There is considerable overlap between left ventricular noncompaction (LVNC) and other cardiomyopathies. LVNC has been reported in up to 40% of the general population, raising questions about whether it is a distinct pathological entity, a remodeling epiphenomenon, or merely an anatomical phenotype.The authors determined the prevalence and predictors of LVNC in a healthy population using 4 cardiac magnetic resonance imaging diagnostic criteria.Volunteers >40 years of age  with no history of cardiovascular disease (CVD), a 10-year risk of CVD < 20%, and a B-type natriuretic peptide level greater than their gender-specific median underwent magnetic resonance imaging scan as part of the TASCFORCE (Tayside Screening for Cardiac Events) study. LVNC ratios were measured on the horizontal and vertical long axis cine sequences. All individuals with a noncompaction ratio of ≥2 underwent short axis systolic and diastolic LVNC ratio measurements, and quantification of noncompacted and compacted myocardial mass ratios. Those who met all 4 criteria were considered to have LVNC.Of 1,480 participants analyzed, 219 (14.8%) met ≥1 diagnostic criterion for LVNC, 117 (7.9%) met 2 criteria, 63 (4.3%) met 3 criteria, and 19 (1.3%) met all 4 diagnostic criteria. There was no difference in demographic or allometric measures between those with and without LVNC. Long axis noncompaction ratios were the least specific, with current diagnostic criteria positive in 219 (14.8%), whereas the noncompacted to compacted myocardial mass ratio was the most specific, only being met in 61 (4.4%).A significant proportion of an asymptomatic population free from CVD satisfy all currently used cardiac magnetic resonance imaging diagnostic criteria for LVNC, suggesting that those criteria have poor specificity for LVNC, or that LVNC is an anatomical phenotype rather than a distinct cardiomyopathy.  Left ventricular noncompaction (LVNC) is characterized as a primary genetic cardiomyopathy by the American Heart Association, but is characterized by the European Society of Cardiologists as an “unclassified cardiomyopathy,” aptly demonstrating some of the controversy that surrounds this condition Since the original CMR criteria was proposed by Petersen et al. Following local ethical committee approval, a cohort of 2,047 volunteers was invited to the imaging arm of the TASCFORCE (Tayside Screening for Cardiovascular Events) study. Volunteers were enrolled into the study if they: 1) were more than 40 years of age; 2) were free from CVD or other indication for statin therapy as recommended by the Scottish Intercollegiate Guidelines Network (SIGN) report 97 for “Risk Estimation and the Prevention of Cardiovascular Disease” published in February 2007; 3) had a serum B-type natriuretic peptide (BNP) level greater than their gender specific median; and 4) had a 10-year risk of coronary heart disease <20% as predicted by the Adult Treatment Panel III algorithm The MRI protocol has been described in detail elsewhere Electrocardiograph (EKG)–gated segmented breath-hold cinematic (CINE) TrueFISP  images were acquired in the horizontal and vertical long axes, and in the short axis from the atrioventricular ring to the left ventricular (LV) apex using a 2-dimensional ECG-gated breath-hold segmented (CINE) TrueFISP sequence. Retrospective ECG gating was used, with 25 cardiac phases reconstructed (25 lines per segment) and 2 image slices acquired per breath-hold. Parallel imaging was also implemented .1.7. For noncompaction assessment, each of the 4 diagnostic criteria was measured as follows (1.Long axis noncompaction (LAX) was measured on the horizontal and vertical LAX cine sequences, which were analyzed at end-diastole. The thickness of the compacted and noncompacted myocardium was measured at the location of maximum noncompaction as described by Petersen et al. 2.DIAS) noncompacted: compacted myocardial ratio ≥3 was considered to meet the SAXDIAS diagnosis of LVNC, whereas a systolic SAX (SAXSYST) noncompacted:compacted myocardial ratio ≥2 was considered to meet the SAXSYST diagnosis of LVNC. The location of maximum noncompaction in both systole and diastole was recorded using the AHA 17-segment model of the left ventricle.Short axis noncompaction (SAX) was performed using the short axis cine images, with the region of highest noncompacted myocardium to compacted myocardium ratio measured both in diastole and systole, as described by Stacey et al. 3.MASS) was measured as described by Jacquier et al. MASS was calculated as the difference between the global LV mass and the compacted LV mass. A noncompacted mass >20% of the global LV mass was considered to meet the NCMASS diagnosis of LVNC.Noncompacted myocardial mass  for each of the 4 measures of noncompaction were determined with ICC >0.75 = excellent, 0.4 to 0.75 = good, <0.40 = poor, and <0.20 = slight. All data were analyzed using SPSS statistical package . Significance was adjusted for multiple comparisons using a Bonferroni correction.Data are expressed as mean ± SD for continuous variables, median (range) for ordinal variables and number of patients (%) for nominal variables. Normality tests were performed; if the test failed, where possible standard transformations such as square root, reciprocal, or logarithmic transforms were used to generate a Gaussian distribution. An independent sample Student Of the 1,528 volunteers who completed the imaging protocol, 48 were excluded due to inadequate image quality. This resulted in 1,480  undergoing complete imaging with diagnostic quality images allowing measurement of all 4 measures of noncompaction.DIAST (35.8%) criterion, with the most common site of maximum noncompaction found in the apical lateral wall (segment 16). A total of 65 (22%) met the SAXSYST criterion, with the most common site of maximum noncompaction found in the apical lateral wall (segment 16). A total of 61 (20.6%) met the NCMASS criteria. Thus, 14.8% of the normal population met at least 1 of the current CMR criteria for LVNC, whereas 1.3% met all 4 of the proposed diagnostic criteria for LVNC.The average maximum LAX noncompacted ratio within the entire cohort was 1.78 ± 0.63. A total of 296 (20.0%) of 1,482 analyzed datasets demonstrated an LAX ratio of ≥2 and were therefore included in subsequent analysis. Of the 296 who underwent all 4 diagnostic tests for LVNC, 219 (74%) met at least 1 diagnostic criterion for LVNC, 117 (39.5%) met 2 criteria, 63 (21.3%) met 3 criteria, and 19 (6.4%) met all 4 diagnostic criteria . A total1.7 vs. 42.5 ± 9.5 g/m1.7, p = 0.004), higher LV end systolic volumes (LVESV) , lower ejection fraction (EF) , and lower LV mass volume ratio (LVMVR)   demonstrated no significant differences in demographics, allometric measures, or cardiovascular risk factors . They di< 0.001) . A signi< 0.001) .DIAS measures was good for intraobserver repeated measures , and good for interobserver measures . Repeatability for the SAXSYST measures was only slight for intraobserver repeated measures , and good for interobserver measures . Repeatability for the NCMASS measures was good for intraobserver repeated measures , and excellent for interobserver measures .Repeatability for the LAX measures was good for intraobserver repeated measures , and poor for interobserver measures . Repeatability for the SAXIn our study, almost 15% of the population meet at least 1 of the current CMR diagnostic criteria for LVNC, and 1.3% of an asymptomatic population free from known CVD meet all 4 current CMR criteria. In addition, we demonstrated that the presence of LVNC is not associated with demographics, body shape, or biochemical markers of CVD.Our findings are comparable with previous work in the MESA (Multi Ethnic Study of Atherosclerosis) population, in which an LAX noncompaction ratio >2.3 was observed in 43% of 323 participants free from cardiac disease and hypertension The observation of high incidence of LVNC in 2 separate population studies indicates 1 of 2 possibilities. One is that the current diagnostic criteria lack specificity for the accurate identification and diagnosis of LVNC, with resultant extensive over diagnosis in normal individuals. Indeed, the poor correlation between the measures, with 15% of the study cohort meeting at least 1 criterion but <2% meeting all 4, suggest that the feature they are measuring is poorly captured by any 1 of the techniques. This is in keeping with previous observations using echocardiographic diagnostic criteria in which, in a population with known heart failure and diagnosis of LVNC, only 29.8% met all 3 criteria, whereas 36.3% fulfilled only 1 criterion The second possibility is that noncompaction is an anatomical phenotype rather than a pathological cardiomyopathy. At 10-year follow-up of the aforementioned MESA study, those who met the Peterson et al. First, we only conducted a full analysis of all diagnostic criteria in those with an LAX ratio of ≥2, and thus may have underestimated the total number who may have met 1 or more of the other 3 diagnostic criteria. However, this is only likely to further strengthen our observation of overdiagnosis if more participants without this criterion happened to meet 1 of the other 3 criteria. Second, a selection criterion for recruitment into the imaging arm of the TASCFORCE study was BNP above the gender specific median. Given the known association between BNP and heart failure, this could bias the results towards detecting a higher prevalence of a phenotype that is traditionally associated with heart failure and a poor clinical outcome. It could also increase the prevalence of those with diastolic dysfunction, which may in turn affect trabeculation quantification if there is a significant remodeling epiphenomenon component to these measures. No previous reports have described an association between these 2, nor is there any evidence of absence of association. However, of some reassurance, we did not observe any correlation between BNP and noncompaction ratios, nor were BNP levels significantly higher in those who met all 4 criteria, suggesting the impact of any potential bias of this is likely to be small.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: The current cardiac MRI criteria for diagnosis of LVNC lead to over-representation of its frequency in asymptomatic patients without other manifestations of heart disease. This suggests that LVNC is an anatomical phenotype rather than a distinct cardiomyopathy.TRANSLATIONAL OUTLOOK: Further studies are needed to validate more specific, comprehensive criteria beyond simple anatomical measures on cardiac imaging that identify patients with LVNC at risk of developing adverse clinical events such as arrhythmia, heart failure, or thromboembolism.A significant proportion of an asymptomatic population free from CVD satisfy all currently used CMR diagnostic criteria for LVNC, suggesting that either these all have poor specificity for LVNC, or that LVNC is an anatomical phenotype rather than a distinct cardiomyopathy.


Mechanistically, HDAC10 overexpression increased extracellular-regulated kinase 1/2 (ERK1/2) activation, whereas depletion of HDAC10 inhibited ERK1/2 activation. Finally, HDAC10 promoted ERK1/2 phosphorylation by deacetylating the promoter of protein tyrosine phosphatase, non-receptor type 22 (PTPN22) and inhibiting the expression of PTPN22, which is a negative regulator of ERK phosphorylation. Collectively, our results identify HDAC10 as a key regulator of angiogenesis and reveal that HDAC10 functions in this process by binding and deacetylating the PTPN22 promoter and subsequently inhibiting PTPN22 expression, which in turn increases ERK1/2 phosphorylation. Our studies suggest that HDAC10 is a potential target for therapeutic intervention to inhibit angiogenesis and tumor growth.Angiogenesis is crucially involved in many physiological and pathological processes including tumor growth, but the molecular mechanisms regulating angiogenesis are incompletely understood. In this study, we investigated the functions and mechanism of histone deacetylase 10 (HDAC10), a member of the HDAC II family, in regulation of angiogenesis. HDAC10 overexpression in human umbilical vein endothelial cells (HUVECs) promoted tube formation, whereas depletion of HDAC10 from HUVECs inhibited tube formation  Angiogenesis is a critical and complex biological event in many physiological and pathological processes, such as tumor growth –3. This αvβ3 [Studies of the signaling pathways in angiogenesis have revealed several potential agents that regulate angiogenesis . Celastrαvβ3 . Additioαvβ3 . AltogetProtein tyrosine phosphatases (PTPases) remove the phosphate group from phosphorylated tyrosine residues on proteins such as SRCs and ERKs . Based oHistone acetylation is highly involved in transcriptional regulation and is catalyzed by two kinds of enzymes: histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) . In genein vitro. Lentiviral particles containing the HDAC10 cDNA or the shRNAs were used to infect HUVECs. Overexpression and knockdown of HDAC10 in the corresponding viral infectants were confirmed by real-time PCR , a control vector (FUGW), two different shHDAC10 vectors or a scrambled shRNA control vector to further investigate the function of HDAC10 in angiogenesis o Figure . We alsoo Figure . In conto Figure . Compareo Figure . Thus, HNext, we performed a microarray analysis using the FUGW, FUW-HDAC10, PLKO and shHDAC10 cells to identify the target genes regulated by HDAC10 in endothelial cells. The heat map shows 1,000 of the differentially expressed genes that exhibit the greatest changes in the microarray data Figure . A PearsTo gain further insight into the mechanism by which HDAC10 regulates angiogenesis, we analyzed the effects of HDAC10 overexpression and knockdown on MAPK activation, which plays crucial roles in the regulation of endothelial cell angiogenesis. HDAC10 overexpression increased the level of activating ERK1/2 phosphorylation, whereas HDAC10 knockdown decreased ERK1/2 phosphorylation Figure . We thenWe next generated viral vectors expressing shRNAs for ERK1 and ERK2 and infected endothelial cells with them to further study the role of ERK1/2 in HDAC10-mediated angiogenesis. Both the ERK1 and ERK2 shRNAs blocked the angiogenic function of HDAC10 in endothelial cells Figure . WesternBased on the microarray data and other results described above, we hypothesize that HDAC10 regulates the expression of PTPN22, which in turn regulates ERK1/2 phosphorylation , 18, 37.To further investigate the function of PTPN22 in the HDAC10 regulation of ERK1/2 phosphorylation and angiogenesis, we generated vectors expressing two different shRNAs for PTPN22 and established PTPN22 knockdown (shPTPN22) Figure  and PTPNin vivo. The shHDAC10-induced inhibition of angiogenesis was markedly reversed by shPTPN22  supplemented with 10% fetal calf serum (GIBCO), 100 U/ml streptomycin and 100 U/ml penicillin (GIBCO). Human embryonic kidney 293T cells (HEK293FT) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum, 100 U/ml streptomycin and 100 U/ml penicillin.The GFP-expressing FUGW lentivirus vector was previously described . The cod5 cells/well of 6-well plate, grown to 30% to 40% confluence and then infected with the virus.To generate the lentivirus, FUGW, FUW-HDAC10 and shRNA constructs were co-transfected with helper plasmids into HEK293T cells with the X-tremeGENE HP DNA transfection reagent (Roche). At 48 h post-transfection, the virus was harvested. HUVECs were plated at a density of 1.2 × 10Total RNAwas isolated from HUVECs using TRIzol (Invitrogen), and 500 ng of RNA from each sample was reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit (TaKaRa). The cDNA was used as the template for real-time PCR.The antibodies used in this study are as follows: HDAC10 , ERK1/2 , p-ERK1/2 (Thr202/Tyr204) , PTPN22 , and GAPDH . For Western blot, HUVECs were lysed with radio-immunoprecipitation assay buffer  on ice for 25 min. The lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the protein concentrations in the samples were determined using the bicinchoninic (BCA) method. Equal amounts of protein were loaded onto SDS-PAGE gels, separated by electrophoresis, transferred to membranes and incubated with the appropriate antibodies. Then, the signals were developed using the Clarity Western enhanced chemiluminescence (ECL) substrate .4 cells/well in 24-well plates  that were pre-coated with 330 μl of Matrigel Growth Factor Reduced Membrane Matrix (BD Biosciences). Tube numbers and crossing points were quantified in 5 random microscopic fields after 8 h.HUVECs (as specified in each experiment) were plated at a density of 4 × 10in vivo was quantified by counting the number of vessels in 5 random microscopic fields.HUVECs (as specified in each experiment) were suspended in Matrigel and subcutaneously injected into the backs of 8-week-old nude mice (each mouse was injected with one Matrigel plug) . For eac2, and 0.5% NP-40) to obtain cell nuclei. The nuclei were lysed in nuclei lysis buffer  for an additional 30 minutes and gently pelleted for 10 minutes on ice. The chromatin was sonicated and sheared using a sonicator . The sheared chromatin samples were used as an input control or incubated with antibodies overnight; 20 μl of beads was added, and the samples were incubated for 1.5 h rotating at 15 rpm. Then, the samples were successively washed 4 times with TSE I , TSE II , wash buffer , and TE buffer (1 mM EDTA and 10 mM Tris-Cl). The DNA samples were dissociated from the immunoprecipitated chromatin and used as templates for real-time PCR with primer pairs targeting the PTPN22 promoter.HUVECs were harvested with 0.25% trypsin (GIBCO) and cross-linked for 10 minutes by directly adding 1% formaldehyde, which was then neutralized with 0.125 M glycine for 5 minutes with gentle shaking. The cells were washed with cold PBS and lysed for 15 minutes on ice in a cell lysis solution . Images were captured from randomly selected fields.All data represent as mean ± SEM. 2-tailed Student’s t test (for parametric data) or Mann-Whitney test (for nonparametric data) were used to compare two groups of samples according to distribution. P values less than 0.05 were considered significant. Prism 5 (GraphPad) was used for statistical analysis.


In addition, during the course of these excitatory processes and inhibition of AChE, a high rate of ATP consumption, coupled with the inhibition of oxidative phosphorylation, compromise the cell's ability to maintain its energy levels and excessive amounts of ROS and RNS may be generated. Pretreatment with N-methyl D-aspartate (NMDA) receptor antagonist memantine, in combination with atropine sulfate, provides significant protection against inhibition of AChE, increases of ROS/RNS, and depletion of high-energy phosphates induced by DFP/carbofuran. Similar antioxidative effects are observed with a spin trapping agent, phenyl-N-tert-butylnitrone (PBN) or chain breaking antioxidant vitamin E. This review describes the mechanisms involved in anticholinesterase-induced oxidative/nitrosative injury in target organs of OPs/CMs, and protection by various agents.Anticholinesterase compounds, organophosphates (OPs) and carbamates (CMs) are commonly used for a variety of purposes in agriculture and in human and veterinary medicine. They exert their toxicity in mammalian system primarily by virtue of acetylcholinesterase (AChE) inhibition at the synapses and neuromuscular junctions, leading into the signs of hypercholinergic preponderance. However, the mechanism(s) involved in brain/muscle damage appear to be linked with alteration in antioxidant and the scavenging system leading to free radical-mediated injury. OPs and CMs cause excessive formation of F


Preanalytic factors such as time and temperature can have significant effects on laboratory test results. For example, ammonium concentration will increase 31% in blood samples stored at room temperature for 30 min before centrifugation. To reduce preanalytic error, blood samples may be placed in precooled tubes and chilled on ice or in ice water baths; however, the effectiveness of these modalities in cooling blood samples has not been formally evaluated. The purpose of this study was to evaluate the effectiveness of various cooling modalities on reducing temperature of EDTA whole blood samples.Pooled samples of canine EDTA whole blood were divided into two aliquots. Saline was added to one aliquot to produce a packed cell volume (PCV) of 40% and to the second aliquot to produce a PCV of 20% (simulated anemia). Thirty samples from each aliquot were warmed to 37.7 °C and cooled in 2 ml allotments under one of three conditions: in ice, in ice after transfer to a precooled tube, or in an ice water bath. Temperature of each sample was recorded at one minute intervals for 15 min.Within treatment conditions, sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling in ice only or transferring the sample to a precooled tube and cooling it on ice. Mean temperature of samples cooled in ice water was significantly lower at 15 min than mean temperatures of those cooled in ice, whether or not the tube was precooled. By 4 min, samples cooled in an ice water bath had reached mean temperatures less than 4 °C (refrigeration temperature), while samples cooled in other conditions remained above 4.0 °C for at least 11 min. For samples with a PCV of 40%, precooling the tube had no significant effect on rate of cooling on ice. For samples with a PCV of 20%, transfer to a precooled tube resulted in a significantly faster rate of cooling than direct placement of the warmed tube onto ice.Canine EDTA whole blood samples cool most rapidly and to a greater degree when placed in an ice-water bath rather than in ice. Samples stored on ice water can rapidly drop below normal refrigeration temperatures; this should be taken into consideration when using this cooling modality. Laboratory error can occur in the pre-, intra-, and postanalytic phases of sample analysis, with 31.6–75% of errors occurring in the preanalytic phase . ExampleLeftover refrigerated canine EDTA whole blood samples were obtained from the University of Tennessee, College of Veterinary Medicine Clinical Pathology Laboratory and used for testing. All samples were seven days old at the time of entry into the study and had been stored at 4 °C. Samples were pooled, and a packed cell volume (PCV) of the resulting pool was measured. The pool was then divided into two aliquots. For aliquot 1 , physiologic saline was added to decrease the PCV to 40%. For aliquot 2 (simulated anemia), physiologic saline was added to decrease the PCV to 20%. Each aliquot was divided into 2 ml samples and 2.5 ml samples that were placed in a 13 × 75 mm, stoppered, EDTA-free sample tubes  and kept refrigerated at 4 °C until testing. In all test conditions, the cooling rate of 2 mls of blood was to be evaluated; however, placement of a warmed blood sample into a precooled tube required transfer of the blood. For those samples, an extra 0.5 ml of blood was included in the storage tubes to ensure a full 2 mls of blood was transferred at the time of testing. Two mls of blood was chosen because it is the standard volume collected in a 13 × 75 mm tube containing 3.6 mg of potassium EDTA; additionally, that volume of blood forms a column less than 30 mm in height within the tube, ensuring that the surface level of the blood will rest several cm below the surface level of the ice or ice/water.The testing apparatus consisted of a lidded 148 ml plastic specimen cup , a temperature probe with an accuracy of ±0.3 °C (Fisher Scientific™ Traceable™ Hi-Accuracy Thermometer), and a support stand with clamps. A whole the diameter of the sample tube was drilled in the lid of the specimen cup to allow the tube to be inserted through the lid and to be held upright and centered in the specimen cup with the bottom of the tube touching the bottom of the cup and the stopper resting above the surface of the lid. A hole was drilled in a tube stopper, which was secured onto the temperature probe and used for all measurements, so that the temperature probe would maintain the same location and depth in each tube and so that each tube remained stoppered for the entirety of the test.Immediately before testing, stoppered blood tubes were placed on a rack in a 38 °C water bath and warmed to 37.7 °C. Samples from each aliquot  were tested under one of three cooling conditions: 1, Ice bath; 2, Precooled (P/C) tube in ice bath; and 3, Ice water bath. For Condition 1 (ice bath), the specimen cup was filled with 70 gm of crushed ice and capped with the prefabricated lid. A 13 × 75 mm stoppered sample tube containing 2 mls of warmed (37.7 °C) blood was inserted through the hole in the lid, and its stopper was replaced with the stopper-temperature probe unit. For Condition 2 (P/C tube in ice bath), an empty 13 × 75 mm stoppered sample tube was placed on ice. After 5 min, 2 mls of blood were withdrawn from a storage tube containing 2.5 mls of warmed (37.7 °C) blood and transferred into the P/C sample tube, which was then positioned in a cup containing 70 g of crushed ice and capped with the stopper-temperature probe unit, as for Condition 1. For Condition 3 (Ice water bath), the specimen cup was filled with 70 g of crushed ice and 45 g of water and covered with the prefabricated lid. Immediately thereafter, a 13 × 75 mm sample tube containing 2 mls of warmed blood was capped and positioned in the specimen cup, as described for Conditions 1 and 2. In each condition, the surface of the blood sample rested several cm below the surface of the cooling agent (ice or ice water). Temperature of each sample was recorded at one-minute intervals for 15 min.Six treatments  were evaluated over 16 time points (0–15 min), with 30 replicates per treatment. Temperature data were evaluated using a one-way ANOVA with repeated measures, with treatment as the between subject factor and time as the within subject factor. Time was analyzed as a categorical variable and a numeric variable, respectively, to estimate the effect of treatment for each time interval and the overall trend of time effect. The rate of cooling (speed) data was analyzed using one-way ANOVA. Rate of cooling was computed using the range of the temperature change divided by the total time interval (15 min). Ranked transformation was applied when data violated ANOVA assumptions such as non-normality and unequal variance. Post hoc multiple comparison was conducted with Tukey’s adjust. To analyze the trend of time, time was included in the model as a numeric factor whereas treatment was included as a categorical variable. Significance was set at p < 0.05, and all analysis was processed in SAS9.4 TS1M1 for Windows x64 .Results are summarized in Within treatment conditions , sample PCV had no significant effect on cooling. Cooling in ice water was significantly faster than cooling on ice only or P/C tube on ice . RegardlFor samples with a 20% PCV, mean temperature at 4 min of cooling in ice water was significantly lower than mean temperature at 14 min for samples cooled on ice only (p = 0.0148) or at 11 min for samples in P/C tube on ice (p < 0.0001). Of samples with a 40% PCV, mean temperature at 4 min of samples cooled in ice water was significantly cooler than mean temperature at 13 min for samples cooled on ice only (p = 0.0335) or at 12 min for samples in P/C tube on ice (p < 0.0001).For samples with a PCV of 40%, there was no difference in mean temperatures or rate of cooling of samples placed on ice and those placed in P/C tubes on ice. For samples with a 20% PCV, however, the rate of cooling was significantly faster for samples in a P/C tube on ice versus those placed on ice. From 4 to 13 min, mean temperatures of samples with a 20% PCV that were placed in a P/C tube on ice were significantly lower than mean temperatures of samples placed on ice without precooling the tube.In this study, we determined blood sample cooling occurred more rapidly and to a greater extent when samples were placed in an ice water bath versus in a cup of crushed ice. With a closed system that lacks motion, as described in this study, cooling of blood samples relies primarily on conduction. Conduction is the flow of heat that reduces the temperature difference between two materials . TransfeIn our study, cooling was performed using closed systems containing crushed ice or ice water. Ice water consists of two major components, ice and water, with thermal conductivity coefficients of 2.1 W/(m K) at 0 °C for ice and 0.6 W/(m K) at 25 °C for water. Crushed ice contains a small amount of liquid water but primarily consists of ice and air, the latter of which has a thermal conductivity coefficient of 0.024 W/(m K) at 0 °C. Because thermal conductivity of water is much greater than air, it serves as a better conductor of heat and thus increases the rate of heat loss from the sample .Surface areas of the cooling materials used in this study cannot be calculated, since pieces of crushed ice varied greatly in size and shape and thus in their ability to contact the surface of the tube. Therefore, a variable amount of air was present within the crushed ice condition. Addition of water to the ice increased the surface area for diffusion of heat via a material with higher conductivity, thereby speeding heat transfer. Temperature of the condition (ice or ice water) was not measured in this experiment. Temperature difference was likely greater between the sample and the ice water, however, since pockets of air within the crushed ice could have maintained higher temperatures than the ice water.A PCV of 40% was chosen as the normal PCV in this study because it was consistent with the mean PCV (39.6% ± 6.4) of 102 dogs presenting for ammonium measurement at our clinic . The samples were diluted with physiologic saline (versus plasma) to maintain consistency, since plasma components can vary among dogs. Between conditions, PCV had no significant effect on rates of cooling. This is not surprising, since the coefficients of thermal conductivity for EDTA blood and EDTA plasma are approximately the same as that of water . HoweverOne concern with use of an ice water bath for cooling of diagnostic whole blood specimens is whether the rapid rate of cooling and attainment of “near-freezing” temperatures will adversely affect samples. In terms of ammonium concentration, sample storage at 0 to −80 °C will reduce, but not halt, spontaneous ammonia generation . Both raCanine EDTA whole blood samples cool most rapidly and to a greater degree when placed in an ice water bath rather than in ice. Use of ice water baths may be important for reducing preanalytical error in the measurement of ammonium and other analytes altered by metabolic processes that continue within blood samples after they are collected; further research is needed in this area. Samples stored on ice water can rapidly drop below normal refrigeration temperatures; this should be taken into consideration when using this cooling modality.10.7717/peerj.2732/supp-1Supplemental Information 1On the X axis is time (0–15 min) for each condition.Click here for additional data file.


Thus, in the current study, we investigated the mechanisms by which hypercapnia effects ENaC cell surface stability in alveolar epithelial cells (AECs). Elevated CO2 levels led to polyubiquitination of β-ENaC and subsequent endocytosis of the α/β-ENaC complex in AECs, which were prevented by silencing the E3 ubiquitin ligase, Nedd4-2. Hypercapnia-induced ubiquitination and cell surface retrieval of ENaC were critically dependent on phosphorylation of the Thr615 residue of β-ENaC, which was mediated by the extracellular signal-regulated kinase (ERK)1/2. Furthermore, activation of ERK1/2 led to subsequent activation of AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK)1/2 that in turn phosphorylated Nedd4-2 at the Thr899 residue. Importantly, mutation of Thr899 to Ala markedly inhibited the CO2-induced polyubiquitination of β-ENaC and restored cell surface stability of the ENaC complex, highlighting the critical role of Nedd4-2 phosphorylation status in targeting ENaC. Collectively, our data suggest that elevated CO2 levels promote activation of the ERK/AMPK/JNK axis in a human AEC line, in which ERK1/2 phosphorylates β-ENaC whereas JNK mediates phosphorylation of Nedd4-2, thereby facilitating the channel–ligase interaction. The hypercapnia-induced ENaC dysfunction may contribute to impaired alveolar edema clearance and thus, interfering with these molecular mechanisms may improve alveolar fluid balance and lead to better outcomes in patients with ARDS.Acute lung injury is associated with formation of pulmonary edema leading to impaired gas exchange. Patients with acute respiratory distress syndrome (ARDS) require mechanical ventilation to improve oxygenation; however, the use of relatively low tidal volumes (to minimize further injury of the lung) often leads to further accumulation of carbon dioxide (hypercapnia). Hypercapnia has been shown to impair alveolar fluid clearance (AFC), thereby causing retention of pulmonary edema, and may lead to worse outcomes; however, the underlying molecular mechanisms remain incompletely understood. AFC is critically dependent on the epithelial sodium channel (ENaC), which drives the vectorial transport of Na This creates a Na+ gradient, which drives paracellular movement of water leading to its clearance from the alveolar space , and recently also the cellular energy sensor, AMP-activated protein kinase (AMPK), have been described as potential modulators of the ENaC/Nedd4-2 interaction by phosphorylating the E3 ligase or the target  were grown in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin, 100 µg/ml streptomycin as previously described . Before each experiment, fresh solutions were prepared with DMEM-Ham’s F-12 medium and Tris base. The buffering capacity of the experimental media was modified by changing the initial pH using Tris base to obtain a pH of 7.4 at 40 and 120 mmHg CO2 . The C-Chamber’s atmosphere was controlled with a PRO-CO2 Carbon Dioxide controller (Biospherix Ltd.). In the chamber, cells were treated with a pCO2 of 40 or 120 mmHg while keeping 21% O2 balanced with N2. Before and after CO2 exposure, pH, pCO2, and pO2 levels in the media were measured using a Rapidlab blood gas analyzer .A549 cells were treated with 40 or 120 mmHg COmmHg CO2 . The desEcoRI/BamH1 and ligated to the multiple cloning site (MCS) of pEYFP-C1 plasmid. The pEYFP-C1 vector contained the epitope-tag eYFP at the N-terminus and a FLAG-tag at the C-terminus. Thus, anti-GFP or anti-FLAG antibodies recognize the α-ENaC construct at a predicted size of 118 kDa, 1,073 amino acids [α-ENaC (90 kDa) plus YFP (27 kDa) and FLAG (1 kDa)]. pcDNA3.1V5/His expressing β-ENaC was constructed by PCR amplifying β-ENaC gene using as a template cDNA transcribed from total mRNA isolated from A549 cells and oligonucleotide primers β-ENaC forward 5′-CTCGGATCCACATGCACGTGAAGAAGTACCT-3′ and β-ENaC reverse 5′-GCACTCGAGGATGGCATCACCCTCACTGT-3′. The resulting amplicon was digested with Xho1/BamH1 and ligated to MCS of pcDNA3.1V5/His plasmid. Finally, E. coli DH5α were transformed using the constructed plasmid. Anti-V5 antibodies recognize the β-ENaC construct at a predicted size of 96 kDa, 872 amino acids [β-ENaC plus V5 at the C-terminus (1 kDa)]. pCMV-HA-C-expressing γ-ENaC was constructed by PCR amplifying γ-ENaC gene using as a template pTNT-γENaC and oligonucleotide primers γ-ENaC forward 5′-AGGCCCGAATTCATGGCACCCGGAGAGAAGAT-3′ and γ-ENaC reverse 5′-GTAGCCGGTACCGAGCTCATCCAGCATCTGGG-3′. The resulting amplicon was digested and ligated to MCS of pCMV-HA-C plasmid. The pCMV-HA-C vector contained the epitope-tag myc at the N-terminus and an HA-tag at the C-terminus. Anti-HA antibodies recognize γ-ENaC at a predicted size of 97 kDa, 881 amino acids [γ-ENaC plus myc (1 kDa) and HA (1 kDa)]. pRK5-HA-ubiquitin was a gift from Ted Dawson [Addgene 17608  and lysed in lysis buffer . Surface proteins were pulled down with streptavidin-agarose beads from Pierce Biotechnology  and analyzed by SDS-PAGE and immunoblot, as described previously (A549 cells were labeled for 20 min using 1 mg/ml EZ-Link eviously .2 (Ctrl) or to 120 mmHg CO2 (CO2) for 15 or 30 min and lysed on ice in lysis buffer , containing a protease inhibitor cocktail from Roche. After lysing the samples, proteins were resolved in 8% polyacrylamide gel, transferred to nitrocellulose membrane  using a semidry apparatus from Bio-Rad . Membranes were blocked in 5% fat-free dried milk powder and immunoblotted with anti-GFP or anti-V5 to detect α- or β-ENaC, respectively. Films were overexposed to detect ENaC ubiquitin conjugates.A549 cells were transfected with ENaC plasmids (2 µg of each) and HA-ubiquitin (3 µg). In some studies, cells were co-transfected with a plasmid coding HA-Nedd4-2  or siRNAs . Cells were exposed to 40 mmHg CO2 concentrations  for the desired times, and then were washed with PBS twice and were lysed on ice in lysis buffer . Samples having the same amount of protein were resuspended in Laemmli sample buffer and boiled for 10 min at 98°C and immunoblotted with specific antibodies.Phosphorylation studies of ERK1/2, AMPK-α1, and c-Jun were performed using antibodies from Cell Signaling . The anti-phospho-β-ENaC (T615) antibody was from Abcam . A549 cells were treated with normal or elevated COp values of less than 0.05 were considered significant. GraphPad prism 6  was used for the analysis and presentation of data.Data are presented as mean ± SEM and were analyzed using one-way analysis of variance (ANOVA) followed by a multiple comparison with the Dunnet test. 2 levels promote endocytosis of ENaC, A549 cells were co-transfected with plasmids encoding the human α- and β-subunit of ENaC and the PM abundance of these proteins was measured after exposure of cells to physiological  or elevated  CO2 concentrations at a pHe of 7.4 for 30 min. Exposure of cells to elevated CO2 levels decreased α- and β-ENaC cell surface abundance by approximately 60%  and exposed the cells to 40 or 120 mmHg CO2 for 15 min. Whereas no ubiquitination of α-ENaC in response to hypercapnia was evident  inhibitor U0126 and exposed to elevated CO2 concentrations for 30 min. Inhibition of ERK prevented phosphorylation and polyubiquitination of the ENaC β-subunit  exposed to hypercapnia , we foun2 Figure A. Moreov2 Figure B. To tes2-induced signaling pattern in AEC, which led to inhibition of the Na,K-ATPase , which cannot be phosphorylated, the level of β-ENaC polyubiquitination significantly decreased (Figure Because JNK activation has been implicated in the COK-ATPase , we nexts Figure A. To furs Figure . A549 ce2 exposure (2 levels (2 levels and observed a significant decrease in polyubiquitination of β-ENaC after CO2 exposure (Figure 2-induced downregulation of ENaC, we transfected A549 cells with α- and β-ENaC and exposed to elevated CO2 levels for 30 min in the presence or absence of the above mentioned siRNA against AMPK-α1 (Figure AMP-activated protein kinase, which has been shown to activate Nedd4-2 and inhibit ENaC , has alsexposure , 33. Fur2 levels . In line2 levels A, which 2 levels B and upse Figure C. As a se Figure D. To fur2 levels initiate a specific signaling pattern leading to ubiquitination-mediated retrieval of ENaC from the PM, thereby reducing cell surface abundance of the channel in a human AEC line. Hypercapnia is associated with a number of acute and chronic pulmonary diseases; however, it is not evident to what extent and by which mechanisms these elevated levels of CO2 may further impact on disease states. While hypercapnia and the associated acidosis have been shown to have anti-inflammatory effects, which might be advantageous at sites of excessive inflammation, recently, it has been clearly demonstrated that by impairing innate immunity, cellular repair, and alveolar epithelial function, elevated CO2 may play a role in the pathogenesis of ARDS and COPD (In the present study, we show that elevated COand COPD , 10, 38.and COPD , 40.2 levels may help us to interfere with the deleterious signals, thereby rescuing or at least not further aggravating lung damage.A major function of the alveolar epithelium is the clearance of excess alveolar fluid, thereby promoting effective gas exchange. This clearance is mediated by the concerted action of various sodium transporters, among which the apically located ENaC and the basolateral Na,K-ATPase have been identified as key players. Indeed, we have previously shown that the Na,K-ATPase is downregulated by hypercapnia; however, a potential regulation of ENaC by carbon dioxide has not been previously investigated. Various factors have been shown to affect ENaC cell surface abundance and function, including interleukin-1β, interleukin-4, transforming growth factor-β, LPS, or hypoxia –45, whicUbiquitination is a posttranslational modification that regulates trafficking and stability of proteins . Numerou2 concentrations affect ENaC cell surface stability by a mechanism involving ubiquitination of the channel and whether the MAPK pathway is involved in the hypercapnia-induced signaling events. Of note, a remarkable and rapid increase in polyubiquitination of β-ENaC and a significant reduction of the cell surface abundance of the α/β-ENAC complex were observed in AEC exposed to hypercapnia, as early as 30 min after CO2 exposure, suggesting that ENaC function is probably sensitive to changes in CO2 levels. In contrast, in the first half an hour after CO2 exposure, total intracellular levels of ENaC remained unchanged, suggesting that CO2 influenced the trafficking of the channel rather than protein degradation. Furthermore, no significant changes in the ubiquitination status of α-ENaC have been detected upon hypercapnic treatment, highlighting and further confirming the central regulatory role of β-ENaC in the trafficking of the channel (Thus, we first investigated whether elevated CO channel .2 levels cause a rapid and time-dependent ERK1/2 activation followed by phosphorylation of β-ENaC at the Thr615 residue. Moreover, genetic inhibition of Nedd4-2, the E3 ubiquitin ligase that drives ubiquitination of the various ENaC subunits (+ transport (Our data demonstrate that elevated COsubunits , by a spsubunits . Anotherransport . Therefo2 responses and that phosphorylation of the kinase is required for the CO2-induced inhibition of the Na,K-ATPase in the alveolar epithelium (2-induced polyubiquitination of β-ENaC although activation of JNK was evident and stabilized α- and β-ENaC at the cell surface. Thus, our data together with the previously published literature suggest that phosphorylation of Nedd4-2 by JNK at the Thr899 residue is critical for the hypercapnia-induced ubiquitination of β–ENaC, which drives endocytosis of the ENaC complex from the PM in AEC.We have previously shown that JNK is also implicated in COithelium . The sigithelium . Of noteithelium . Remarkaithelium . To asse−/− mice (2-induced β-ENaC polyubiquitination and endocytosis of α-, and β-ENaC, which is consistent with previous findings showing that in human embryonic kidney cells, AMPK activation promoted Nedd4-2/ENaC association (2 on Nedd4-2 and ENaC are indirect and mediated by JNK. Moreover, although AMPK is an early element of the CO2-induced signaling pattern, its activation appears to be downstream of ERK upon hypercapnic exposure. This is of particular importance as ERK appears two have a dual role in the hypercapnia-induced downregulation of ENaC. On the one hand, it rapidly phosphorylates the β-subunit of the channel and by activating AMPK and JNK, it indirectly promotes phosphorylation of the E3 ligase Nedd4-2 as well. Of note, both of these phosphorylation events seem to be critically required for the CO2-induced ubiquitination and subsequent endocytosis of ENaC, probably by enhancing the association of the E3 ligase and the target molecule.We have previously shown that AMPK, a cellular metabolic sensor that inhibits several ion transporters including the cystic fibrosis transmembrane conductance regulator, Na,K-ATPase, and ENaC, is rapidly activated by hypercapnia , 29, 54.−/− mice . Interes−/− mice , 29. In ociation . Furtherociation , we alsoin vivo investigations will be necessary to establish the role of the hypercapnia-induced signaling events identified in the current manuscript in ENaC-driven AFC and alveolar epithelial barrier dysfunction in an animal model of hypercapnic acute lung injury.Our study has some clear limitations. Although we show a rapid activation of ERK and a subsequent phosphorylation of β-ENaC at the Thr615 residue, which is a known target of ERK, we have not investigated the potential rescue of β-ENaC ubiquitination or trafficking of the ENaC complex after preventing phosphorylation at this specific site. A mutation of this residue will be necessary to definitely prove that ERK-promoted phosphorylation of β-ENaC at this residue drives the downregulation of the channel upon hypercapnia. Moreover, the current study was performed exclusively in AECs and further 2 levels, ENaC cell surface abundance is rapidly downregulated in a human AEC line by a specific, CO2-induced and ERK-, AMPK-, and JNK-mediated signaling pathway, which promotes phosphorylation of both β-ENaC and Nedd4-2, leading to ubiquitination of β-ENaC and subsequent internalization of the α/β-ENaC complex. This novel signaling pathway may contribute to the persistence of alveolar edema and thus, interfering with these molecular mechanisms may improve alveolar fluid balance and lead to better outcomes in patients with ARDS and hypercapnia.Taken together, our study shows for the first time that upon exposure to elevated COConception or design of the work: PG and IV; acquisition, analysis, or interpretation of data: PG, BB, KM, SH, RM, WS, and IV; drafting the work: PG and IV; revising it critically for important intellectual content: PG, BB, KM, SH, RM, WS, and IV. All the authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reconstructing the extent, flow and decay of the Laurentide Ice Sheet (LIS) on the continental shelves of North America during the last glaciation provides paleoglaciological analogues that are essential for understanding and predicting how modern marine-based ice-sheets will respond to future climate change and sea level fluctuations. The geometry of the LIS during Marine isotope stage 2  is one key element for ice-sheet modelling. The maximum extent of the LIS during this stage is well constrained for most sectors of the ice sheet, but major uncertainties remain, especially along the continental shelves of Arctic Canada. Despite a series of recent papers, the extent of the LIS in Western Baffin Bay, an area draining large volumes of glacial ice through multiple ice streams and likely characterized by ice shelves, remains highly speculative. Here we present unequivocal marine geophysical evidence that during the MIS-2 the LIS extended to the edge of the continental shelf, seaward of the previously proposed limits and subsequently retreated episodically westward during deglaciation. These data support interpretations of deep glacial ice grounding in Western Baffin Bay. The Flint (extensive) scenario1 was supported by modelling4 but, however, lacked field data while the minimalist scenario has been challenged by recent dating of moraines6 and by the identification of terrain preserved under cold-based ice on coastal forelands8. Cosmogenic nuclides exposure dating on terrestrial moraines as well as lacustrine and marine sediment cores data were used to resolve the enigma of the maximum extent of the LIS by proposing a scenario where most of LIS outlet glaciers reached the continental shelf at fjord mouths, with ice margins terminating inland in-between fjords 9. This interpretation was largely used in most paleogeographical reconstructions and in ice-sheet modelling studies15. More recent studies have, however, challenged this model, suggesting a more extensive LIS margin on the northeastern Baffin Island shelf during the Last Glacial Maximum (LGM) based on the observation of (1) an ice-contact seismostratigraphic unit extending on most of the continental shelf (the Baffin Shelf Drift16) and (2) a till wedge at 1300 m water depths at the southeast end of Lancaster Sound deposited at the ice grounding-zone17  are aligned in the trough axis and have low amplitudes (<20 m). Their very good state of preservation and the weak attenuation of their morphology due to a thin cover of postglacial sediments suggest that they relate to the last stage of ice occupation. Crosscut by grounding-zone wedges, MSGLs are interpreted to reflect time-transgressive ice-flows occurring during the landward retreat of an ice stream. In Pond and Scott troughs, MSGLs extend onto the shelf break  as the limit of the LIS margin during MIS-2.Four cross-shelf troughs that were glacially eroded and moulded during Quaternary glaciationsghs Fig. . These meak Figs . A smalleak Figs . These l24 which restricts vertical accommodation space for sediments in favor of sediment progradation, leading to the formation of low-amplitude and horizontally extensive landforms 23. GZWs are observed in the four studied troughs from swath bathymetry imagery and/or seismic data  that is coherent with postglacial sedimentation rates observed in Scott Trough16. Also, all the GZWs observed in Scott, Buchan and Pond troughs overprint sets of MSGLs formed during a previous phase of ice-flow  are formed by the accumulation of subglacial sediments at the grounding zone of an ice stream during temporary standstills of an ice margin e.g. ata Figs . On swatata Figs . On seisata Figs . The tra33. TMFs generally consist of stacked acoustically transparent lenses of glacigenic-debris flows (GDFs) separated by acoustically stratified units representing suspension-settling sediments, turbidites and/or contourites37. GDFs are mostly the product of instabilities at the grounding zone during the occupation of the trough by an ice stream, while the stratified units relate to deglacial to interglacial conditions43. Stacked GDFs interbedded with stratified units observed on seismic reflection profiles in Scott Trough could represent such a succession of glacial debris flows and interglacial sedimentation, but of unknown age age Fig. . At the ank Fig. . A hummoank Fig. . These l46. Multiple ISLMs are observed on both sides of Buchan and Scott troughs  on the outer continental shelf. Similarly to GZWs it could also indicate the presence of a constraint for vertical accumulation 47.Ice stream lateral moraines (ISLMs) indicate fast-flowing ice surrounded by either slower ice-flow or ice-free terrainghs Fig.  and 2, fghs Fig. . This di48. The chaotic acoustically semi-transparent unit extends laterally from the ridges and parallel to the shelf break; it is interpreted as ice-contact sediments correlated to the Baffin Shelf Drift16.Swath bathymetry imagery reveals ridges parallel to the shelf break on the banks seaward of Cape Adair Fig. . These r9 is located ~100 km eastward from the MSGLs terminus. Ice flowing through Pond Trough likely converged into the Lancaster Ice Stream in Bylot Trough and from there flowed eastward to reach the LGM grounding-zone on the Lancaster TMF17. Subglacial sediments delivered to Bylot Trough by the Pond Trough ice stream would have been taken in charge by Lancaster ice that fed the Lancaster Fan. In Sam Ford Trough, GZWs constructed from the same till/ice-contact seismic unit in both Buchan and Scott troughs extends at least to the middle of Sam Ford Trough to form a distinct transverse-to-flow ridge  the ice-contact sediments and the TMFs reported here could be of diachronic origin; (2)14C dates from these deposits are lacking; and (3) the LGM is a diachronic position corresponding to a time-period rather than a precise event in time. This time-period along eastern Baffin Island is yet to be resolved50.Ice-contact deposits observed at the shelf edge are interpreted as MIS-2 because (1) they occur down-ice (eastward) from series of GZWs deposited during an episodic westward retreat of the LIS margin; and (2) the grounding-zone wedges are overlain by only one sedimentary sequence of deglacial-postglacial deposition corresponding to the last glacial retreat. The thickness of the upper sedimentary sequence along with the sedimentation rates observed in Scott Trough support a deglacial origin for the GZWs. A deglacial age for the GZWs therefore implies a seaward position of the LIS margin during MIS-2. The LIS margin could have reached its maximum extent prior to the LGM53. The presence of cold-based ice in the inter-ice stream areas is in accordance with the preservation of older deposits than MIS-2 on coastal forelands in between the fjords7. The basal thermal regime of the LIS in the region seems therefore complex and spatially variable with low-gradient outlet fast-flowing ice being constrained to the fjords axis as proposed by Miller et al. (2002). In Sam Ford Trough, the absence of ice-flow morphologies and the presence of perpendicular-to-trough ridges suggest that ice-flow was slow in the trough. This slow ice-flow was probably due to the transport of ice discharge from Sam Ford Fjord into the Scott Inlet Ice Stream. The ice-flow route from Sam Ford Fjord to Scott Inlet can be inferred from the northward orientation of MSGLs and drumlins at the junction of Sam Ford Fjord and Hecla & Griper Trough. The location of the GZWs suggests that the retreat of the LIS margin from the shelf edge to the fjords head has been marked by several phases of stabilization.The revised ice-margin and the distribution of highly elongated bedforms (MSGLs) indicate that ice streams were operating along Buchan, Scott and Pond troughs during MIS-2. The presence of ISLMs suggests that the LIS was experiencing spatial variations in ice-flow velocities, suggesting that inter-trough areas, were submitted to slow ice-flow or cold-based ice50 and is in accordance with inferred LGM extents recently raised18.The swath bathymetry and geophysical data presented in this paper provide firm evidence that the LIS extended across the northeast Baffin Island continental shelf during MIS-2, reaching the continental shelf break between Scott Trough and Pond Trough. These data also confirm the presence of grounded glacial ice on the Lancaster Fan east of Pond Trough during MIS-2. The proposed maximum extent of the LIS during MIS-2 is located at least 36 km seaward of the previously proposed limits16. Finally, these results indicate that the LIS deglaciation was driven by a complex spatially varying thermal regime with low-gradient ice-streams in the troughs and cold-based/slow-flowing ice on the inter-trough areas. An LIS deglaciation from an outer seaward position implies differences in the LIS geometry, dynamics, flow-mechanisms and feedbacks to post-LGM relative sea-level rise in Western Baffin Bay. This revised ice margin and its retreat dynamics should therefore be taken into account to increase the accuracy of LIS reconstructions or modelling, which have implications on how we can understand the long-term evolution of a marine-based ice-sheet during deglaciation.Fast-flowing ice streams occupied Pond, Buchan and Scott troughs while slow ice-flow or cold-based ice occupied the shelf and Sam Ford Trough. The presence of GZWs in the four major cross-shelf troughs indicates an episodic retreat style punctuated by ice-margin stabilization rather than a rapid/catastrophic retreatwww.caris.com/products/hips-sips/) and MB-System (https://www.ldeo.columbia.edu/res/pi/MB-System/) softwares. Data visualization and mapping was realized using ESRI ArcGIS 10.4 software (http://www.esri.com). Seismic reflection data were analyzed and extracted using the LizardTech GeoViewer software (https://www.lizardtech.com/geoviewer-pro/overview). Maps and seismic reflection data were transferred to the Adobe Photoshop CS5 software (www.adobe.com/photoshop) for figure production and editing. Seismic reflection data were enhanced using the Brightness/Contrast tool in Adobe Photoshop CS5 for a clearer visualization.Multibeam data were collected using Kongsberg Simrad EM-300 (12 kHz) and EM-302 (30 kHz) echosounders onboard the CCGS Amundsen by the Ocean Mapping Group (University of New Brunswick) and the Laboratoire de Géosciences Marines  for the ArcticNet program. The multibeam data were processed using the Caris HIPS&SIPS (http://ftp.maps.canada.ca/pub/nrcan_rncan/raster/marine_geoscience/Seismic_Reflection_Scanned/).The multibeam bathymetry dataset can be visualized on the Université Laval Géoindex + website . The seismic reflection data along with the acquisition specifics are available on the Geological Survey of Canada website (


The double-stranded RNA mimetic poly(I:C) and lipopolysaccharide (LPS) activate Toll-like receptors 3 (TLR3) and TLR4, respectively, triggering the activation of TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1) complexes, the phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of the interferon β (IFNβ) gene. Here, we demonstrate that the TANK–TBK1 and optineurin (OPTN)–TBK1 complexes control this pathway. The poly(I:C)- or LPS-stimulated phosphorylation of IRF3 at Ser396 and production of IFNβ were greatly reduced in bone marrow-derived macrophages (BMDMs) from TANK knockout (KO) mice crossed to knockin mice expressing the ubiquitin-binding-defective OPTN[D477N] mutant. In contrast, IRF3 phosphorylation and IFNβ production were not reduced significantly in BMDM from OPTN[D477N] knockin mice and only reduced partially in TANK KO BMDM. The TLR3/TLR4-dependent phosphorylation of IRF3 and IFNβ gene transcription were not decreased in macrophages from OPTN[D477N] crossed to mice deficient in IκB kinase ε, a TANK-binding kinase related to TBK1. In contrast with the OPTN–TBK1 complex, TBK1 associated with OPTN[D477N] did not undergo phosphorylation at Ser172 in response to poly(I:C) or LPS, indicating that the interaction of ubiquitin chains with OPTN is required to activate OPTN–TBK1 in BMDM. The phosphorylation of IRF3 and IFNβ production induced by Sendai virus infection were unimpaired in BMDM from TANK KO × OPTN[D477N] mice, suggesting that other/additional TBK1 complexes control the RIG-I-like receptor-dependent production of IFNβ. Finally, we present evidence that, in human HACAT cells, the poly(I:C)-dependent phosphorylation of TBK1 at Ser172 involves a novel TBK1-activating kinase(s). IFNβ gene and stimulates transcription . Cell lysates were clarified by centrifugation at 14 000×6 cells) were plated onto a 10 cm diameter tissue culture grade plate and transfected the next day with 1 µg each of guide (g) RNA plasmid targeting the gene of interest. FuGene® HD transfection reagent (5 µl per 1 µg of plasmid DNA) was used for transfection. At 24 and 48 h post-transfection, fresh media containing 2 µg/ml puromycin was added to the cells, while 72 h after transfection the puromycin-containing medium was replaced by medium lacking puromycin. After a further 48 h, cells were single-cell plated onto 96-well plates and left for 2–3 weeks until colonies began to form. The colonies were then analyzed for the expression of TRIF and TAK1 by immunoblotting.HACAT cells . To immunoprecipitate TANK and OPTN, 1.0 mg of cell extract protein was incubated for 2 or 4 h with 3.0 µg of anti-TANK or 6.0 µg of anti-OPTN. Protein-G-Sepharose beads (20 µl) were added and, after end-over-end rotation for 1 h at 4°C, the beads were collected by centrifugation, washed three times with cell lysis buffer, denatured in SDS, subjected to SDS–PAGE, transferred to polyvinylidene fluoride membranes and immunoblotted.Total RNA was extracted from macrophages using the MicroElute Total RNA kit (Omega bio-tek). RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer's instructions. Polymerase chain reaction (PCR) mixes were assembled using the SsoFast™ EvaGreen® Supermix (Bio-Rad). Reactions were performed with the SYBR Green  programme on the C1000 thermal cycler quantitative PCR system (Bio-Rad). All reactions were performed in duplicate. The concentration of IFNβ released into the cell culture medium was determined by using the LegendMax mouse IFNβ ELISA kit (Biolegend). The primer sequences used in the present paper are given in Supplementary Table S1.This was performed using the Image J software and normalized to immunoblots obtained with antibodies that recognize all forms of IRF3.P < 0.05; *P < 0.05, **P < 0.01, ***P < 0.001. n.s. means not significant.Statistical analyses were performed using the GraphPad Prism software. Quantitative data were presented as the arithmetic mean ± SEM. Statistical significance of differences between wild-type and the other genotypes was assessed using two-way ANOVA with Bonferroni post-test to compare each genotype with the control wild-type sample, unless stated otherwise. Differences in means were considered significant if OPTN is the protein with the greatest amino acid sequence similarity to NEMO, the regulatory component of the canonical IKK complex. The proteins have similar ubiquitin-binding domains and, like NEMO, OPTN binds to Met1-linked ubiquitin (M1-Ub) and Lys63-linked ubiquitin (K63-Ub) chains, but not to Lys48-linked ubiquitin chains . To inveTLR3 signals specifically via the adaptor protein TRIF, whereas TLR4 signals via MyD88 and TRIF. The decreased phosphorylation of TBK1 at Ser172 observed 15 min after stimulation with LPS in BMDM from TANK KO and TANK KO × OPTN[D477N] mice is explained by the TANK–TBK1 complex making a major contribution to the MyD88-dependent activation of TBK1 , which oThe mRNAs encoding TLR3, TLR4 and TRIF were not decreased in BMDM from the TANK KO, OPTN[D477N] or TANK KO × OPTN[D477N] mice , indicating that the decreased phosphorylation of IRF3 and reduced production of IFNβ mRNA in BMDMs from TANK KO × OPTN[D477N] mice were not explained by decreased expression of these receptor and adaptor molecules that are situated ‘upstream’ of IRF3 in this signalling pathway. The expression of UNC93B, which interacts with TLR3 and localizes it to endosomal membranes , was alsIKKε, the protein kinase most closely related to TBK1, also forms a complex with TANK  and has LPS stimulation decreased the electrophoretic mobility of TANK and OPTN . This isifnβ gene transcription in mouse BMDM, since the JNK-dependent activation of the transcription factors c-Jun and ATF2 has been reported to co-ordinate the adenovirus-mediated induction of primary IRF3-responsive transcripts in conjunction with activated IRF3  and wild-type mice up to 1 h. However, the poly(I:C)-dependent activation of these MAP kinases then decreased much more rapidly between 1 and 4 h in TANK KO × OPTN[D477N] BMDM compared with wild-type BMDM , we studied Sendai virus-induced IFNβ production in BMDM. These experiments showed that IRF3 phosphorylation at Ser396 and IFNβ secretion were similar in BMDM from TANK KO, OPTN[D477N], TANK KO × OPTN[D477N] and wild-type mice . Therefore, in contrast with the TLR3- and TLR4-signalling networks, the TANK–TBK1 and OPTN–TBK1 complexes were not rate-limiting for RLR-dependent IFNβ production under the conditions that were studied.The interleukin-1 (IL-1)-dependent phosphorylation of TBK1 at Ser172 in mouse embryonic fibroblasts (MEFs) is catalyzed by IKKβ and by TBK1 itself, and is therefore prevented by incubating IKKα-deficient MEFs with an IKKβ inhibitor plus a dual TBK1/IKKε inhibitor, but not by either inhibitor alone. Since the IL-1-dependent activation of IKKα and IKKβ in MEFs is catalyzed by the protein kinase TAK1 , the IL-1-dependent phosphorylation of TBK1 can also be suppressed by a TBK1 inhibitor in MEFs from knockin mice expressing a kinase-inactive form of TAK1 . In contMEFs do not respond robustly to either poly(I:C) or LPS, and TAK1 KO/knockin mice display early embryonic lethality. To investigate which protein kinases activate TBK1 in the TLR3/TLR4–TRIF signalling network, we therefore used human keratinocyte HACAT cells, which respond to poly(I:C)  44]. Th. Th44]. Interestingly, although the poly(I:C)-dependent phosphorylation of IRF3 at Ser396 was unimpaired in TAK1 KO HACAT cells , IFNβ geifnb gene transcription - or LPS-induced phosphorylation of Ser172 in the OPTN–TBK1 complex is prevented in the OPTND477N]–TBK1 heterodimer , implyin7N–TBK1 h


BB significantly increased CREB activity, dependent on increased CREB phosphorylation at serine-133 but not on CRTC-2/3 activation. CREB silencing significantly inhibited basal and PDGF induced proliferation. These data demonstrate that cAMP activation of CREB, which is CRTC2/3 dependent and serine-133 independent, is anti-mitogenic. Growth factor activation of CREB, which is serine-133-dependent and CRTC2/3 independent, is pro-mitogenic. Hence, CREB plays a dual role in the regulation of VSMC proliferation with the mode of activation determining its pro- or anti-mitogenic function.Vascular smooth muscle cell (VSMC) proliferation has been implicated in the development of restenosis after angioplasty, vein graft intimal thickening and atherogenesis. We investigated the mechanisms underlying positive and negative regulation of VSMC proliferation by the transcription factor cyclic AMP response element binding protein (CREB). Incubation with the cAMP elevating stimuli, adenosine, prostacyclin mimetics or low levels of forksolin activated CREB without changing CREB phosphorylation on serine-133 but induced nuclear translocation of the CREB co-factors CRTC-2 and CRTC-3. Overexpression of CRTC-2 or -3 significantly increased CREB activity and inhibited VSMC proliferation, whereas CRTC-2/3 silencing inhibited CREB activity and reversed the anti-mitogenic effects of adenosine A2B receptor agonists. By contrast, stimulation with serum or PDGF Vascular smooth muscle cell (VSMC) proliferation and migration contributes towards the development of various vascular diseases characterised by pathological intima formation, including atherosclerosis, transplant vasculopathy and pulmonary hypertension. Increased VSMC proliferation and migration also contributes towards restenosis after angioplasty and late vein graft failure. Interruption of this cycle can slow disease progression and limit pathological intima formation. This is exemplified by medications that lower low density lipoprotein (LDL) cholesterol or block cell proliferation, which have revolutionized preventive therapy for cardiovascular disease (CVD) and ameliorated the problem of angioplasty restenosis, respectively. However, these do not fully normalize CV risk and additional treatments are therefore needed.s protein-coupled receptor stimulation. In VSMC, elevated cAMP inhibits mitogen stimulated proliferation in vitro and after injury in vivo5. Inhibitory effects of cAMP on VSMC migration also help reduce intimal lesion formation in experimental vascular injury models7. Moreover, altered or aberrant cAMP signalling is implicated in the development of many vascular pathologies9, including late vein-graft failure12, angioplasty restenosis13 cardiac hypertrophy14, atherogenesis15 and pulmonary hypertension. For example, the ability of VSMC to synthesise cAMP is reduced in response to hypertension16, vein grafting12, hypoxia and oxLDL17. cAMP levels are also reduced in blood mononuclear cells in CVD patients18. This suggests that deregulation of cAMP signalling is a common feature of many vascular diseases. Moreover, stimulation of cAMP signalling has numerous beneficial effects including reductions in neointima formation19, VSMC migration20, VSMC proliferation19, endothelial permeability21, inflammation, ROS generation, plasma triglycerides and monocyte recruitment. Therefore, treatments that elevate or mimic the action of cAMP carry significant therapeutic promise because they improve endothelial function, inhibit VSMC cell proliferation and migration and reduce inflammation.The second messenger 3′-5′ cyclic adenosine monophosphate (cAMP) is synthesized in cells by adenylyl cyclase enzymes in response to vasoactive G22. However, the mechanisms downstream of PKA and EPAC are incompletely characterised. Activation of the cAMP sensitive transcription factor CREB may represent one important mechanism underlying cAMP-dependent cellular responses. Some studies have suggested anti-mitogenic properties of CREB in VSMC. For example, CREB levels are high in differentiated VSMC but decrease during phenotypic modulation20. Stimuli that enhance cAMP-signalling and inhibit VSMC proliferation and are associated with increased CREB activity23. Consistent with this, CREB inhibition with siRNA or dominant-negative mutants is sufficient to induce VSMC proliferation25, whereas overexpression of constitutively-active mutants of CREB arrests cell-cycle progression25. However, several other studies have reported pro-mitogenic properties of CREB in VSMC. For example, VSMC proliferation induced by angiotensin II26, thrombin27 or the interferon-γ inducible protein-1028 is dependent on CREB activation. These observations suggest that CREB activation may play a role in both mitogenic and anti-mitogenic responses in VSMC.The anti-mitogenic effects of cAMP are mediated via activation of cAMP sensitive Protein Kinase A (PKA) and Exchange Protein Activated by cAMP (EPAC), which synergise to regulate levels of several cell-cycle proteins29. PKA is implicated since this residue is part of a consensus PKA phosphorylation site (RXXS) and its phosphorylation can be blocked by PKA inhibition30. However, selective PKA activation fails to inhibit VSMC proliferation2, suggesting that PKA-mediated phosphorylation of CREB at serine-133 may not be sufficient for the anti-mitogenic effects of cAMP in these cells. Furthermore, several studies have also suggested that phosphorylation of CREB at serine-133 may not be essential for CREB-dependent gene expression, implicating alternative mechanisms. For example, T-cell receptor stimulation of Jurkat cells induces high levels of CREB serine-133 phosphorylation without inducing target gene expression31. Induction of CREB activity by voltage-sensitive Ca2+ channels is markedly reduced in a PKA-deficient PC12 cells, despite robust CREB phosphorylation at serine-13332. Expression of mouse hippocampal CREB target genes in response to fear conditioning is unaffected in CREB-S133A transgenic mice33. Importantly, CREB can be activated independently of serine-133 phosphorylation by binding of CREB Regulated Transcription Coactivators 34. However, the role of CRTC-regulated CREB activity in VSMC is unknown. Here, we investigated the relative importance of CREB serine-133 phosphorylation and nuclear translocation of CRTCs in pro- and anti-mitogenic effects on VSMCs.Phosphorylation of CREB at serine-133 is one important mechanism of CREB-mediated transcriptional activationwild-type or GAL4-CREBS133A, in which serine-133 is mutated to an alanine. Stimulation with 0.5 µM forskolin significantly induced GAL4 reporter activity in cells expressing GAL4-CREBwild-type or GAL4-CREBS133A to a similar extent  significantly induced CREB-dependent reporter gene activity after 4 hours Fig. , whereasent Fig. . Taken t2. 6-BNZ-cAMP-AM (BNZ-AM) is an extremely membrane-permeant precursor of the specific PKA agonist 6-BNZ-cAMP. Selective activation of PKA by this agonist has been validated previously35. 8-pCPT-2′-O-Me-cAMP-AM (CPT-AM) is a membrane-permeant precursor of the specific EPAC agonist 8-pCPT-2′-O-Me-cAMP. Selective activation of EPAC but not PKA by this agonist has been validated previously36. In this study, stimulation with BNZ-AM or CPT-AM alone did not significantly increase CREB activity . In plasmid transduced, serum starved cells, GFP-tagged CRTC1, CRTC2 and CRTC3 were all predominantly cytoplasmic Fig. . CRTC1 r22. We therefore investigated the role of cAMP/CRTC-dependent CREB activation on VSMC proliferation and migration. We utilised three approaches to activate and inhibit CRTCs: adenovirus mediated expression of constitutively-active CRTC2 (Ad:CRTC2S171A) or CRTC3 (Ad:CRTC3S161A); pharmacological inhibition of Salt Inducible Kinase (SIK), which phosphorylates CRTC and represses activation; and siRNA-mediated silencing of CRTC2 and CRTC3. Infection of VSMC with Ad:CRTC2S171A or Ad:CRTC3S161A resulted in equal expression of FLAG-tagged CRTC2 and CRTC3 protein -induced proliferation  and this persisted for 60 minutes , which induce supra-physiological levels of cAMP in VSMC4. Consistent with these findings, we were also able to detect increased serine-133 phosphorylation in VSMC stimulated with 25 µM forskolin , phospho S133 CREB (#9196), VASP (#3132), CRTC3 (#2720) and Lamin A/C (#4777) were from Cell Signaling Technologies. Antibodies to GAPDH (#MAB374) and CRTC2 (#ST1099) were from Merck-Millipore. Anti-BrdU antibody (B2531) was from Sigma Aldrich. Silencer® Select siRNA targeting CRCT2 and CRTC3 (#4390791 and #4390771 respectively) and negative control (#4390843) were purchased from Thermo Fisher. ON-TARGETplus SMARTpool CREB siRNA (#L-092995-02-0005) and ON-TARGETplus Non-targeting Control Pool (#D-001810-10-05) were purchased from Dharmacon. UO126, LY294002, SB 203580 were from Merck-Calbiochem. Bisindolylmaleimide I  was purchased from Tocris Bioscience. MK-2206 was from Selleckchem. HG-9-91-01 was a gift from Dr Kris Clark (University of Dundee)51 Stimulations were performed in serum free DMEM unless otherwise stated. Cells were serum starved for 18 hours or 4 hours, as indicated in figure legends. Proliferation was measured by culture in the presence of 10 µM bromodeoxyuridine (BrdU) for 6 hours. Following fixation in 70% ethanol, incorporated BrdU was detected by immune-histochemical staining as previously described2. Typically, all cells (at least 200) in five to ten fields of view were manually counted using ImageJ software. For nuclear and cytosolic fractionation, cells were lysed with ice-cold hypotonic lysis buffer  and phosphatase (PhosTOP) inhibitor mix (Roche Diagnostics Ltd)). After incubation for 20 min on ice with regular vortexing, nuclei were pelleted at 2000 g at 4 °C and the supernatant was collected as the cytosolic fraction. After washing the pellet in lysis buffer containing 0.1% NP40, nuclear proteins were extracted in SDS lysis buffer .All experiments were performed using different batches of cells that were prepared from different animals. Male Sprague Dawley rats were killed by cervical dislocation in accordance with the Directive 2010/63/EU of the European Parliament, under schedule 1 of the United Kingdom Home Office Animal Scientific Procedures Act 1986 and performed in accordance with guidelines, regulations and approval of the University of Bristol. Approval was granted by the University of Bristol ethical review board. Cultures of rat aortic VSMCs (RaVSMCs) were prepared as previously described4 cells/well for into ImageLock-96 well plates (Essen Bioscience). Wells were scratched using a WoundMaker® tool and phase contrast images of cell migration into the wounded area acquired hourly for 24 hours. Relative wound confluence was calculated using the Cell Migration Image analysis module of the IncuCyte® ZOOM software.Real-time analysis of migration was performed using a IncuCyte® ZOOM live cell imaging system (Essen BioScience) according to the manufacturer’s instructions. Briefly, RaVSMCs were seeded at 2 × 1052. FLAG-CRTC1, FLAG-CRTC2 and FLAG-CRTC3 plasmids were a gift from Dr Nina Balthasar (University of Bristol). GFP-CRTC-1, -2 and -3 plasmids were constructed by sub-cloning CRTC-1, -2 or -3 cDNAs into pEGFP-N1 expression vector (Clontech). For adenoviruses expressing constitutively active CRTC2 (S171A) or CRTC3 (S162A), cDNAs were cloned into pDC515 (Microbix) and adenoviruses generated as previously described2. GAL4-Nano-luciferase plasmid (GLA4-NLUC) was created by sub-cloning the 5xGAL4 binding elements from plasmid pG5E1b-LUC (a gift from Ugo Moens) into the Nhe1 and Xho1 sites of pNL3.3[secNluc/minP] (Promega). pcDNAI-GAL4-CREBwild-type and pcDNAI-GAL4-CREBS133A expression vectors were obtained from Addgene (#46769 and #46771 respectively)30.CREB reporter plasmid  was a gift from Prof Stan McKnight2. Total RNA, extracted using Ambion Pure-Link kits (Thermo Fisher) and was reverse transcribed using QuantiTect RT kit (Qiagen) and random primers. Quantitative PCR was performed using Roche SYBR Green using a Qiagen Roto-Gene Q PCR machine . Primers sequences are described in Supplement Table Quantification of mRNA and protein levels was performed by RT-qPCR and western blotting respectively, as described previously53. Plasmid transfection was performed by electroporation using an Amaxa Nucleofector-1.5. 1 × 106 VSMCs were transfected with 5 μg of DNA or 100 pmoles of siRNA using the standard Nucleofector program A033. Cells were stimulated with the indicated agents 24 hours post transfection followed by lysis in Promega cell culture lysis buffer or for secreted nano-luciferase reporters cell culture media collected and assays using NanoGlow assay system (Promega). Luciferase and Renilla activity were quantified using the dual reporter assay kit (Promega) according to the manufactures instructions using Glomax Discover luminometer (Promega).CREB activity was determined by quantifying the luciferase reporter activity in cells transfected with CREB-LUC (α-168), which contains 168 bp of the endogenous alpha polypeptide glycoprotein hormones gene (CGA) promoter containing two identical repeats of the 8-bp core CREStatistical analysis was performed using two-way ANOVA, one-way ANOVA with Student-Newman-Keuls post-test or where appropriate a paired student’s t-test, as indicated. *Indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001.Supplementary Information


An approximation to the molecular mechanism of the inhibition has been proposed, suggesting that indolic compounds might interact with the cell surface components of the endothelial membrane in a way that prevents VEGF from activating the receptor. Additionally, wound-healing assay revealed that exposure of HUVECs to melatonin and 3-indolacetic acid in the presence of VEGF significantly inhibited cell migration by 87% and 99%, respectively, after 24 h. These data demonstrate that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin would be good molecules for future exploitation as anti-VEGF signaling agents.Excessive concentrations of vascular endothelial growth factor (VEGF) trigger angiogenesis, which causes complications such as the destabilization of atherosclerotic plaques and increased growth of tumors. This work focuses on the determination of the inhibitory activity of melatonin and other indolic related compounds on VEGF-induced VEGF receptor-2 (VEGFR-2) activation and an approximation to the molecular mechanism underlying the inhibition. Quantification of phosphorylated VEGFR-2 was measured by ELISA. Migration wound-healing assay was used to determine cell migration of human umbilical vein endothelial cells (HUVECs). This is the first time that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin are proved to significantly inhibit VEGF-induced VEGFR-2 activation in human umbilical vein endothelial cells and subsequent angiogenesis. 3-Indolacetic acid showed the highest inhibitory effect (IC Angiogenesis, the physiological process in which new blood vessels are formed from pre-existing ones, plays a critical role on tumor progression and development, as well as in the development and destabilization of atherosclerotic plaques ,2. AngioMelatonin is an ubiquitous molecule, which has been found not only in the human pineal gland but also in vegetables and their fruits and seeds, medicinal herbs, and fermented products such as nuts, tomatoes, beetroots, cucumber, banana, strawberry, cherry, apple, walnut, pistachio, bread, cocoa powder, green coffee, mustard seeds, feverfew, St John’s Wort, olive oil, wine, beer, etc., at concentrations varying between 5 pg/g or mL to 230 µg/g or mL ,27,28,29Melatonin has received a great deal of attention not only because it synchronizes circadian rhythms ,40 but aTryptophan is the essential precursor molecule for melatonin synthesis in animals and plants . In addiThe original aim of the present study was to determine the inhibitory effect of melatonin, serotonin, 5-hydroxytryptophol, and 3-indolacetic acid on VEGF-induced VEGFR-2 activation in human umbilical vein endothelial cells as a direct mechanism of action of angiogenesis inhibition by indolic compounds and to provide insight into the possible molecular mechanism. 2. All experiments were performed with HUVECs between passages 4 and 5.Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza . They were maintained in Endothelial Cell Growth Medium-2 (EGM-2) . The cells were cultured at 37 °C in an atmosphere at 5% COMelatonin, serotonin, and 3-indolacetic acid were purchased from Sigma-Aldrich . 5-hydroxytryptophol was obtained from Cayman Chemical . 165, R&D Systems, Minneapolis, MN, USA) for 5 min. After the treatments, the cells were lysed with radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors . The protein content of the lysates was determined through bicinchoninic acid assay . For IC50 value determination, five different concentrations were tested (0.1–2.5 mM) at 4 h of incubation.Confluent HUVECs were washed two times with warm phosphate-buffered saline (PBS) before the addition of either vehicle controls (≤0.1% DMSO) or indolic compounds  at 0.001–1 mM concentrations using endothelial basal medium (EBM) . The treatments were 4 and 24 h of incubation, with and without a washing step, prior to stimulation with 25 ng/mL VEGF  sandwich ELISA kit , following the manufacturer’s instructions.® 20 (TBST). The immunoreactive bands were detected using SuperSignal West Pico chemiluminescent substrate  and visualised on an Amersham Imager 600 station .The protein lysates were mixed with NuPAGE lithium dodecyl sulfate (LDS) sample buffer, NuPAGE DTT , and denatured by heating at 70 °C for 10 min. The protein contents were then subjected to electrophoresis on NuPAGE 4%–12% Bis-Tris gels (Invitrogen) before being transferred to 0.2 μm nitrocellulose membranes . Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) buffer and incubated overnight at 4 °C with antibodies directed against phospho-VEGFR-2 (Tyr 1175) and VEGFR-2 . The membranes were then incubated for 1h at room temperature with anti-rabbit IgG-HRP antibodies  in 5% bovine serum albumin (BSA) in tris buffered saline with TweenHUVECs were seeded onto 50 mm imaging dishes . When the cells achieved 100% confluence, a straight lesion was made in the center of the monolayer using a sterile 200-μL pipette tip. The wells were subsequently washed twice with PBS to remove the dead cells and incubated with EBM, containing 1 mM melatonin , 1 mM 3-indolacetic acid , or vehicle controls (≤0.1% DMSO) for 4 h before being treated with VEGF (25 ng/mL) for 24 h. The wounds were photographed using phase contrast microscopy on an inverted microscope . Initial and final wound sizes were determined using the Nis-Elements BR v.4.30.02 software , and the difference was used to determine the migration distance using the following formula; initial wound size minus final wound size divided by two. Three independent experiments were carried out. t test and one-way ANOVA (Dunnett’s multiple comparisons test) were used to test significant differences between samples.Statistical analyses were carried out using Graphpad Prism software . Student’s Five different experiments were designed to explore the mechanism of action resulting in the inhibition of VEGF-induced VEGFR-2 activation by melatonin . The firSecondly, we incubated HUVECs with melatonin, at the concentrations which had been proved to have a higher inhibitory effect (0.1 and 1 mM), or with vehicle control for 4 h. Subsequently, the cells were rinsed twice with PBS, and new media containing only VEGF (25 ng/mL) was added for 5 min. The third experimental design consisted of pre-mixing melatonin with VEGF prior to being exposed to HUVECs to determine whether melatonin binds directly to VEGF molecules, as occurs with other bioactive compounds such as polyphenols ,67. MelaSince pre-incubation of melatonin with HUVEC for 4 h and subsequent stimulation with VEGF for 5 min were the most effective conditions for the inhibition of VEGF-induced VEGFR-2 activation, we extended the pre-incubation time to 24 h to check whether the inhibitory effect was time-dependent. However, 0.1 mM melatonin did not show inhibition, and 1 mM melatonin showed only 14% of inhibition E, which Melatonin membrane receptors MT1 and MT2 have been described to be involved in mediating melatonin antiproliferative properties in HUVECs; melatonin binds to these receptors and inhibits the activation of the intracellular cascade ERK/PI3K/Akt/PKC/ NF-κB . To exclAll the results that originated from the abovementioned experiments support the notion that the interaction of melatonin with the cell surface components of the endothelial membrane, in a way that prevents VEGF from activating the receptor, might be the dominant mechanism, resulting in the highest inhibition of VEGF-induced VEGFR-2 activation by melatonin.We also evaluated the inhibitory effect of other related indolic compounds derived from tryptophan metabolism, such as 3-indolacetic acid, 5-hydroxytryptophol, and serotonin, on VEGF-induced VEGR-2 activation and whether they share the same mechanism of action. For comparative purposes, these compounds were tested following the same experimental conditions and concentrations (0.1 and 1 mM) mentioned above for melatonin.50 value of 0.9704 mM  . MoreoveIn addition, 0.1 and 1 mM 3-indolacetic acid and 5-hydroxytryptophol and 1 mM serotonin showed similar inhibition percentages at 24 h  than at Finally, the pre-mixing experiment of 0.1 and 1 mM 3-indolacetic acid, the most effective indole, with VEGF prior to exposure to HUVECs showed that only 1 mM 3-indoleacetic acid was effective in reducing VEGFR-2 activation (22% of inhibition) E. HoweveThese data reinforce the putative mechanism proposed above for melatonin and support that the dominant mechanism resulting in the highest inhibition of VEGFR-2 activation activity of VEGF by indolic compounds might be the interaction of indolic compounds with the surface components of the endothelial cell membrane in a way that prevents VEGF from activating the receptor.Melatonin and 3-indolacetic acid, which were two of the most effective inhibitors of VEGF-mediated VEGFR-2 activation, were selected to evaluate whether they are capable of inhibiting the cell migration of HUVECs in the presence of VEGF (25 ng/mL) by wound-healing assay. After stimulation of HUVECs with VEGF (25 ng/mL) for 24 h, the wound was no longer visible and had healed completely . HoweverThis is the first time that melatonin and other related indolic compounds derived from tryptophan metabolism  are proved to inhibit VEGF-induced VEGFR-2 activation and subsequent angiogenesis. Additionally, we propose that the dominant mechanism resulting in the highest inhibition of VEGF-induced VEGFR-2 activation by indolic compounds might be the interaction of indolic compounds with the surface components of the endothelial cell membrane, without the involvement of melatonin MT1 and MT2 receptors. Additionally, we proved that melatonin and 3-indolacetic acid inhibited HUVEC cell migration in the presence of VEGF. Thus, the results of the present study provide new evidence of the anti-angiogenic mechanism of the action of melatonin and other related indolic compounds on HUVEC cells.The ability of melatonin to inhibit the angiogenic effect on endothelial cells has been previously described ,49,57. Á50 value of 970.4 µM), followed by 5-hydroxytryptophol (35% of inhibition at 0.1 mM), melatonin (30% at 1 mM), and serotonin (24% of inhibition at 1 mM). A previous report has shown that polyphenol compounds inhibit VEGF-induced VEGFR-2 activation at lower concentrations, showing higher inhibition potency  [50 value for the strongest indolic inhibitor. Hence, the data presented in this study show the potential for melatonin and its derivatives to inhibit VEGFR-2 activation in vitro, although the effects are likely to be undetectable in vivo after the consumption of melatonin-rich food. However, melatonin is available as a food supplement at 1.95 mg doses [3-Indolacetic acid was the most effective inhibitor of VEGF-induced VEGR-2 activation (IC14.7 µM) . However14.7 µM) ,33,34,69r = 0.3–0.42) [50 = 970.4 µM) as another alternative to target cancer cells.Previous studies have shown the controversial effects of 3-indoleacetic acid on endothelial and cancer cells ,77,78,79.3–0.42) ,74. At t.3–0.42) . On the .3–0.42) , T24 bla.3–0.42) , Pc-3 pr.3–0.42) . The resThe pro/anti-angiogenic effects (migration and proliferation) have been described for serotonin on endothelial cells depending on cell type and concentration (0.1 nM–1 mM) ,81,82,83Existing data support both the stimulatory ,89,90,915-Hydroxytryptophol has been previously related to the antiproliferative effect on cultured tumor cell lines such as sarcoma, choriocarcinoma, and macrophage-like cell lines . HoweverSince endothelial cell migration is essential for the formation of new blood vessels during angiogenesis, we also evaluated the anti-angiogenic effect of melatonin and 3-indolacetic acid, two of the most effective indoles for the inhibition of VEGF-mediated VEGFR-2 activation, on HUVECs migration in the presence of VEGF, following the same experimental conditions that showed the highest inhibitory effect (pre-incubation with melatonin and 3-indolacetic acid for 4 h prior to stimulation with VEGF). Melatonin and 3-indolacetic acid (1 mM) significantly inhibited HUVEC migration by 87% and 99%, respectively, after 24 h in the presence of VEGF . ÁlvarezIn conclusion, the data presented in this study show, for the first time, that indolic compounds derived from tryptophan metabolism, such as melatonin, 3-indolacetic acid, serotonin, and 5-hydroxytryptophol, inhibit VEGF-induced VEGFR-2 activation and subsequent angiogenesis. Our data presented in this report suggest that the mechanism proposed here for the first time, which suggests the interaction between indolic compounds and surface components of the endothelial cell membrane in a way that prevents VEGF from activating the receptor, could be proposed as an additional mechanism of the anti-angiogenic effect of indolic compounds.


Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP -2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1’-b-d-2’-deoxyribofuranosyl)-imidazo-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo.One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them  The reaction was initiated by adding a fixed concentration of enzyme followed by incubating at 37°C and terminated by heating at 95°C for 5 min. Aliquots were collected at various times and applied to polyethyleneimine-cellulose F TLC plates (Merck). Chromatography was performed with isobutyric acid:NH4OH: H2O (66:1:33) (v/v) as the mobile phase for 8–12 h. The products were visualized by autoradiography. The apparent KM and Vmax values were determined according to the Eadie-Hofstee plot. All of the experiments were performed in triplicate using the same batch of purified enzymes. Data represent the mean ± SD (n = 3).The adenosine 5’-triphosphate transfer assay was conducted to measure the enzymatic activities under multiple turnover conditions , 26. Anakcat and KM were derived from the Eadie-Hofstee plots (kcat/KM [The phosphorylation activities of DmdNK and DmdNK-Q81E with different deoxyribonucleosides (dC / dG and dZ / dP) were examined under multiple turnover conditions. The kinetic parameters ee plots , and the, whereas for the dC analog dZ, its phosphorylation was approximately 3,000-fold less efficient than that for dC . This difference was mainly because of a sharp decline in the Kcat value . Compared with dC, the affinity of DmdNK for dZ was also decreased more than 10-fold . Previous research has shown that C-glycosides influence the enzymatic recognition and acceptance of dZ [As shown in ce of dZ , 28.kcat/KM = 5.9 × 106 vs. 3.5 × 103 M-1 min-1 for DmdNK-Q81E and DmdNK, respectively). The substitution of GLn81 with Glu resulted in a greater than 10-fold increase in catalytic rate  and an approximately 150-fold increase in affinity . As for dP phosphorylation, the catalytic efficiency of the mutant was also increased compared with the wild type  because of a slight increase in catalytic rate  and an approximately 4-fold decrease in affinity .To improve the catalytic efficiency of DmdNK towards dZ, the DmdNK mutant DmdNK-Q81E was designed and tested for its phosphorylation activity towards the unnatural nucleosides. As shown in Drosophila melanogaster (DmdNK) is known to efficiently phosphorylate all four natural nucleosides and many nucleoside analogs [The deoxyribonucleoside kinase from  analogs –26. Beca analogs –34. Addi analogs , 35–38.Based on these advantages, we applied DmdNK to the biological phosphorylation of the Z-P UBP in this research. Our results showed that the wild-type DmdNK was able to efficiently phosphorylate the natural dC and dG and unnatural dP nucleosides, whereas the phosphorylation of the dZ nucleoside was less efficient. To overcome this obstacle, we engineered a DmdNK mutant by rational design. The crystal structure of the DmdNK:dC complex has been reported , which revealed that DmdNK has an α/β mixed architecture that consists of eight α-helices and a central five-strand parallel β-sheet , 32. TheKM value . The Gln81, C and G in the experimentally determined crystal structures were manually modified to Glu, Z and P in the corresponding models. The modified structures were evaluated and optimized by Amber10 software. Then, the manually modified models describing the interactions between DmdNK/DmdNK-Q81E and the two unnatural deoxyribonucleosides were subject to energy minimization. The figures were generated by Discovery Studio Visualizer 3.5 . As shown in KM value . Indeed,in vivo.In this work, rational design and a detailed structure analysis were used to construct a mutant of DmdNK (DmdNK-Q81E) that could efficiently phosphorylate the unnatural deoxyribonucleosides dZ and dP. Therefore, this study provides a groundwork for the biological phosphorylation and synthesis of an unnatural base pair S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.