with zinc oxide-eugenol cement  in rocking motion and held in place with constant finger pressure until the cement set. The excess cement was removed using an explorer. A hole was made in the location of screw access channel in group 2 and 3 by using a 2-mm fissure bur on a high speed handpiece, and was filled with photo-polymerized composite resin .°C for 30 seconds with 12-second intervals according to the literature.[Finally, all specimens were thermocycled for 500 cycles between 50-65terature.-32 Each The force was applied perpendicular to the occlusal surface in the central part of the restoration with a cross-head speed of 2mm/min. In order to simulate the contact established by the opposing tooth, the rounded edges of the loading pin simultaneously contacted the triangular ridges of both buccal and lingual cusps of the crowns . The spep= 0.491).The mean fracture resistance value was 2190±738 N in group 3 , 1928±539 N in group 2 and 1947±487 N in control group . No statp> 0.05).With respect to the obtained results of the present study, the null hypothesis cannot be rejected. The fracture resistance of specially designed cement-retained crowns with screw access was higher than the other two groups; this difference was not significant though (et al.[Zarone et al. evaluateet al.[In another study, Karl et al. observedSeveral studies , 32-34 ret al., screw access hole in cement-retained implant restoration had not any negative influence on the retention.[The especially cement-retained design proposed in the present study offered the advantages of cement-retained restorations in conjunction with the likelihood of the restoration retrievability. Furthermore, according to the study of Rocha etention.There were some limitations in the present study, one of which was the single static force that was used to load the specimens and it differed from the dynamic load in the oral environment. In mouth, the restoration may also fracture due to fatigue loading. In the pFuture researchers are recommended to investigate larger sample sizes under physiologic fatigue loading. Employing different types of cements for luting the specimens is also advised. Within the limitations of this study, the following conclusions are drawn:Presence of screw access opening in cement-retained implant restoration would not compromise the fracture resistance. Designing a ledge in the framework of cement-retained implant restorations in the location of screw access opening could be a useful method to retrieve restoration, without compromising its strength.


In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca2+ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9–39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gαs coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gαs coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca2+ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B.The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca Cells were transiently transfected for 48h using JetPrime transfection reagent  according to the manufacturer’s instructions.Human embryonic kidney (HEK)293 cells were maintained at 37°C in a 5% CO2. Where indicated, cells were incubated with antagonist for 30min prior and during stimulation with agonist at 37°C/5% CO2. Cells were then fixed with 4% paraformaldehyde  for 10min, blocked with 1% BSA made in TBS (1% BSA/TBS) for 45min and probed with the anti-GLP-1R mouse antibody (diluted 1:15000) in 1% BSA/TBS for 1h. Cells were washed with TBS and then incubated with the HRP-conjugated anti-mouse IgG (diluted 1:5000) in 1% BSA/TBS for 1h. Cells were washed and developed using 1-step Ultra TMB-ELISA substrate  for 15min and the reaction stopped by adding an equal volume of 2M sulphuric acid. The optical density was read at 450nm using a plate reader.This assay was carried out as described previously with unpermeabilised cells to quantify cell surface expression of the hGLP-1R , 28. Bri2 and then with the anti-GLP-1R mouse antibody (diluted 1:5000) in 1% BSA/DMEM for 1h at 4°C. Cells were stimulated without or with agonist in the absence or presence of antagonist at 37°C/5% CO2, fixed with 4% PFA for 30min, permeabilised with 0.2% Triton X-100 made in PBS for 10min, blocked in blocking buffer (1% BSA made in wash buffer [0.1% Triton X-100 in PBS]) for 30min and then incubated with the Cy3-conjugated anti-mouse antibody (diluted 1:200 in blocking buffer) for 1h. Cells were then washed 3 times with wash buffer and incubated with DAPI  diluted 1:2000 in PBS to stain nucleus. Coverslips were mounted on glass microscopic slides using mounting solution  containing 2.5% DABCO . Immunofluorescence staining was visualised using Zeiss LSM710 confocal microscope fitted with a 63x oil immersion lens . GL. GL2+ acmulation  and ERK mulation  in a con2+ accumulation and ERK phosphorylation are required for GLP-1 stimulated hGLP-1R internalisation ). The addition of 33nM GLP-1 to cells pre-incubated with compounds 2 and B reduced ERK phosphorylation from 48.59 ± 2.5% to 18.33 ± 2.51% and 20.2 ± 4.95% (p<0001) respectively. ERK phosphorylation induced by 100nM GLP-1 was also significantly reduced by pre-incubation with compounds 2 or B . These results suggest compounds 2 and B either bind the hGLP-1R and cause a conformational change that either reduces GLP-1 access to the orthosteric binding site in a non-competitive manner or prevents GLP-1 occupied GLP-1R coupling to the Gαq pathway [Allosteric small molecule agonists have the potential benefit of binding to a site on the receptor that is distinct from the site used by the orthosteric agonist. Therefore, allosteric agonists can act upon the receptor at the same time as the endogenous orthosteric agonist and alter affinity and/or efficiency of the orthosteric agonist, potentially providing more ‘physiological’ regulations . Recentl pathway . In this2+ accumulation or ERK phosphorylation and as a result they did not induce hGLP-1R internalisation. Compounds B and 2 have previously been reported to induce a small or no increase in intracellular Ca2+ accumulation [1 muscarinic acetylcholine receptor, which results in ERK phosphorylation and intracellular Ca2+ mobilisation but not internalisation of the receptor [2+ accumulation and ERK phosphorylation are essential [In agreement with our previous study , both thmulation , 25, 39,mulation . The μ-omulation . Additioreceptor , 42. Thissential .s subunit [s coupling, although they bind at different sites on the hGLP-1R. While this manuscript was being prepared, a recent study showed that compounds 2 and B covalently modifies Cys347 in intracellular loop 3 of GLP-1R [In this study, antagonists Ex9–39) [–39 9, 1 and JANT subunit , 37, red2+ accumulation and ERK phosphorylation. Reducing hGLP-1R internalisation prevents dampening of the receptor activity [2 (GLP-1 metabolite) by sensitising the GLP-1R for activation by this metabolite [1 receptor, because their binding to the receptor results in a conformation change that increases the affinity of the orthosteric agonist to the receptor [2 muscarinic acetylcholine receptor [q pathway required for intracellular Ca2+ accumulation and thereby ERK phosphorylation [In this study, compounds 2 and B have been shown to reduce GLP-1 induced hGLP-1R internalisation, intracellular Caactivity . Therefoactivity –36-NH2  and as a result these agonists did not induce hGLP-1R internalisation (q pathway [s coupling, similar to that induced by the orthosteric agonist binding to the receptor. Additionally, compounds 2 and B reduced, in a non-competitive manner, GLP-1 induced GLP-1R coupling to Gαq and internalisation (In summary, small molecule agonists, compound 2 and compound B, were pharmacologically analysed in this study for their effect on hGLP-1R internalisation, cAMP production, intracellular Calisation . We have pathway . With thlisation . Therefo


Screw speed, barrel temperature, and their interaction had a significant influence on sugar recovery from the selected biomasses. A maximum of 28.2, 66.2, and 49.2% of combined sugar recoverywasachieved for switchgrass, big bluestem, prairie cord grass when pretreated at a screw speed of 200, 200, and 150 rpm and at a barrel temperature of 75, 150, and 100°C, respectively, using cellulase and β-glucosidase at a ratio of 1 :  4. Extrusion pretreatment of these biomasses used only 28–37% of the rated extruder power.Biofuels from biomass have the potential to reduce the dependency on fossil fuels. An efficient pretreatment method is required to accomplish the target of the Energy Act 2005. Extrusion could be a viable continuous pretreatment method to be explored. The objectives of the current study were to investigate the influence of screw speed and barrel temperature on sugar recovery from the selected warm season grasses and to select a suitable enzyme combination and dose for enzymatic hydrolysis. The ground, moisture-balanced biomasses were pretreated using a single screw extruder at various screw speeds  and barrel temperatures . Cellulase or multienzyme with  Renewable energy from biomass has the potential to reduce dependency on fossil fuels, in addition to combat the environmental issues. The Energy Policy Act of 2005 mandates blending of 7.5 billion gallons of alternative (biofuel) fuels by 2012 . LignoceThe focus of pretreatment research is to develop processes that would reduce the bioconversion time, lower the cellulase enzyme usage, and increase ethanol yields . Severalβ-glucosidase) supplementation with cellulase enzyme was necessary to eliminate the inhibition effect of cellobiose [β-glucosidase results in the absence of product inhibition [β-glucosidase whereas it was only 57.8% without complementation of β-glucosidase when corn stover was pretreated using wet oxidation. A wide range of enzyme dosage and hydrolysis conditions has been reported in literature for different biomasses depending on pretreatment. Due to variation in enzyme activity and its dose, making meaningful comparison between pretreatment methods is very difficult. Hence, there is a need to determine the suitable enzymes and their doses for economic reasons.Enzyme loading is an expensive input in biomass conversion –19; hencllobiose , 21. Thehibition , 23, thuhibition  reportedThe objective of the current study is to address the two most expensive steps such as pretreatment and enzymatic hydrolysis in the biomass conversion process. The first objective is to evaluate the effect of screw speed and barrel temperature on sugar recovery from warm season grasses such as switchgrass, big bluestem, and prairie cord grass. The second objective is to select a suitable enzyme combination and their ratio for the enzymatic hydrolysis of pretreated switchgrass, big bluestem, and prairie cord grass.Switchgrass, big bluestem, and prairie cord grass are three major warm season grasses among the big four native grasses (other: indiangrass) in South Dakota with potential as biomass feedstocks . Switchgl/d) of 20 : 1. In order to have a smooth biomass (plug) flow into the die section, the screw discharge end was fitted with conical metal piece. The single screw extruder was fitted to a 7.5 hp motor, which had a provision to adjust the screw speed from 0 to 210 rpm. The screw speed of the extruder was maintained at 100, 150, and 200 rpm during the extrusion of biomass samples. The extruder barrel had provisions to control the temperature of the feed and transition zone in both barrel and die section. The transition zone and die section temperature of barrel was maintained between 50, 75, 100, 150, and 200°C for different screw speeds depending upon the biomass. The temperature inside the barrel and the speed of the screw were controlled by a computer; and feeding to the extruder was done manually. Compressed air was supplied as a cooling agent along the barrel length. About 500 g of prairie cord grass was extruded under each pretreatment condition, divided into two batches accounting for variations due to extruder operation, and considered replicates. The mean residence time varied between 30 and 90 sec depending upon the screw speed. The power consumption for extrusion pretreatment of different feedstocks was measured clamp meter .Extrusion was performed in a single screw extruder , which had a compression ratio of 3 : 1, barrel length to screw diameter ratio (−1) to maintain constant pH and to inhibit microbial contamination, respectively. In order to select an enzyme combination and ratio, multienzyme (activity 100 FBGg−1), cellulase (activity 70 FPUg−1) with β-glucosidase (activity 250 CBUg−1) in the ratio of 1 : 1 and 1 : 4 was added to the pretreated corn stover. The amount of cellulase was maintained at 15 FPU/g of dry matter. Multienzyme complex consists of arabinose, β-glucanase, cellulase, hemicellulase, pectinase, and xylanase, which has the ability to liberate bound materials and can degrade a variety of nonstarch polysaccharides. Hydrolysis was carried out for 72 hours at 50°C and 150 rpm as described by Selig et al. [2SO4 at a flow rate of 0.6 mL/min at 65°C and a sample volume of 20 μL as mentioned by Sluiter et al. [The enzymatic hydrolyses were conducted in hungate glass tube  with 0.3 g dry weight of pretreated biomass in a solution of citrate buffer  and sodium azide  of 0.05.The moisture balanced big bluestem and prairie cord grass were extruded using a screw with 3 : 1 compression ratio at varying screw speeds of 100, 150, and 200 rpm and barrel temperatures of 100, 150, and 200°C, which resulted in nine treatment combinations  per each biomass. The switchgrass was pretreated at three different screw speeds  and the barrel temperatures of 50, 75, 100 and 150°C. Each treatment run was divided into two batches and the samples collected were considered as replicates. The collected data were analyzed with PROC GLM procedure to determine the main, interaction and treatment effect in SAS 9.1  using a type I error  were determined and given in β-glucosidase was used with different ratios during hydrolysis. In general, the screw speed had a positive influence on sugar recovery from switchgrass regardless of enzyme combinations and their ratios used during hydrolysis as seen from β-glucosidase was used with 1 : 1 ratio  resulted in a lower sugar recovery among the enzyme combinations and ratios studied, and it was almost less than 50% of cellulase with β-glucosidase (1 : 1). These results showed that higher screw speed is required to disturb the cell wall structure of the big bluestem.As observed from was used . As the drolysis  whereas, 200 rpm . Multienβ-glucosidase was used during hydrolysis, whereas sugar recovery increased with screw speed when multienzyme with β-glucosidase was used. The sugar recovery slightly increased as the screw speed was increased from 100 to 150 rpm; further increase in screw speed decreased the sugar recovery from prairie cord grass . The barrel temperature had a negative impact on sugar recovery from cord grass. However, the temperature effect was not significant on sugar recovery across the temperatures studied. When multienzyme and β-glucosidase was used at a ratio of 1 : 1, the sugar recovery decreased considerably when compared to cellulase and β-glucosidase at 1 : 1 ratio. A different sugar recovery pattern was observed with 1 : 1 multienzyme and β-glucosidase was employed during hydrolysis  of sugar recovery with an increase in screw speed, whereas the sugar recovery pattern was different for prairie cord grass. The sugar recovery from switchgrass showed an increasing trend with screw speed and it was similar to corn stover . SwitchgKeshwani . −1, respectively, recorded for switchgrass at a barrel temperature of 50°C with screw speeds of 100 and 150 rpm. The maximum concentration of glycerol and acetic acid was 0.03 and 0.04 gL−1, respectively, and was recorded for big bluestem at a screw speed of 150 rpm and barrel temperature of 100°C. The concentration of glycerol and acetic acid was similar to switchgrass but occurred at higher temperature and screw speed. In contrary to other extrusion pretreatments of corn stover, miscanthus and Douglas fir wood, acetic acid and glycerol were found in this study. Interestingly no furfural and HMF were found in any of the pretreatment conditions studied, which was in agreement with other extrusion pretreatments performed on different biomasses [Glycerol and acetic acid were the byproducts found in a few pretreated switchgrass, big bluestem, and prairie cord grass samples. The concentration of glycerol and acetic acid were in the range of 0.02–0.05 and 0.02–0.04 gLiomasses , 13, 16.l/d extruder wattage is 2000 W. The total rated power to run this extruder for an hour is 7595 Watt-hour. The ideal/no load drive and heater power consumption are 1.4 and 0.8 amps, respectively. The actual drive power consumption is 3.5, 4.4, and 4.1 amps and heater power consumption is 1.6, 2.4, and 2 amps, respectively, for big bluestem, prairie cord grass, and switchgrass. For three phase alternate current, W = V is voltage (240 V) and I is current in amps. The total energy consumption for extrusion pretreatment of big bluestem, prairie cord grass, and switchgrass is 2120, 2826, and 2515 W, respectively, which is 27.9, 37.2, and 33.1% of the rated drive and heater power.It consists of two components, namely, drive and heaters. The extruder has a power source of 7.5 hp (5595 W). There are 4 heaters per extruder zone at 250 W each, so Zone 1 = 1000 W and Zone 2 = 1000 W. Thus, the total 20 : 1 −1 depending on the types of feedstock. Amount of ethanol production can be calculated based on the availability of glucose in the raw feedstock and glucose recovered through extrusion pretreatment. Assumping a thumb rule is 50% of the glucose will be converted into ethanol during fermentation with an efficiency of 90%. Ethanol density and its energy content are 0.79 g/mL and 21.1 MJL−1, respectively (1 MJ = 278 Watt-hour). For example, glucose recovery of 57.5, 38.7, and 41.1% recorded for big bluestem, switchgrass, and prairie cord grass, respectively, would result in 1349, 909, and 809 Watt-hour. Researchers across the world are working on utilizing hemicellulose fraction; once the technology is developed, one can expect an additional energy of 60% that would narrow down the difference between input and output. However, extruder used in this study is a lab-scale one; the efficiency would improve as a result of scale-up process, and thereby one can expect net positive energy balance. In conclusion, technology development for hemicellulose utilization and scale-up process would be need of the hour.This extruder can pretreat 2-3 kghβ-glucosidase at a ratio of 1 : 4. The big bluestem could be pretreated at a screw speed of 200 rpm with 150°C, and prairie cord grass could be pretreated at a screw speed of 150 rpm and 100°C while cellulase and β-glucosidase was employed at a ratio of 1 : 4. Pretreatment of these biomasses used 28–37% of the rated extruder power. Further studies will be conducted to improve the sugar recoveries from this biomass.This experiment was conducted to understand the influence of screw speed and barrel temperature on sugar recovery from switchgrass, big bluestem, and prairie cord grass. When different enzyme combinations and ratios were employed during hydrolysis, it was confirmed that screw speed, barrel temperature and their combinations significantly influenced the sugar recovery. Based on the highest glucose, xylose, and combined sugar recovery, switchgrass could be pretreated at a screw speed of 200 rpm with barrel temperature of 75°C with cellulase and


The mechanism of cellular [18F]FLT retention has been assigned primarily to alteration of the strict transcriptionally regulated S-phase expression of thymidine kinase 1 (TK1). This, however, does not explain how anticancer agents acting primarily through G2/M arrest affect [18F]FLT uptake. We investigated alternative mechanisms of [18F]FLT cellular retention involving post-translational modification of TK1 during mitosis.3′-Deoxy-3′-[18F]FLT cellular retention was assessed in cell lines having different TK1 expression. Drug-induced phosphorylation of TK1 protein was evaluated by MnCl2-phos-tag gel electrophoresis and correlated with [18F]FLT cellular retention. We further elaborated the amino acid residues involved in TK1 phosphorylation by transient transfection of FLAG-pCMV2 plasmids encoding wild type or mutant variants of TK1 into TK1 negative cells.[18F]FLT cellular retention and TK1 protein expression were associated. S-phase and G2/M phase arrest caused greater than two-fold reduction in [18F]FLT cellular retention in colon cancer HCT116 cells (p<0.001). G2/M cell cycle arrest increased TK1 phosphorylation as measured by induction of at least one phosphorylated form of the protein on MnCl2-phos-tag gels. Changes in [18F]FLT cellular retention reflected TK1 phosphorylation and not expression of total protein, in keeping with the impact of phosphorylation on enzyme catalytic activity. Both Ser13 and Ser231 were shown to be involved in the TK1 phosphorylation-modulated [18F]FLT cellular retention; although the data suggested involvement of other amino-acid residues.Baseline [18F]FLT cellular retention and hence reporting of antiproliferative activity, with implications especially for drugs that induce a G2/M cell cycle arrest.We have defined a regulatory role of TK1 phosphorylation in mediating [ However, this does not account for the impact of some drugs on [18F]FLT uptake, particularly those that induce G2/M arrest. In this regard, alternative mechanisms of TK1 regulation during mitosis are hypothesized to be relevant. Chang and co-workers reported TK1 hyperphosphorylation during G2/M phase 18F]FLT cellular retention during anticancer drug treatment.To date the mechanism of cellular [− human osteosarcoma cells were kindly provided by Prof. Vera Bianchi  and cultured as described previously 18F]FLT was purchased from Siemens PETNET Solutions .Aphidicolin, nocodazole and roscovitine were purchased from Sigma . Paclitaxel was obtained from Calbiochem . Calf-intestinal alkaline phosphatase (CIP) was purchased from New England Biolabs . Lipofectamine 2000 and RNAiMAX transfection reagents were from Invitrogen . Phos-tag Acrylamide was initially synthesized by small modifications of a previously reported method . Before electrotransfer, gels were washed 3 times in 0.5 M EDTA pH 8.0 to remove metal complexes. Blots were probed with specific primary antibodies, and proteins of interest were visualized via enhanced chemiluminescence after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Mouse anti-thymidine kinase 1 and mouse anti-thymidylate synthase antibodies were purchased from Abcam . Rabbit anti-Cdk1 and anti-Cdk2 were obtained from Cell Signaling Technology . Rabbit anti-actin antibody was purchased from Sigma. Anti-mouse and anti-rabbit HRP-conjugated antibodies were from Santa Cruz Biotechnology .Cells were lysed in RIPA buffer and proteins denatured in SDS-containing sample buffer. Equal amounts of proteins were subsequently resolved on 12% SDS-PAGE and transferred on polyvinylidene fluoride membranes. For MnClCell cycle distribution was assessed using propidium iodide  and DNA-based flow cytometric analysis in fixed, permeabilized cells, as previously described via immunoblot analysis.FLAG-pCMV2 plasmids encoding for Δmet wild type TK1, Δmet S13A TK1, Δmet S13D TK1 or Δmet S231A TK1 were kindly donated by Dr. Chang, University of Taiwan. Adherent cells were incubated with Lipofectamine 2000 transfection reagent and the selected plasmid, according to the manufacturer's instructions. The transfection mixture was removed 6 hours post transfection and replaced with fresh media, containing nocodazole as required. Transgene expression was tested 24 hours after transfection Human Cdk1 and Cdk2 ON-TARGETplus SMARTpool siRNA and ON-TARGETplus non-targeting pool (scrambled) were purchased from Dharmacon . HCT116 cells were transfected with Cdk1, Cdk2 or scrambled siRNA for 48 hours using wet reverse transfection with RNAiMAX transfection reagent, according to manufacturer's instructions. Validation of knockdown was done by immunoblot and qPCR.2 humidified incubator at 37°C. Cells were subsequently scraped in media, and spun at 6,500 rpm at 4°C for 2 min. Cell pellets were washed twice with PBS, before being resuspended in 200 µl RIPA buffer, transferred into counting tubes and analyzed for radioactivity counts on a Cobra II Auto-Gamma counter .Cells were seeded in 6-well plates in triplicates to achieve ∼60% confluency, and treated with cell-cycle arresting agents 24 h prior the experiment. About 0.37 MBq (10 µCi) of radioactivity was added to each well and incubated for 1 h in a 5% COData were analyzed using GraphPad Prism, version 5 , applying the Student's two-tailed t-test to calculate statistically significant differences between data sets, and are represented as mean ± Standard Error of the Mean (S.E.M.). Significant differences were expressed as * (p<0.05), ** (p<0.01) or *** (p<0.001).de novo pathway for thymidine production. Cells lacking TK1 (Ost TK1−) showed high TS expression signifying an ability to synthesize DNA via the de novo pathway. For cells that were proficient for TK1, TS expression ranged from low to high , treatment with the DNA polymerase inhibitor aphidicolin (S-phase arrest), microtubule interfering agents nocodazole and paclitaxel (G2/M arrest), and roscovitine . The effect of these agents on the cell cycle – compared to vehicle (DMSO) or untreated controls - was confirmed by flow cytometry in HCT116 cells  also supported this assertion.Phosphorylated and unphosphorylated TK1 proteins were separated on 12% SDS-PAGE containing 75 µM MnCl-protein . As posi-protein . Band #2-protein . Further18F]FLT cellular retention (p<0.0001) compared to control (18F]FLT cellular retention by 90% (p<0.0001) suggesting that the protein formed was likely less active (mixture of non-phosphorylated and phosphorylated proteins). This finding is perhaps also explained by the negative feedback generated by thymidine triphosphate (dTTP) accumulation during S-phase arrest; dTTP is not incorporated into the DNA due to inhibition of DNA polymerase, therefore it interacts with TK1 to modulate its activity 18F]FLT cellular retention (p<0.0001) similarly indicating inactive protein. This is in line with the notion that at the end of DNA replication, TK1 enzymatic activity is no longer needed for the production of thymidine monophosphate, and is therefore down-regulated 18F]FLT cellular retention (p<0.0001), in spite of its unremarkable cell cycle arrest profile  and Ser231 to alanine  was intended to eliminate phosphorylation, while mutation of Ser13 to aspartate (S13D) was intended to mimic phosphorylation. Transfection with wild-type TK1  is a direct measure of TK1 enzymatic activity, was performed to determine if the specific phosphorylation profiles were linked to differential enzymatic activity. As shown in 18F]FLT cellular retention in Ost TK1− cells transfected with TK1 vectors  compared to untransfected Ost TK1− cells. For each transfection, treatment with nocodazole for 24 hours led to lower [18F]FLT cellular retention compared to the corresponding vehicle-treated samples . Finally, nocodazole treatment of cells expressing S231A mutated TK1 caused a 3-fold lower [18F]FLT cellular retention compared to the vehicle treated S231A cells. via S13A substitution was responsible for an 80% lower [18F]FLT cellular retention compared to WT (p = 0.0005), whereas mimicking TK1 phosphorylation (S13D mutation) showed only a 20% lower (p<0.05) cellular retention. Eliminating phosphorylation via S231A resulted in lower radiotracer retention, although not as marked as with S13A, being 50% of the WT retention value (p = 0.0033). − cells, indicating that the effects on [18F]FLT cellular retention were not caused by differences in protein expression between the TK1 variants, but were generated by the specific mutations.Identifying the functional consequence of phosphorylation on different amino acid residues was deemed important for characterizing the oscillation of TK1 activity during cell-cycle progression. For this reason, [18F]FLT uptake was reduced after substitution with alanine; this is in contrast to the previously described effect of Ser13 substitution on TK1 activity during G2/M 18F]FLT cellular retention. This result is in contrast with the report by Li et al.18F]FLT uptake, and 2) there might be residues, different from the ones investigated in this study, which are phosphorylated on TK1 protein; in fact, there was still a decrease in [18F]FLT uptake after nocodazole treatment, in contrast to the expected result, i.e., no difference between nocodazole treated and non-treated S231A samples.Taken together, the results presented here suggests that Ser13 phosphorylation might be important for activating the TK1 enzyme, since compared to WT, FLT cellular retention are also of interest. We approached identification of phosphorylated amino acid residues by transfecting TK1-deficient cells (Ost TK1−) with DNA plasmid vectors encoding mutant variants of TK1 cDNA. A previous report 18F]FLT uptake, i.e. TK1 activity, close to that of wild type TK1 protein, even after nocodazole treatment, assuming that Ser13 was specifically phosphorylated during G2/M. On the contrary, S13A substitution caused a significant decrease in [18F]FLT uptake compared to control, suggesting a role in increasing the activity of the enzyme. Moreover, nocodazole treatment of cells transfected with the S13A variant of TK1 showed a significant decrease in radiotracer retention compared to untreated samples, suggesting that the phosphorylation of a different residue might in part be responsible for the TK1 activity reduction. Ser231 appeared to be an important residue phosphorylated during mitosis in our studies; this finding is supported by the identification of TK1 phosphorylation on Ser231 in proteomic studies 18F]FLT cell uptake studies following nocodazole treatment. In this regard, other amino acid residues on TK1 including Ser30 and Ser194 have been suggested to play a role in G2/M arrest The specific amino acid residues involved in TK1 phosphorylation-modulated [18F]FLT cellular retention and hence reporting of antiproliferative activity, with implications especially for drugs that induce G2/M cell cycle block. We demonstrated the presence of at least two phosphorylated residues on TK1 sequence, which are responsible for modulating the activity of the enzyme in relation to [18F]FLT cellular retention. Indeed, changes in [18F]FLT cellular retention following G2/M-arresting drug treatment reflected TK1 phosphorylation and not expression of total protein, in keeping with the impact of phosphorylation on enzyme catalytic activity when DNA synthesis has already terminated 18F]FLT PET for evaluating treatment of patients with drugs that inhibit G2/M including taxanes In conclusion, we have defined a regulatory role of TK1 phosphorylation in mediating [Figure S1Validation of Akt dephosphorylation as a control for protein dephosphorylation by CIP. HCT116 cells were grown in complete medium (10% FBS) or serum-deprived medium (0% FBS) for 24 h. Insulin treatment was performed for 1 h to enhance AKT protein phosphorylation, after which cells were lysed and incubated with CIP for 1 h.(TIF)Click here for additional data file.Figure S2TK1 phosphorylation profile in synchronized HCT116 cells. HCT116 cells were serum starved for 24 h, and subsequently incubated in fresh medium, containing 10% FBS and 0.5 µg/ml nocodazole (NOC) or 250 nM paclitaxel (PAX) as required, for 18 h (O/N), 24 h or 48 h. Cells were lysed for western blotting or analyzed via flow cytometry. A) Representative resolution of TK1 phosphorylated isoforms on 12% acrylamide+75 µM MnCl2-phos-tag after cell synchronization. B) Representative cell cycle analysis of DNA content measured after propidium iodiode (PI) staining. O/N = overnight; NT = non-treated; NOC = nocodazole; PAX = paclitaxel.(TIF)Click here for additional data file.Protocol S1Synthetic details for the preparation of phos-tag acrylamide.(DOCX)Click here for additional data file.