Saccharomyces cerevisiae have provided mechanistic insights on the regulation of TORC1 signaling in response to nutrients. Using a highly specific antibody that recognizes phosphorylation of the bona fide TORC1 target ribosomal protein S6 (Rps6) in yeast, we found that nutrients rapidly induce Rps6 phosphorylation in a TORC1-dependent manner. Moreover, we demonstrate that Ypk3, an AGC kinase which exhibits high homology to human S6 kinase (S6K), is required for the phosphorylation of Rps6 in vivo. Rps6 phosphorylation is completely abolished in cells lacking Ypk3 (ypk3Δ), whereas Sch9, previously reported to be the yeast ortholog of S6K, is dispensable for Rps6 phosphorylation. Phosphorylation-deficient mutations in regulatory motifs of Ypk3 abrogate Rps6 phosphorylation, and complementation of ypk3Δ cells with human S6 kinase restores Rps6 phosphorylation in a rapamycin-sensitive manner. Our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that the use of a phospho-S6 specific antibody offers a valuable tool to identify new nutrient-dependent and rapamycin-sensitive targets in vivo.The target of rapamycin complex 1 (TORC1) is an evolutionarily conserved sensor of nutrient availability. Genetic and pharmacological studies in the yeast  TOR gene, several yeasts such as S. cerevisiae and S. pombe have two TOR genes. In budding yeast, both TOR1 and TOR2 can be part of TORC1, but TORC2 contains only TOR2 [Target of rapamycin (TOR) is a highly conserved serine/threonine kinase that is found in two structurally and functionally distinct complexes termed TOR complex 1 (TORC1) and TORC2 of which TORC1 is uniquely sensitive to rapamycin [nly TOR2 . TORC1 anly TOR2 ,5.In mammals, protein kinases of the AGC (protein kinase A/protein kinase G/protein kinase C) family are direct targets for mammalian TORC1 (mTORC1) and mTORC2. The ribosomal S6 kinase (S6K) is the best characterized target of mTORC1. S6K requires mTORC1-mediated phosphorylation in the hydrophobic motif Thr389) as well as phosphorylation by phosphoinositide-dependent kinase 1 (PDK1) in its activation loop to be fully active . Thus, S9 as wellS. cerevisiae, the AGC kinases Sch9 and Ypk1/2 are direct targets for TORC1 and TORC2, respectively. Sch9 is considered to be the functional ortholog of mammalian S6K [RPS6A and RPS6B, which arose from genome duplication. Two phosphorylation sites (Ser232 and Ser233) exist at the C-terminal region of Rps6a and Rps6b that correspond to Ser235 and Ser236 in the human protein. Mutation of the Ser232/233 residues to alanine abolishes Rps6 phosphorylation status [in vitro [in vivo. Interestingly, two studies performed in fission yeast showed that: a) TORC1 regulates phosphorylation of S6 in response to nutrients [In n status ,11, suggutrients , and b) utrients .S. cerevisiae. We also demonstrate that the AGC kinase Ypk3 is limiting for Rps6 phosphorylation, and propose Ypk3 as an S6K ortholog. Our data emphasize the significance of the phospho-S6 specific antibody as a valuable tool to study TORC1 activity.Using a highly specific commercial antibody that recognizes the mammalian phospho-S6 sites, we have now found that nutrients rapidly induce Rps6 phosphorylation at Ser232/233 in a TORC1-dependent manner in E. coli DH5α cells were used as plasmid DNA host and were grown at 37°C in LB broth supplemented with 50 μg/ml ampicillin when required. Yeast and bacterial cells were transformed using standard methods.Yeast cells were incubated at 30°C in YPD medium  or in synthetic medium containing 20 g/l glucose and the appropriate selection requirements (SC). For nitrogen starvation, nitrogen-free (-N) medium (1.7 g Yeast Nitrogen base without amino acids and without ammonium sulfate and 20 g/l glucose) was used. The composition of YMM medium containing only one source of nitrogen at a final concentration of 0.5 g/l was previously described . E. coliKanMX deletion mutants in the BY4741 or BY4742 background were obtained from EUROSCARF. The rps6S232A S233A strain in the BY4741 background was generated by integrating a linear PCR fragment (obtained by PCR using primers 5’-CCAAGGATAAAGTGGTTAATGCTTATTCGTC-3’ and 5’-GAAAGGAGTTTTTGAACAAAAGAATTTATCTTTTACTGCTTTCTAACTTTAAGCCTTCAA AGCAGCAGCTCTTC-3’) containing the mutant form of RPS6 (rps6S232A S233A) into BY4741 rps6AΔ strain carrying URA3 at the genomic locus of RPS6A (rps6A::URA3). Transformants were selected for loss of URA3 marker by plating on standard 5-fluoroorotic acid (5-FOA) plates. Successful integration of rps6S232A S233A was confirmed by sequencing. The resulting strain (rps6AS232A S233ARPS6B) was then crossed to BY4742 rps6BΔ cells to obtain the BY4742 rps6AS232A S233Arps6BΔ strain by standard tetrad dissection techniques. BY4741 rps6AΔ strain was generated using pUG72-based method described by [The relevant genotypes of the strains described in this work are shown in ribed by . Point mribed by .Rapamycin (LC Laboratories) was dissolved in dimethyl sulfoxide (DMSO). 2-nitro-5-thiocyanatobenzoic acid  was dissolved in water. 5-FOA was purchased from Thermo Scientific (R0812). Antibodies are as follows: phospho-Ser235/Ser236-S6 , RPS6 , S6K , RSK , anti-HA , actin , peroxidase-Goat Anti-Mouse IgG , and peroxidase-Monoclonal Mouse Anti-Rabbit IgG .3, 10 mM p-nitrophenyl phosphate, 10 mM sodium pyrophosphate, and 10 mM β-glycerophosphate), 1 mM phenylmethylsulfonyl fluoride (PMSF), and EDTA-free protease inhibitor cocktail (Roche). One volume of glass beads was added, and cells were broken by vigorous shaking in a Fastprep  at 4°C. Unbroken cells and debris were removed by centrifugation at 500 ×g for 3 min. The protein concentration of the cleared lysate was determined by Bradford. 10 μg of protein were fractionated by SDS-PAGE in 10% polyacrylamide gels and transferred to Protran BA85 nitrocellulose membranes . Membranes were blocked with 5% BSA for 1 h at 24°C, and incubated for 1 h at 24°C or overnight at 4°C with the respective antibodies followed by the secondary antibodies at 1:10000 dilution. Immunocomplexes were visualized using Pierce ECL Western blotting substrate (Thermo Scientific). Chemiluminescence was detected using CL-Xposure films (Thermo Scientific). Preparation of cell extracts for detection of Sch9 phosphorylation was performed as described previously [Whole yeast cell lysates were obtained by resuspending the cells in lysis buffer . For the spot assays, four serial 1:5 dilutions starting at OD600 of 0.5 were spotted onto the indicated plates. Yeast cell size was quantified using a BD FACSCanto II analyzer (BD Biosciences).Yeast cell growth was monitored by measuring ODAll experiments were repeated at least two times with similar results and a representative experiment is shown. Statistical analysis was performed by using two-way analysis of variance (ANOVA). A p-value less than 0.05 was considered statistically significant.S. cerevisiae, Ser232 and Ser233 correspond to Ser235 and Ser236 in human S6  abolished Rps6 phosphorylation  cells but failed to decrease Rps6 phosphorylation in a rapamycin-resistant TOR1–1 TOR2–1 strain  into fresh, complete medium  in a TORC1-dependent manner [S. pombe S6K, mutation of Ser321 (T-loop) or Ser513 to alanine abolished Rps6 phosphorylation under normal growth conditions, while mutation of Thr490 in the TM had no impact on Rps6 phosphorylation (pkh1Δ pkh2ts mutant. Rps6 phosphorylation was completely abolished in cells shifted to non-permissive temperature (37°C) for 1 h  which is located in the catalytic domain, the hydrophobic motif (HM) which is located in a non-catalytic region following the kinase domain, and the turn motif (TM), another phosphorylation site that promotes S6K integrity. mTORC1 phosphorylates S6K on the HM and TM whereas PDK1 phosphorylates S6K on the T-loop ,22. All t manner . To inverylation . Altoget for 1 h . These r3Δ cells . In cont3Δ cells . Interes3Δ cells , indicatrps6bΔ, and phosphorylation-deficient rps6aS232A S233Arps6bΔ cells. In agreement with previous observations [rps6bΔ cells proliferated slower than WT cells, indicating that Rps6b is important for growth. However, the phosphorylation-deficient mutant resembled rps6bΔ cells . This suggests that TORC1 controls Rps6 phosphorylation via Ypk3 but independently of PKA. Further experiments are required to elucidate the crosstalk of TORC1 and PKA towards Ypk3 activity.Ypk3 is phosphorylated by PKA in vitro ,28 and ind Ser90 . This inS. cerevisiae. Mutation of Ser232/233 to alanine in Rps6 had no detectable effect on growth on different carbon sources, sporulation, or sensitivity to heat shock [ypk3Δ mutants as well as ypk3S321A and ypk3S513A cells proliferated like WT cells (data not shown). Knock-in mice in which the five S6K target serine residues in S6 are mutated to alanine (S6P−/− mice) are viable, fertile, and show no detectable differences in body size. Global translation is not impaired, but rather enhanced, in S6P−/− MEFs and liver [S6K1−/−S6K2−/− cells maintain global translation [S6K1−/−S6K2−/− and S6P−/− mice showed more phosphorylation and inhibition of 4E-BP, leading to the release of 4E-BP from eIF4E and thus upregulating protein synthesis [S. cerevisiae, if any, will shed light on the role of Rps6 phosphorylation in cell growth and proliferation.What is the functional relevance of Rps6 phosphorylation? The biological function of Rps6 phosphorylation in yeast is obscure. The first attempts to elucidate the physiological role of Rps6 phosphorylation were carried out in at shock ,11. In and liver ,30, whicnslation . It is tynthesis . Identif


Oleaginous microalgae contain a high level of lipids, which can be extracted and converted to biofuel. The lipid-extracted residue can then be further utilized through anaerobic digestion to produce biogas. However, long-chain fatty acids (LCFAs) have been identified as the main inhibitory factor on microbial activity of anaerobic consortium. In this study, the mechanism of LCFA inhibition on anaerobic digestion of whole and lipid-extracted algal biomass was investigated with a range of calcium concentrations against various inoculum to substrate ratios as a means to alleviate the LCFA inhibition.Nannochloropsissalina represents high lipid content algal biomass while lipid-extracted residue represents its low lipid counterpart. The anaerobic digestion experiments were conducted in a series of serum bottles at 35 °C for 20 days. A kinetic model, considering LCFA inhibition on hydrolysis, acidogenesis as well as methanogenesis steps, was developed from the observed phenomenon of inhibition factors as a function of the LCFA concentration and specific biomass content or calcium concentration. The results showed that inoculum to substrate ratio had a stronger effect on biogas production than calcium, and calcium had no effect on biogas production when inoculum concentration was extremely low. The microbial community analysis by high-throughput Illumina Miseq sequencing indicated that diversity of both bacterial and methanogenic communities decreased with elevation of lipid concentration. Hydrolytic bacteria and aceticlastic methanogens dominated bacterial and archaea communities, respectively, in both high and low LCFA concentration digesters.Whole algal biomass of This study demonstrated that inoculum concentration has a more significant effect on alleviating LCFA inhibition than calcium concentration, while calcium only played a role when inoculum concentration met a threshold level. The model revealed that each functional microbial group was subject to different levels of LCFA inhibition. Although methanogens were the most susceptible microbes to LCFA inhibition, the inhibition factor for hydrolytic bacteria was more highly affected by inoculum concentration. The microbial community analysis indicated that the bacterial community was affected more than the methanogenic community by high LCFAs concentration. Syntrophic acetogens were sensitive to high LCFA concentrations and thus showed a decreased abundance in such an environment. Oleaginous microalgae offer a promising option for sustainable production of renewable transportation fuels while reducing lifecycle greenhouse gas emissions relative to fossil fuels . SeveralDuring the AD process, lipids are initially hydrolyzed to long-chain fatty acids (LCFAs) and glycerol in a fast step by extracellular lipases excreted by hydrolytic bacteria. LCFAs then adsorb to and are transported within microbial cell membranes. Once inside, LCFAs are further degraded to acetic acid and hydrogen through β-oxidation by syntrophic acetogenic bacteria. In lipid-containing substrates, degradation of LCFAs via β-oxidation is the slowest conversion step and controls the overall kinetics of the digestion process , 5. The The inhibitory effect of LCFAs on microbial activity of hydrolytic bacteria, acidogens, acetogens, and methanogens within anaerobic consortium has been well documented –11. MethMicrobial cell membranes, where various essential processes occur, are a primary target of LCFAs. Although the inhibition mechanism of LCFAs on microbial cell membranes is not completely clear, it can be categorized as biochemical and physical in nature. Biochemical inhibition of LCFAs is correlated with its amphipathic structure. Due to detergent properties, LCFAs act as detergent and solubilize the lipid bilayer or membrane proteins, leading to cell lysis , enzyme The physical absorption of LCFAs to the surface of microbial cell membranes can lead to mass transfer limitation , 17, 18.Efforts to reduce the inhibitory effect of LCFAs are needed to maintain an efficient and stable digestion process. Various strategies, including co-digestion , additioThe purpose of this research is to investigate LCFA degradation during AD, focusing in particular on the effect of the calcium:LCFA ratio against the LCFA:biomass ratio. A kinetic model was developed with the consideration of an inhibition factor as a function of LCFA concentration and specific biomass content or calcium ion concentration. Individual inhibition factors for each function group were considered rather than a lumped parameter. Both hydrolytic bacteria and methanogen community structure were characterized by high-throughput sequencing technology Illumina Miseq to evaluate the community structure shift under LCFA inhibition.I/S) had a significant effect on biogas production  digester. The calcium dosed digester showed enhanced biogas production by 10 % as well as accelerated reaction rate at I/S ratio of 1 in both the NS1 and low lipid concentration (NS2) digesters. At I/S ratio of 1, calcium dosing with calcium to LCFAs ratio of 0.5 noticeably increased methane production while further increase of calcium to LCFAs ratio had barely any effect on biogas production. Actually, a single calcium ion could bond with two LCFA molecules, so that the calcium to LCFA ratio of 0.5 would be ideal if calcium and LCFAs were completely mixed. Further increase in calcium concentration could not bond more LCFAs, leading to no effect on free LCFA concentration.Inoculum to substrate ratio . Palatsi et al. [The modeled relationship of specific methane production (SMP) with inoculum:LCFA ratio and calcium:LCFA ratio is illustrated in Fig. i et al.  confirm I/S ratio and proper calcium dosing. It seems that high inoculum concentration could be used as a mean of alleviating the inhibition mediated by LCFAs. Calcium ion could also be an effective way to bond LCFAs and thus keep microbial cells from being tightly wrapped by LCFAs. The impact of calcium ion, however, is dependent on the concentration of inoculum, in which a minimum inoculum concentration is required.In this research, the LCFA concentration in NS1 and NS2 digesters was 9.9 g COD/L and 3.1 g COD/L, respectively, noticeably higher than the approximate inhibitory threshold range (~0.5–1.5 g COD/L) mentioned in literature , 26, 27.I/S ratio and calcium concentration were simulated with the developed kinetic model , Levilinea , Proteiniclasticum , and Stenotrophomonas . Acinetobacter was reported to be the main strain among several pure cultures degrading lipid-containing wastewater with efficient lipase secretion capability [Acinetobacter in the NS1 digester. The bacterial community in the NS2 digester was dominated by Levilinea , Tissierella , Proteiniclasticum , Clostridium , and Parabacteroides . The population analysis demonstrates that a clearly different microbial community structure was formed in the two digesters due to different lipids loading, although hydrolytic/acidogenic bacteria dominated both NS1 and NS2 digesters.Two samples from digesters fed with NS1 and NS2, respectively, at pability , 32. ThiStenotrophomonas is responsible for the hydrolysis and fermentation of carbohydrate and amino acids [Clostridium, Smithella, Tissierella, Syntrophorhabdus, Sedimentibacter and Sporacetigenium, also presented in the two digesters, although the concentrations were low. Interestingly, the abundance of syntrophic acetogens in the NS1 digester (10.8 %) was significantly lower than that in the NS2 digester (28.2 %), suggesting that syntrophic acetogens were more sensitive to high lipid concentration.no acids . SyntropMethanolinea , a strict hydrogenotrophic genus, and Methanosaeta , a strict aceticlastic methanogen genus. However, Methanosaeta (blue) prevailed in both of the communities of the NS1 and NS2 digesters , followed by two hydrogenotrophic genera, Methanobacterium  and Methanomethylovorans  in both digesters, indicating that aceticlastic methanogenesis was the main pathway for methane formation in the two digesters, regardless of the different lipids content. Dominance of Methanosaeta was also found in the anaerobic reactors treating microalgal biomass which was attributed to the low levels of acetate [Methanogenic archaea communities were analyzed in the three samples, with a total of 14,220 reads affiliated to 15 genera and 3 orders. The genus level identification of the archaea communities is illustrated in Fig.  acetate .Fig. 6ArInhibition of LCFAs could be mainly attributed to physical attachment on the surface of microbial cells. As microbes are coated by LCFAs, limitations on transportation hinder substrate access and subsequent biogas release . CalciumIt is foreseeable that multivalent ions, ferric ion for example, could bond more LCFAs according to its charge and give rise to a more sophisticated steric hindrance effect, leading to a stronger effect on alleviating LCFA inhibition while using a reduced amount. The optimal multivalent ion to LCFA ratio would be reciprocal to the value of its charge. However, the same rule as calcium still applies, in that it could not relieve LCFAs inhibition after LCFAs attached to the surface of microbial cells.High inoculum concentration is the key for a healthy process when digesting high concentration of LCFAs. Inoculum concentration had a more pronounced effect on overcoming the inhibition of LCFAs than that of calcium ion, while calcium ion plays a role only when inoculum concentration met a threshold level. Calcium ion could bond with free LCFAs available to the surface of microbial cells and reduce half of the original LCFAs concentration. Kinetic modeling revealed a remarkable difference among the inhibition factors for each function group of microorganisms. Although methanogens were the most susceptible microbes to LCFA inhibition, the inhibition factor for hydrolytic bacteria was more highly affected by inoculum concentration. The bacterial community was affected more than the methanogenic community by high concentration of LCFAs. Diversity of both bacterial and methanogenic communities decreased with elevation of lipid concentration in the digester. Hydrolytic bacteria and aceticlastic methanogens dominated bacterial and archaea communities, respectively, in both high and low LCFA concentration digesters. Syntrophic acetogens were sensitive to high LCFA concentrations and thus showed a decreased abundance in such environment.Nannochloropsissalina (Solix BioSystems) was selected as it was the algal biomass with greatest availability and had lipid content emblematic of industrial strains. Whole algal biomass of Nannochloropsissalina (NS1) represents high lipid content algal biomass while lipid-extracted residue (NS2) represents its low lipid content counterpart. Freeze-dried solid biomass of both NS1 and NS2 was provided by Solix BioSystems, Inc. . Lipid in NS1 was extracted with a 3:2 mixture of hexane/isopropanol at 70 °C and 1500 psi [esidue NS represensalina NS represenI/S) on methane production from NS1 and NS2. The experimental design considered treatments with calcium  at concentrations of 0.5, 1 and 2 times that of the algal lipid concentration (mole/mole) against I/S ratios of 0.1, 0.4 and 1.0 (gVS/gVS). All BMP assays were conducted in serum bottles with working volume of 150 mL and headspace of 100 mL. No additional external nutrients/trace elements were added to the BMP bottles as it was assumed that basic nutrient requirements for anaerobic microorganisms were provided by the wastewater-based inoculum [2·2H2O at designed calcium concentration, before inoculum was added according to the respective I/S ratio. Before experiments were initiated, each bottle was flushed with N2 gas for 15 min to induce anaerobic conditions, and then incubated in a 16-cell automated Challenger AER System  maintained at 35 ± 1 °C and mixed continuously with a magnetic stirrer set to 200 rpm. Daily methane production was monitored via scrubbing of carbon dioxide with sodium hydroxide pellets containing color indicator.A series of biochemical methane potential (BMP) assays were set up to investigate the effect of calcium addition and inoculum to substrate ratio  equipped with a thermal conductivity detector [The analysis for TS, VS and COD was done according to the standard methods . The voldetector .2SO4 acid hydrolysis followed by HPLC measurement of monosaccharides [Lipids were analyzed as fatty acid methyl esters after a one-step acid catalyzed in situ trans-esterification reaction using a GC-FID (Agilent 6890N) equipped with an HP-5 ms capillary column (30 m × 0.25 mm id × 0.25 µm) according to the procedure of Laurens et al. . Proteincharides .I/S ratio of 1 with no calcium addition were collected. Genomic DNA was extracted and purified using the PowerSoil DNA isolation kit  according to the manufacturer’s instructions.At the end of the digestion experiment, samples of initial saved inoculum and mixed liquor from the two digesters fed with NS1 and NS2 at The V4–V5 hypervariable region of the 16S rRNA gene was amplified with region-specific primers designed to include Illumina adaptor and barcode sequences  , 42. GenThe concentrations of amplicons were determined using a picogreen assay and a Fluorometer (SpectraMax GeminiXPS 96-well plate reader) and then pooled in equal amounts (~100 ng) into a single tube. The amplicon pool was then cleaned to remove short undesirable fragments using the following procedure. First, the pool was size selected using AMPure beads (Beckman Coulter), the product was then run on a 1 % gel, gel cut and column purified (Qiagen MinElute PCR purification kit), and size selected again with AMPure beads. To determine the final quality, we PCR amplified the resulting amplicon pool with Illumina adaptor-specific primers and ran the PCR product on a DNA 1000 chip for the Agilent 2100 Bioanalyzer. The final amplicon pool was deemed acceptable only if no short fragments were identified after PCR. Otherwise, the procedure was repeated again. The cleaned amplicon pool is then quantified using the KAPA 454 library quantification kit (KAPA Biosciences) and the Applied Biosystems StepOne plus real-time PCR system. Finally, sequences were obtained using an Illumina MiSeq paired-end 300 bp protocol  .https://github.com/msettles/dbcAmplicons) by both expected barcode and primer sequences. Barcodes were allowed to have at most 1 mismatch (hamming distance) and primers were allowed to have at most 4 mismatches (Levenshtein distance) as long as the final 4 bases of the primer matched the target sequence perfectly. Reads were then trimmed of their primer sequence and merged into a single amplicon sequence using the application flash [http://www.ncbi.nlm.nih.gov/sra) under project SRP052619.Raw DNA sequence reads from the Illumina MiSeq were demultiplexed and identified with the custom python application dbcAmplicons  was used to measure the relative change in methane production for a ± 100% change in kinetic parameters and stoichiometric parameters by Eq.  was uThe parameters, is study , 52–54,


Precooling methods such as ice vests and cold water immersion are employed before competition, to lower Tc and increase heat storage before a critical Tc is reached. However, there are numerous practical limitations meaning pre-existing methods are impractical for use in a field setting. Ice slurry ingestion has been investigated as a more practical precooling method and has enhanced endurance performance [Major competitions are often staged in hot environments. However, the body's ability to dissipate heat is compromised during heat stress resulting in a concomitant rise in core temperature  either ingested 7.5 g.kg−1 ice slurry (IS) or acted as a control (C). After ingestion a 45 minute intermittent shuttle test  was performed, followed by an alternating run to exhaustion  in 33°C 59% rh. Tc, skin temperature (Tsk), heart rate (HR), rating of perceived exertion (RPE), thermal sensation and blood lactate (BLa) were measured during intermittent activity and Tc and HR were recorded throughout the run to exhaustion. Gross sweat loss, heat storage and body temperature (Tb) was calculated for the entire protocol. A student paired t-test was performed upon run time and sweat rate. A 2 × 2 repeated measures ANOVA was conducted upon Tc, Tsk, Tb, HR, RPE, thermal sensation and BLa. Significance was set at P ≤ 0.05.In a counterbalanced order, ten female footballers . Tc remained significantly lower on IS compared with C during the first 30 minutes of intermittent activity (P = 0.05). Heat storage was significantly higher (P = 0.003), Tsk, Tb and RPE were significantly lower (P ≤ 0.05), and there was a trend for a reduction in thermal sensation throughout intermittent activity in the IS trial (P = 0.064). There were no significant differences between conditions in BLa (P = 0.743), HR (P = 0.113) and sweat rate (P = 0.278). At the end of the run to exhaustion Tc was 0.3 °C higher on the IS trial (P = 0.004).Time to exhaustion was significantly longer after ice slurry ingestion when compared to control (P = 0.0005). Prior to exercise ice slurry reduced Tc and promoting greater heat storage without any changes in blood lactate accumulation. Previous research indicates this could be due to numerous mechanisms including the reduction of inhibitory afferent feedback to the brain and stimulation of sensory receptors leading to improved perceptual responses.Ice slurry ingestion improved run time by 2 minutes 20 seconds (93 seconds), through reducing TIce slurry ingestion appears to be an effective precooling strategy for use in sports characterised by intermittent activity. Ice slurries are a practical alternative to existing methods and could be incorporated into pre-match hydration strategies. Future research should focus on determining the optimal volume and timing of ice slurry ingestion.


PIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs.Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway.These results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members. Auxin plays important roles in plant growth and development. Directional cell-to-cell transport and the formation of concentration gradients of auxin are pivotal for its biological function. Diffusive cellular auxin efflux is not effective because of ionization of intracellular auxin, so asymmetrically localized auxin efflux carriers in the plasma membrane (PM) play a critical role in polar auxin transport .Arabidopsis PIN family consists of eight members, among which six PINs  have a long central hydrophilic loop  connecting the five transmembrane (TM) helices on each end. These ‘long’ PINs mainly localize to the PM. Conversely, the other two PINs (PIN5 and 8) have a very short (27–46 residues) HL and localize to the internal compartment  or to both the internal compartment and the PM (PIN8) [PIN-FORMED (PIN) auxin efflux carrier proteins show a distinctive asymmetrical subcellular distribution, which is dynamically regulated by environmental and developmental cues, and their mutations cause defects in auxin gradient formation and plant growth and development . The Ara PINs PIN and 8 haLong PINs show diverse polar localization in the PM of diverse cell types -11. Althin vitro and in a protoplast phosphorylation assay, and that the phosphorylation sites of the PIN-HL are required for PINs’ polarity and biological functions [Protein phosphorylation and dephosphorylation have been implicated in auxin transport and PIN trafficking . There iunctions -24.Recently, we reported a phosphorylatable motif, named M3, in the PIN3-HL that includes five S/T (four S and one T) residues and is necessary for the polarity and intracellular trafficking of PIN3 . Root hahttp://www.cbs.dtu.dk/services/NetPhos/, [To examine the functional conservation of the M3 motif among long PINs, we chose PIN7 , as areEXPANSIN A7 promoter  in A in AEXPA  causes internal accumulation of PIN proteins into so-called ‘BFA compartments’, which are indicative of PM-localization of PINs ,32. To cUnlike phosphorylation-defective PIN1 and PIN7, the mutant PIN2 proteins showed completely different trafficking behavior. While wild-type PIN2 was clearly localized to the PM, phosphorylation-defective 3 m1- or M3-PIN2 proteins were barely detectable in root hair cells; this was consistent in all five independent transgenic lines observed for each mutant construct  did not have much effect on the PM-localization of wild-type PIN2 or restored 3 m1- or M3-PIN2 proteins in the root hair cell  show distinctive structures  PIN2. This proteasome inhibitor was found to inhibit the internalization or vacuolar targeting of PIN2 from the PM ,35, and Overall, our results suggest that the M3 motif in PIN2 may have specialized to modulate protein degradation by lytic vacuolar trafficking. The aforementioned fact that PIN2 protein degradation is frequently observed under various conditions may reflect that PIN2 is more susceptible to the protein trafficking machinery for degradation. The different behavior of M3- and 3 m1) PIN2 from that of other M3-PINs leads us to hypothesize that the specialized function of the PIN2 M3 motif could be due to its specific structure. The M3 motif of PIN2 not only has additional amino acid residues between S212 and S215, but also includes additional serine residues in the M3 motif other than the five S/T residues conserved in the long PIN M3 motif  or partial (for PIN1 and PIN7) failure of PM targeting and facilitation of vacuolar lytic trafficking (for PIN2). The dynamicity of subcellular PIN polarity is important to modulate local auxin gradient formation and ultimately diverse developmental processes, which are subject to perpetual changes in environmental and developmental stimuli. Phosphorylation of the PIN-HL domain provides PINs the ability to change their subcellular trafficking behavior. Furthermore, multiple phosphorylation sites in the PIN-HL domain and their divergence in specific structure may generate diverse phosphorylation codes to drive dynamic PIN trafficking depending on internal and external stimuli.Arabidopsis thaliana) Columbia ecotype (Col-0) was used as the wild-type plant in this study. All seeds were grown on agarose plates containing half-strength Murashige and Skoog (MS) nutrient mix (Sigma-Aldrich), 1% sucrose, 0.5 g/L MES (pH 5.7), and 0.8% agarose. All seeds were cold treated (4°C) for 3 days and germinated at 22°C under a 16-h-light/8-h-dark photoperiod. Arabidopsis transformation was accomplished by Agrobacterium tumefaciens (strain C58C1)-mediated infiltration [Arabidopsis  was  was Arab  and FM4-64 (red) fluorescence were observed using a LSM700 confocal laser scanning microscope (Carl Zeiss). Green and red fluorescence were detected by 488/490–555 nm and 555/640 excitation/emission filter sets, respectively. Localization of PIN:GFP fusion proteins was observed in 4-day-old seedlings. For observation of the subcellular localization of PIN:GFP after BFA, MG132, auxin, or wortmannin treatment, seedlings were incubated in half-strength liquid MS medium for the indicated time periods. The control (mock) liquid medium included the same amount of the solvent dimethylsulfoxide (0.04%). FM4-64 (2 μM), BFA (25 μM), CHX (50 μM), MG132 (25 μM), NAA (5 μM), or wortmannin (20 μM) was applied to the seedlings before observation as described in the results. The relative intensity of PIN2:GFP signals with or without wortmannin was estimated from the root hair cells of 7–9 seedlings using the Adobe Photoshop histogram menu as described previously ,48.The Arabidopsis Information Resource (TAIR) accession number for the genes mentioned in this article are At1G12560 (EXPANSIN A7), At1G73590 (PIN1), At5G57090 (PIN2), At1G70940 (PIN3), At2G01420 (PIN4), At5G16530 (PIN5), At1G77110 (PIN6), At1G23080 (PIN7), At5G15100 (PIN8).BFA: Brefeldin A; CHX: Cycloheximide; HL: Hydrophilic loop; NAA: 1-naphthaleneacetic acid; PIN: PIN-FORMED; PM: Plasma membrane; TM: Transmembrane.The authors declare that they have no competing interests.DS performed most experiments. DS and HTC designed experiments, analyzed the data, and wrote the manuscript. AG and MP participated in the confocal work. All authors read and approved the final manuscript.Figure S2. Root hair length of independent transgenic lines. Figure S3. Confocal images of M3 phosphorylation-defective PIN1 lines. Figure S4. Confocal images of M3 phosphorylation-defective PIN7 lines. Figure S5. Confocal images of root hair cells from BFA-treated M3 phosphorylation-defective PIN7 lines. Figure S6. Confocal images and expression analysis of M3 phosphorylation-defective PIN2 lines. Figure S7. Confocal images of auxin-treated M3 phosphorylation-defective PIN2 lines. Figure S8. Confocal images of MG132-treated M3 phosphorylation-defective PIN2 lines. Figure S9. Confocal images of wortmannin-treated M3 phosphorylation-defective PIN2 lines. Table S1. Primer list.Conservation of the M3 motif in long PINs. Click here for file


Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose . The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS) was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia.Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds  Type 2 diabetes is a chronic metabolic disease characterized by defects in insulin secretion and action, and has developed into a widespread global health threat. Currently, 20.9 million people in the United States are diagnosed with diabetes; nearly 8% of the adult population . PostpraControl of serum glucose by modulating the activity of α-amylase and α-glucosidase is one strategy in the management of diabetes ,7. SeverIn vivo assays using diabetic mice have demonstrated the efficacy of seaweed in improving fasting serum glucose levels −2. Mass spectrometry identification was achieved through the accurate mass parent ion and daughter ions (The larger phlorotannins (DP > 11) manifested distinguishable peaks in the negative mode mass spectra as [M − H]ter ions . The master ions , allowinProton NMR yielded two groupings of signals. One represented the meta-distributed aromatic proton signals directly bound to the aryl rings (5.8–6.1 ppm). The second, broader due to proton exchanges, was seen in the hydroxyl proton range (9 ppm). The shift and multiplicity of these proton groupings was similar to other studies of phlorotannins , providiUnless otherwise noted, all chemicals were of reagent or spectroscopic grade and obtained from Sigma-Aldrich .et al. [m/z 150–2500. Shimadzu’s LCMS Solution software was used for system control and data analysis. NMR spectra were recorded on a Bruker Avance 700 MHz spectrometer .LC–MS analysis was performed using a Shimadzu LC–MS-IT-TOF instrument  equipped with a Prominence HPLC system . The LC separation was performed using a method adapted from Grace et al.  with a DFucus distichus,  Saccharina latissima, Saccharina groenlandica, and Alaria marginata); one species of Rhodophyta  ; and a species of Chlorophyta  (Ulva lactuca)  species (lactuca) . Freshly40 g of each freeze-dried sample were powdered using a grinding mill  and the powder was suspended in 1 L 80% aqueous methanol and shaken overnight at room temperature, then filtered through Whatman #1 filter paper and the powder re-extracted a second time. The extract was evaporated under reduced pressure to remove excess solvent, and the resulting aqueous residue was diluted to 400 mL with deionized water and sequentially partitioned with hexane, ethyl acetate, and 1-butanol (3 × 400 mL), yielding 4 crude fractions including the aqueous residue . Solvents were removed via rotary evaporation, and all fractions were lyophilized and held at −80 °C until analysis and subfractionation.254; Sigma-Aldrich, St. Louis, MO, USA) stained with vanillin-HCl.Active fractions were separated by flash chromatography using either silica gel  eluted with hexane: ethyl acetate:methanol (100:0:0 → 0:50:50) to collect 20 subfractions, or Sephadex LH-20 columns  eluted with methanol: chloroform 4:1 followed by 70% aqueous acetone to obtain 24 subfractions. The progress of separation was monitored by TLC  was diluted to a working concentration of 0.005 mg/mL in 100 mM phosphate buffer, pH 7 (PB). To each well of a 96-well plate, 20 µL of inhibitor, extract, or solvent (control) was mixed with 100 µL of the substrate p-nitrophenyl-α-d-glucopyranoside  and incubated at 30 °C for 5 min. 100 µL of the enzyme working solution was charged to each well, and absorbance was measured at 405 nm for 30 minutes using a Molecular Devices M3 microplate reader at 30 °C . Blank wells (without enzyme) were subtracted from each well and results were compared versus a control (no inhibition). The commercial pharmaceutical acarbose was utilized as a positive control. Half-inhibitory concentration (IC50), where 50% of the enzyme activity was inhibited, was determined using serial dilutions of active extracts or fractions (n ≥ 6), and the percentage inhibition was plotted against the log10 sample concentration.A rapid, multi-well plate system was used to assay seaweed extracts and fractions for inhibitory activity against α-glucosidase. α-glucosidase from con is the absorbance of the uninhibited enzyme, Aext is the absorbance of the enzyme treated with the extract, and Ablank is the absorbance of the extract with substrate (no enzyme present).For each sample, the inhibitory activity was calculated as follows:t = 6 min. The inhibition constant Ki of the competitive inhibitor was calculated using the following equation:The enzyme-inhibitor reaction was performed using the above method at both 0.25 and 0.125 μg/mL of the inhibitor and at substrate concentrations from 0.25 to 2 mM PNPG. The type of inhibition was determined by Lineweaver-Burk plot, using the double reciprocal of the substrate concentration and velocity of inhibition at 2O) was added, and inhibition was determined by reading absorbance at 595 nm on a microplate reader. Blank wells without amylase were subtracted from each well to account for innate color of the sample. As with the α-glucosidase assay above, acarbose was utilized as the positive control, and IC50 values were generated from serially diluted sample solutions.To determine the inhibition of α-amylase, the α-amylase from porcine pancreas  was dissolved in 20 mM Tris-HCl buffer pH 7.5  to a working concentration of 25 μM. Each well of a 96-well plate was charged with 35 μL of the extract, fraction, or inhibitor, with 5 μL of the starch substrate , and incubated at 37 °C for 5 min. Ten microliters of the working enzyme solution was charged to each well, and the resulting solution was incubated for 10 min at 37 °C. 150 μL of diluted Lugol’s solution .For each sample, the inhibitory activity was calculated as follows:p < 0.05 or p < 0.01 level. Half maximal inhibitory concentration (IC50) data was calculated after logarithmic transformation and expressed as the geometric mean with 95% confidence intervals. Lineweaver-Burk plots and kinetic data were obtained using biochemical algorithms in Prism.All assays were performed at least in triplicate. Results are presented as mean of triplicate runs ± SEM. Statistical analysis was conducted using repeated measures analysis of variance (ANOVA) followed by Tukey’s test , with statistical significance determined at the et al. [The main approach of anti-diabetic treatments is the maintenance of circulating glucose concentrations to a near constant level by delaying absorption of glucose via inhibition of the two primary enzymes responsible for carbohydrate digestion, α-glucosidase and α-amylase. Due to undesirable side effects of synthetic inhibitors , there iet al.  correlatA. marginata and F. distichus, demonstrated sufficiently high activity against α-glucosidase and α-amylase to warrant further analysis using bioassay-guided fractionation techniques. From each of these two species, one fraction (AM-E-17 and FD-E-22) displayed efficacy as potential anti-hyperglycemic agents, and IC50 values for both demonstrated strong potential for the inhibition of α-glucosidase and α-amylase. The IC50 values for inhibition of α-glucosidase by FD-E-22 (0.89 ± 0.08 µg/mL) were significantly lower than reported values for isolated phlorotannin derivatives; one published study reported IC50 values for fucophloroethol and eckol oligomers ranging from 8.0 to 24.5 µg/mL [50 values from 1.2 to 17.8 µg/mL [In this study, the organic fractions from six different seaweed species harvested from the southeastern coast of Alaska were evaluated for their potential to inhibit carbolytic enzyme activity. All species exhibited stronger inhibitory activity against α-glucosidase than α-amylase, which may suggest a more selective inhibition profile by seaweed extracts, with the potential to regulate the digestion and metabolism of complex carbohydrates without incurring the side-effects of non-specific enzyme inhibitors. Milder α-amylase inhibition has been shown to prevent abnormal gut microfloral fermentation of undigested carbohydrates when they enter the colon . The fou.5 µg/mL , while a.8 µg/mL . FD-E-22Fucus, especially in the widely studied Fucus vesiculosus and F. spiralis [Alaria marginata that demonstrated significant levels of enzyme inhibitory activity, AM-E-17, remained inconclusive. Originally hypothesized to also contain phlorotannins, none were observed after NPLC-MS analysis. This could be due to the initial use of silica gel for sub-fractionation of A. marginata, potentially leading to the oxidative breakdown of labile phlorotannins [Laminaria and Alaria, it is possible that the concentration, degree of polymerization or configuration of phlorotannins were not sufficient to inhibit the assayed enzymes. Variations in structure of the polysaccharide fucoidan, isolated from both Ascophyllum and Fucus, displayed marked differences in the resulting enzyme inhibition of α-glucosidase and α-amylase, suggesting the potential for wide variation in these polymeric phytochemicals between genera [The dominant phytochemicals in the active extract FD-E-22 were phlorotannins, polyphenolic oligomers of phloroglucinol. These polyphenolic polymers have been revealed in other members of the genus spiralis ,32. Phytotannins ,39, whern genera .An NPLC-MS methodology allowed for the separation and subsequent identification of phlorotannins up to 18 monomer units in size. Previous studies had reported the elution of phlorotannins greater than 1200 Da in size (a degree of polymerization 10 and higher), as a single, broad peak, with many isomers co-eluting simultaneously. This conglomeration of phlorotannins impeded accurate detection and characterization , and it Fucus distichus were even more effective in their inhibition efficacy than the known pharmaceutical inhibitor acarbose, suggesting their potential to delay the absorption of digested carbohydrates in the gastrointestinal tract post-consumption. Thus, these species have potential as natural sources of anti-diabetic agents that could reduce post-prandial hyperglycemia.In summary, traditionally consumed Alaskan seaweed, particularly brown kelps, evidenced a pronounced inhibitory effect on carbolytic enzyme activity. The phlorotannins found in


Iron > Cobalt > Copper > Cadmium > Lead. Cadmium, a potential carcinogen was found in concentrations higher than permissible limits in many vegetables from all sites. Highest level of cadmium (1.20 mg/kg) and copper (81.33 mg/kg) was reported in site which was in vicinity of waste water drain but irrigated with ground water. Concentration of copper and lead in vegetable samples from different sites exhibited no statistically significant difference with respect to different sites.Irrigation of agricultural land with wastewater leads to continuous buildup of metals at these sites which gets accumulated in the vegetables and crops growing on these sites. Not just the crops irrigated with wastewater are hazardous, in present study, we have found that vegetables growing in vicinity of wastewater drain are also not safe for human consumption. The risk associated with consumption of vegetables was assessed by calculating hazard quotient and results revealed that the hazard quotient for leafy and tuberous vegetables was higher than the safe limits in all the sites irrespective of mode of irrigation. Spinach was the most hazardous among all as the hazard quotient with respect to cobalt and copper was highest in spinach. Uptake trend of metals in all vegetables:  Injudicious use of resources and haphazard urbanization have over-exploited the natural resources and caused detrimental effects on the environment. Unplanned economic development has led to pressure on cultivable land and suitable water for irrigation. To meet the food demands of exponentially growing human population, cultivation of food crops is carried out at places which are not suitable for agriculture like along wastewater drains or other polluted sites. To address water crisis, irrigation using the large amount of wastewater discharged from the rapid growing industries is being carried out in many parts of the world. Wastewater used for irrigation has many contaminants mainly heavy metals depending upon the source of discharge . The discharge of municipal waste and effluents from various industries  are released into the drain. This site was nearest to the industrial discharges. Agricultural fields at this site are irrigated with ground water.Site 2 Also, located across Tung-Dhab drain and downstream of Village Mahal. The site was 15 km downstream of Site 1. Main source of irrigation at this site was also ground water.Site 3 Site on the southern side of city. Municipal wastewater drain flows across this area and the agricultural fields at this site are irrigated with municipal wastewater.Three sites under vegetable cultivation were selected on the basis of exposure to wastewater. From each site the samples were collected from different fields under vegetable cultivation. The sites are represented in Fig. Edible parts of vegetables growing across each site were coded and collected in triplicate. They were brought to laboratory and wrapped in absorbent paper after sufficient washing with distilled water and initial air-drying. Samples were then oven-dried at 70 °C to remove all moisture content. Dried samples were crushed using pestle and mortar.3:H2SO4:HClO4 = 5:1:1) till we obtained transparent fumes. Samples were cooled and filtered using Whatman filter paper no. 1. The final volume was made 50 ml using double distilled water. Heavy metal content of samples was analyzed using Atomic Absorption Spectrophotometer (Model: 240FSAA Make: Agilent Technologies).0.5 g of dried vegetable sample was digested using Tri-Acid mixture (HNOStandard solution (1000 mg/l) of different metals viz. copper, cobalt, iron, lead and cadmium were procured from Agilent technologies. Standard curve was prepared using various concentrations made from standard solution. Digested samples were then analyzed for the metal content.Content of heavy metals in vegetable samples was estimated. Apart from content, following parameters were assessed to estimate risk associated with uptake of metals:n = Concentration of metal n in sample.Assessment of overall load of metals in each vegetable growing at each site was made by computing metal pollution index (MPI) , RfD = reference oral dose of metal (mg/kg of body weight/day) and BO = Body weight (kg). Daily intake of vegetables was taken as 0.100 kg for adults, as this is the minimum vegetable requirement for a balanced diet, it is prescribed that for a balanced diet, one must have a minimum serving of 100 g for 3 times  entclass1pt{minimaStatistical significance of variation in content of heavy metals in vegetables from different sites was tested using two way ANOVA followed by post hoc Tukey HSD test for metal concentration in vegetables from different sites. All the statistical calculations were done using SPSS 17.0.Iron > Cobalt > Copper > Cadmium > Lead. The concentration of all metals was found to be higher in the leafy and tuberous vegetables as compared to fruit vegetables with an exception of iron in vegetable samples from site 1. This trend was found to be in accordance with previous studies estimating heavy metals in vegetables  is significant with respect to site and type of vegetables Table . VariatiMetal pollution in samples is reliably estimated using metal pollution index (MPI). Figure Health risk associated with any pollutant is dependent upon the level of exposure and amount of absorption by human body. Thus, hazard quotient is a valid tool to assess the level of risk associated with particular pollutant. If level of Hazard quotient is less than 1, the risk associated with exposure of metal is negligible. However if level of hazard quotient is higher than 1, the metal may pose serious health hazards. The estimation of hazard quotient of metals in different vegetables from various sites demonstrated alarming results. Though the concentration of some metals was significantly higher in vegetable samples from wastewater irrigated sites but the health risk associated with these metals was also found to be very higher. Cadmium identified as potential carcinogen by US EPA , was fouHazard quotient of cobalt was found more than one for children in almost all vegetables except for fruits and bulb vegetables. Cobalt is component of Vitamin B12 and is important for various physiological functions. But toxicity of higher concentration is widely reported (Simonsen et al. Apart from various other toxicity affects, US EPA  identifiFigure The healthy and balanced diet is considered to be one which is having many servings of vegetables in a day but due to injudicious agricultural practices this can cause a serious threat to human population. The sites irrigated with wastewater due to lack of water resources are the most risky ones but the sites which are in close vicinity with wastewater drains are also of significant consideration due to percolation of water. No significance difference was observed in copper and lead uptake by vegetables growing in sites irrigated with wastewater and those irrigated with ground water but are in close vicinity with wastewater drain. The regular consumption of vegetables grown in sites can cause detrimental effects to the human population. The authors strongly recommend that consumption of tuberous and leafy vegetables from these sites should be restricted and continuous monitoring of vegetables from these sites should be done. Efforts should be made to amend the soil to reduce the uptake of metals in vegetable crops or these sites should be used for cultivation of non-food crops.


Sde3396, the first ICE identified in the β-hemolytic Streptococcus dysgalactiae subsp equisimilis (SDSE) and demonstrated its transfer to single isolates of Streptococcus pyogenes  and Streptococcus agalactiae . While molecular studies found the ICE in multiple SDSE and GBS isolates, it was absent in all GAS isolates examined.Integrative conjugative elements (ICEs) are mobile genetic elements (MGEs) that possess all genes necessary for excision, transfer and integration into recipient genome. They also carry accessory genes that impart new phenotypic features to recipient strains. ICEs therefore play an important role in genomic plasticity and population structure. We previously characterised ICESde3396:km is transferable from SDSE to multiple SDSE, GAS and GBS isolates. However not all strains of these species were successful recipients under the same growth conditions. To address the role that host factors may have in conjugation we also undertook conjugation experiments in the presence of A549 epithelial cells and DMEM. While Horizontal Gene Transfer (HGT) occurred, conjugation efficiencies were no greater than when similar experiments were conducted in DMEM. Additionally transfer to GAS NS235 was successful in the presence of DMEM but not in Todd Hewitt Broth suggesting that nutritional factors may also influence HGT. The GAS and GBS transconjugants produced in this study are also able to act as donors of the ICE.Here we demonstrate that ICEWe conclude that ICEs are major sources of interspecies HGT between β-hemolytic streptococci, and by introducing accessory genes imparting novel phenotypic characteristics, have the potential to alter the population structure of these species. Streptococcus pyogenes , S. agalactiae  and S. dysgalactiae subsp. equisimilis (SDSE) are related Gram-positive β-hemolytic bacteria that can cause a number of potentially fatal diseases in humans [n humans –4. Two oGenetic and genomic studies have revealed an extensive history of intra-species horizontal gene transfer (HGT). Although genetic evidence for interspecies HGT is also present, the mechanisms of this HGT have yet to be full elucidated. Indeed, comparative genomics of GAS and GBS, the two major pathogens, have revealed the importance of HGT and Mobile Genetic Elements (MGEs) to the genomic plasticity of these two species –11. ThesSde3396 encodes 66 open reading frames, and possesses the genes encoding machinery necessary for excision, transfer and integration into the recipient chromosome [Sde3396:km is transferable from both SDSE and GBS to GAS, and that the ICE is stably integrated into the chromosome of all three species. The transconjugant GAS and GBS are also able to act as donor of the ICE. Our data support a model where ICEs are the major MGE involved in inter-species dissemination of new genes throughout the β-hemolytic streptococcal population.We have previously reported the identification and characterisation of the first ICE in SDSE. ICEromosome . The ICESde3396:km as the donor strain . The assay also utilised SDSE NS3396 ICESde3396 in the GAS isolates, and suggests that genetic or environmental factors may inhibit the transfer or maintenance of ICESde3396 in GAS. It has been reported that growth of GAS in the presence of pharyngeal cells results in bacteriophage induction [The inability to transfer the ICE to GAS in this model is consistent with our epidemiological data  demonstrnduction , theoretSde3396:km, GAS NS235str::ICESde3396:km and GBS RBH05str::ICESde3396:km, transconjugants produced in earlier experiments were used as donors in a new round of conjugations. As these donor strains were also streptomycin-resistant, the inherent bacitracin resistance present in SDSE and GBS was used as the selectable marker for these recipients. To monitor transfer of the ICE into GAS, a spectinomycin resistant GAS NS235 was generated. For both donors, we were able to transfer the ICE to recipients of all three species, as determined on the basis of a double antibiotic resistance phenotype. Figure To determine whether GAS and GBS transconjugants were also capable of acting as donors of ICESde3396 differed between GAS, GBS and SDSE, Southern blots were performed on NdeI digested DNA recovered from transconjugants of each species. The R3P probe used for these assays distinguishes between episomal and integrated forms of the ICE. In its episomal form, R3P reacts with a 8.0 kb fragment flanked by NdeI sequences present in the ICE, and which span the att site. When integrated into the chromosome, one NdeI site of the reactive band is present in the ICE, and other is present in the chromosome. The size of the reactive band when the ICE was integrated into the chromosome is therefore dependent on the location of the chromosomal NdeI site adjoining the attachment site (attB). For all GAS and SDSE transconjugants examined, the reactive band observed was greater than 8.0 kb, indicating that the ICE was chromosomally integrated , we cannot exclude the possibility that other MGEs are present in the rplL integration site, a known hotspot for ICE integration [Sde3396 in the natural GAS population [While our success in transferring the ICE to multiple GAS isolates appears contradictory to our previous epidemiological results, our inability to transfer the ICE to all recipients suggest that strain-specific factors may play a role in acquisition of the ICE. While none of the GAS recipient strains used in this study possessed ICEegration . Alternaegration , or propegration  have alspulation .Although we successfully transferred the ICE in the presence of A459 cells, conjugation frequency was no greater than that observed when fresh or spent DMEM, suggesting that the eukaryotic cells play no part in promoting ICE mediated HGT. Interestingly in the case of GAS NS235, we were able to demonstrate transfer from SDSE GGS 3396:km in the presence of DMEM, but not THB, providing some evidence that environmental factors influence ICE transfer, at least for this strain. Of note when we replaced the Todd Hewitt broth with DMEM in the microfuge model, transfer of the ICE was observed (data not shown). From these experiments, it is not clear whether the addition of DMEM results in increased induction of the ICE, or increased permissiveness of the recipient.Su32457 [Sde3396 and ICESu32457 are almost identical. The major difference between the two ICEs is the 15 kb region and Region 2 which both carry accessory genes. Unlike ICESu32457 all transconjugants examined in this study retained arsenate resistance (data not shown), indicating that firstly, arsenate resistance is not lost through gene mutation or deletion, at least within the scale of this study. Additionally, our results indicate that unlike ICESu32457 and the 15 kb mobile genetic element, Region 2 is stably integrated into the chromosome.Palmieri recently reported a 15 kb mobile genetic element harbouring antibiotic resistance genes that could exist either independently in episomal form, or integrated into a larger ICE, ICESu32457 . The 15 Sde3396 is transmissible from multiple β-hemolytic species, and has the necessary attributes to be able to act as an efficient vehicle for dissemination of genes through the β-hemolytic streptococcal population. The presence of similar ICEs in S. suis[S. suis harbour antibiotic resistance genes. Acquisition of antibiotic resistance cassettes by ICESde3396, or alternatively, transfer of the ICEs from S. suis to β-hemolytic streptococci represent a potential mechanism by which antibiotic resistance may become fixed in specific strains in these populations.In summary we have shown that ICE S. suis, suggestSde3396:km is a recombinant strain [Sde3396 marked with a kanamycin resistance gene in Region 2  and rplLr (5′-gtgaaatcacaggcgaaggt-3′) under standard conditions. Southern hybridisations were performed using standard protocols. Briefly, 2 μg of NdeI restricted chromosomal DNA was electrophoresed in 0.7% agarose gel in 1×TAE buffer and transferred to nitrocellulose membrane . The blots were incubated with digoxigenin-UTP labelled probes, and hybridisation detected using anti-DIG Alkaline Phosphatase conjugated antibody (Roche Diagnostics) and CDP-Star detection reagent (Tropix). The R3P probe used for hybdrisation here was amplified using forward  and reverse  primers.Chromosomal DNA was extracted using the RBC HiYield Genomic DNA Mini kit, modified by the addition of 10 U mutanolysin (Sigma) and 0.5 mg proteinase K (Promega) in the lysis steps. Primers and conditions for amplification of discrete regions of Region 1 and Region 3 have been previously described . In this8 CFU/ml) were mixed in 1.5 ml microcentrifuge tubes. Controls were prepared by mixing 500 μl of the donor or recipient culture mixed with 500 μl of THB. The suspensions were centrifuged at 3000 g, supernatant removed and pellet resuspended in 1 ml of THB. The samples were centrifuged again to facilitate contact between bacteria. After incubation at 37°C for between 10 min and 3 hours the samples were centrifuged, washed, plated onto THA or CBA containing kanamycin, selective for ICESde3396:km, and a second antibiotic, selective for the recipient strain. The plates were incubated overnight at 37°C and colony forming units (CFU) determined. Serially diluted samples were also plated onto agar containing a single antibiotic to score for the recovery of donors and recipients. Conjugation efficiency was calculated by dividing the number of transconjugants by the number of donors recovered per unit volume.Unless otherwise stated, conjugation experiments were performed using a microfuge method developed for streptococcal conjugation in this study. Five hundred microliters of overnight cultures of donor and recipient (~1 × 102. The wells were then inoculated with donors (~106 CFU) and recipients (~107 CFU). For control experiments streptococci were added to fresh DMEM or cell culture supernatant (spent medium). The plates were incubated for six hours at 37°C under 5% CO2. Post incubation, the supernatant was transferred to a new microfuge tube. To collect bacteria that might have adhered to the eukaryotic cell surface each well was also treated with 0.25% trypsin and 0.0125% Triton X-100 and detached cells added to the above tubes. The resulting suspension was centrifuged, resuspended in PBS and plated onto agar containing appropriate antibiotics.Eukaryotic A549 epithelial cells were grown to confluence in six-well plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) at 37°C under 5% CO


Leucine-rich repeat kinase 2 (LRRK2) is extensively phosphorylated in cells within a region amino-terminal to the leucine-rich repeat domain. Since phosphorylation in this region of LRRK2, including Ser910, Ser935, Ser955, and Ser973, is significantly downregulated upon treatment with inhibitors of LRRK2, it has been hypothesized that signaling pathways downstream of the kinase activity of LRRK2 are involved in regulating the phosphorylation of LRRK2, although the precise mechanism has remained unknown. Here we examined the effects of LRRK2 inhibitors on the phosphorylation state at Ser910, Ser935, and Ser955 in a series of kinase-inactive mutants of LRRK2. We found that the responses of LRRK2 to the inhibitors varied among mutants, in a manner not consistent with the above-mentioned hypothesis. Notably, one of the kinase-inactive mutants, T2035A LRRK2, underwent phosphorylation, as well as the inhibitor-induced dephosphorylation, at Ser910, Ser935, and Ser955, to a similar extent to those observed with wild-type LRRK2. These results suggest that the kinase activity of LRRK2 is not involved in the common mechanism of inhibitor-induced dephosphorylation of LRRK2. LRRK2 gene has been identified as a causative gene for PARK8, an autosomal-dominant form of FPD LRRK2 locus have been reported to be associated with the risk for sporadic PD in two independent genome-wide association studies, implicating LRRK2 in the pathogenesis of PARK8 as well as of sporadic PD Parkinson’s disease (PD) is one of the most common neurodegenerative disorders pathologically characterized by neuron loss in the substantia nigra accompanied by formation of Lewy bodies in vitro, it has been believed that these phosphorylations do not represent autophosphorylation but are executed by other kinases It has been repeatedly shown that LRRK2 is phosphorylated in cells at multiple sites amino-terminal to the leucine-rich repeat (LRR) domain Interestingly, it has been shown that phosphorylation of LRRK2 at these sites are rapidly downregulated upon treatment of Swiss 3T3 cells with inhibitors of LRRK2 However, LRRK2 harboring mutations either inactivating (e.g. K1906M) or increasing (e.g. G2019S) its kinase activity is phosphorylated at these sites at similar levels to those in wild-type (WT) LRRK2 The expression plasmids encoding full-length human LRRK2  cloned in the p3×FLAG-CMV-10 vector (Sigma) were constructed as described previously 2 atmosphere. Transient expression in HEK293 cells was performed by transfecting the plasmids using FuGENE6 (Roche) or polyethylenimine (Sigma) according to the manufacturer’s instructions. LRRK2-IN-1 was provided by Professor Dario Alessi (University of Dundee). Sunitinib and H-1152 were purchased from Sigma and Calbiochem, respectively. LRRK2-IN-1 and sunitinib were dissolved in dimethyl sulfoxide (DMSO). H-1152 was dissolved in sterilized distilled water (SDW). Thirty-six hours after transfection, cells were treated with inhibitors or an equivalent volume of solvents (DMSO or SDW). The final concentrations of solvents were 0.1% (v/v) for DMSO and 1% (v/v) for SDW.HEK (human embryonic kidney) 293 cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% (v/v) fetal bovine serum and 100 units/ml penicillin/100 µg/ml streptomycin at 37°C in 5% CORabbit monoclonal antibodies for human LRRK2 , for the phosphorylated form of LRRK2 (anti-pSer910 LRRK2 (#5098-1); anti-pSer935 LRRK2 (#5099-1)), and autophosphorylated form of LRRK2 (anti-pThr1410 LRRK2 (#7125-1); anti-pThr1491 LRRK2 (#7058-1)) were purchased from Epitomics. A rabbit monoclonal antibody recognizing anti-pSer955 LRRK2 was purchased from Abcam (#ab169521). A rabbit monoclonal antibody recognizing phosphorylated LRRKtide was purchased from Cell Signaling Technology (#3726). A rabbit polyclonal antibody recognizing phosphorylated Thr1357 of LRRK2 was generated as described previously 2PO4, 137 mM NaCl, 8.1 mM Na2HPO4, 0.05% (v/v) Tween-20 (pH 7.4)]. Phosphorylation-specific antibodies (anti-phospho-LRRKtide) diluted in the buffer C  were added to the plates followed by incubation at 4°C overnight. The plates were washed four times with DPBS-T, and secondary antibodies conjugated with horseradish peroxidase  diluted in the buffer C were added to the plates followed by incubation for 1 h at room temperature. The plates were washed eight times with DPBS-T and developed using TMB Microwell Peroxidase Substrate System (KPL). For detecting in vitro autophosphorylation of LRRK2, immunoprecipitated LRRK2 was incubated in 20 µl of the reaction buffer, and the reaction was stopped by addition of 20 µl of 2×SDS-PAGE sample buffer and boiling. Samples were analyzed by immunoblotting using antibodies recognizing autophosphorylation sites of LRRK2.An amino-terminally biotin-tagged LRRKtide   was used as a substrate. Transfected HEK293 cells in a 10 cm dish were lysed in 1 ml of the lysis buffer  for 30 minutes at 4°C. Cleared lysates were incubated with 10 µl of 50% (v/v) M2-agarose (Sigma) for 1 h at 4°C. The beads were washed three times with the wash buffer 1  and then twice with the wash buffer 2 . The beads were incubated for 15 minutes in 10 µl of the reaction buffer  containing 300 µM bLRRKtide. The reactions were stopped by addition of 990 µl of the ice-cold stop buffer , and the beads were removed by passing through empty spin columns. Fifty microliter of the diluted reaction mixtures were applied onto a 96-well plate coated with streptavidin (Thermo Fisher Scientific) and incubated for 1 h at 37°C. The plates were washed four times with DPBS-T [2.68 mM KCl, 1.47 mM KHPrior to examining the statistical significance of differences, a normal distribution of the data was examined by Shapiro-Wilk test using IBM SPSS Statistics Version 21. The statistical significance of differences between data following a normal distribution was examined by the Student’s t-test or one-way/two-way ANOVA followed by Bonferroni test as indicated in the figure legend. The statistical significance of differences between data not following a normal distribution was examined by the Kruskal-Wallis test if possible. Otherwise Student’s t-test or ANOVA followed by Bonferroni test was carried out and asterisks showing the statistical significance were marked with parentheses. Statistical tests were done using Prism 6 (GraphPad), and differences were considered to be statistically significant when p<0.05.Given that there exists equilibrium between phosphorylation and dephosphorylation of proteins within cells, the currently prevailing understanding of the molecular mechanism underlying the inhibitor-induced dephosphorylation of LRRK2 is based on an assumption that the equilibrium is disrupted by inhibition of the kinase activity of LRRK2 . Based oWe first confirmed the inhibitor-induced dephosphorylation of wild-type (WT) LRRK2 by overexpressing amino-terminally 3×FLAG-tagged full-length (FL) LRRK2 into HEK293 cells and treating them with LRRK2-IN-1, sunitinib, or H-1152 To investigate whether inhibition of the kinase activity of LRRK2 is involved in the inhibitor-induced dephosphorylation of LRRK2, we systematically characterized the inhibitor-induced dephosphorylation of LRRK2 mutants harboring kinase-inactive mutations. We utilized LRRK2 harboring K1906A, K1906M, D1994A, D1994N, D2017A, S2032A, and T2035A mutations as potentially kinase-inactive mutants, which have already been described in literature We next examined the inhibitor-induced dephosphorylation of LRRK2 harboring the kinase-inactive mutations using three LRRK2 inhibitors. Assuming that the hypothesis depicted in Given that the kinase activity of LRRK2 is involved in the regulation of phosphorylation of LRRK2 in cells, hyperactive mutations should also affect the phosphorylation of LRRK2. To address this question, we examined the phosphorylation of LRRK2 at Ser910, Ser935, and Ser955 harboring G2019S or T2031S mutation which has been reported to upregulate the kinase activity in vitro assays. Transfected and immunoprecipitated 3×FLAG-LRRK2 was incubated with LRRKtide in the presence of ATP for 15 min at 30°C, and phosphorylation of LRRKtide was detected by enzyme-linked immunosorbent assay (ELISA). We also examined the autophosphorylation of LRRK2 at Thr1357, Thr1491, and Thr1503 by immunoblotting using their respective phosphorylation-specific antibodies in vitro. We also confirmed that the hyperactive mutants exhibited increased levels of kinase activity compared with WT LRRK2  are supposed to be required for binding ATP (or ATP-competitive inhibitors) with proper orientation, whereas Asp1994 and Thr2035 which are equivalent to Asp166 and Thr201 in PKA are supposed to be required for positioning the catalytic loop and not directly involved in binding of ATP In summary, we identified a clear counterexample (T2035A) to the hypothesis that the phosphorylation of LRRK2 is regulated by the kinase activity of LRRK2, showing that inhibition of the kinase activity of LRRK2 is dispensable for the inhibitor-induced dephosphorylation of LRRK2 in cells. Further investigation into the conformational change of LRRK2 would elucidate the mechanism of the inhibitor-induced dephosphorylation of LRRK2 and that of the familial Parkinson mutations.


In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, reactive oxygen species (ROS) are produced by different systems which are involved in endothelial cells injury, ultimately leading to severe organ dysfunctions. The aim of this work was to study the effect of ROS produced by hypoxanthine-xanthine oxidase (Hx-XO) on the adhesion of human umbilical vein endothelial cells (HUVEC) and on the signaling pathways involved. Results show that Hx-XO-derived ROS induced an increase in HUVEC adhesion in the early stages of the process (less than 30 min), followed by a decrease in adhesion in the later stages of the process. Interestingly, Hx-XO-derived ROS induced the same biphasic effect on the phosphorylation of the focal adhesion kinase (FAK), a nonreceptor tyrosine kinase critical for cell adhesion, but not on ERK1/2 phosphorylation. The biphasic effect was not seen with ERK1/2 where a decrease in phosphorylation only was observed. Wortmannin, a PI3-kinase inhibitor, inhibited ROS-induced cell adhesion and FAK phosphorylation. Orthovanadate, a protein tyrosine phosphatase inhibitor, and Resveratrol (Resv), an antioxidant agent, protected FAK and ERK1/2 from dephosphorylation and HUVEC from ROS-induced loss of adhesion. This study shows that ROS could have both stimulatory and inhibitory effects on HUVEC adhesion and FAK phosphorylation and suggests that PI3-kinase and tyrosine phosphatase control these effects. In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, large amounts of reactive oxygen species (ROS) are produced by different enzymatic systems such as xanthine oxidase, mitochondria, and the phagocyte NADPH oxidase NOX2. These ROS are believed to be involved in endothelial cell injury leading to severe tissue and organ dysfunctions , 2.Oxidative stress, resulting from an imbalance between oxidant production and antioxidant systems, has been reported to induce alterations in signaling pathways leading to modulation of cellular functions, apoptosis, and necrosis , 4. EndoPhosphorylation and dephosphorylation events play a critical role in the signal transduction pathways that regulate various processes in living cells. ROS have been reported in various cells to alter the phosphorylation of several key proteins involved in signaling pathways –17, some2+ and Mg2+ (HBSS or HBSS wo), was obtained from GIBCO . Bacto-gelatin was from DIFCO . Reagents for cell culture were supplied by GIBCO ; 100 mm dishes and 6- and 24-well plates were from Costar . Mouse monoclonal FITC-conjugated antihuman factor VIII, mouse monoclonal antibody anti-human focal adhesion kinase (anti-p125FAK), and rabbit polyclonal antibodies anti-human ERK1 and anti-ERK2 were from Santa Cruz Biotechnology . Mouse monoclonal antibody anti-human active phosphorylated ERK1/2 was from New England Biolabs . Mouse monoclonal anti-phosphotyrosine (anti-Tyr(P)) antibody was from Upstate Biotechnology . The rainbow markers (high range), sheep anti-mouse IgG conjugated horseradish peroxidase antibody, and ECL Western blot detection system were from Amersham Biosciences . Alkaline phosphatase conjugated goat anti-rabbit IgG antibody was from Jackson Laboratories. Nitrocellulose membranes were from Schleicher and Schuell . PD098059, wortmannin, and LY294002 were from Calbiochem . Protein G Sepharose 4 fast flow media were provided by Amersham Biosciences . Calcein-acetoxymethyl  was purchased from Molecular Probes . Resveratrol was kindly donated by Dr. Bagchi . All other products were from Sigma .Hank's balanced salt solution, with or without Ca2. At confluence adherent cells were harvested following trypsin-EDTA treatment . Primary-passage HUVECs were seeded into 2% gelatin-coated 100 mm petri dishes and 6- and 24-well tissue culture plates. Culture medium was replaced every 2 days. Adherent cells were identified by their cobblestone appearance at confluence and positive labelling with mouse FITC conjugated antihuman factor VIII. Only cells of secondary cultures were used for the studies.Endothelial cells from human umbilical cords veins (HUVECs) were harvested by 0.05% collagenase treatment for 15 minutes at 37°C, as previously described , 19. The−4 M) and xanthine oxidase  for various times (5 to 90 minutes) in HBSS buffer. Hypoxanthine oxidation by xanthine oxidase, generated around 3 nmol/mL/minutes of superoxide anion (O2•−) and 50 nmol/mL of hydrogen peroxide (H2O2) over a 30-minute period.HUVECs were washed with HBSS and incubated with hypoxanthine  using a 480 nm excitation wavelength and recording of green fluorescence at 520 nm. Values were expressed as mFU units per well. Each experiment was performed in triplicate.Adhesion to the matrix was measured with a Calcein-AM assay as previously described . FollowiμL of ice-cold immunoprecipitation (IP) buffer . Samples were sonicated and centrifuged at 15,000 ×g for 10 minutes to remove cellular and nuclear debris. Protein content in clear supernatants was measured using the Bio-Rad protein assay reagent. Samples were incubated with anti-FAK antibody (5 μg/4 × 106 cells) for 2 h at 4°C. Protein was immunoprecipitated using gamma-bind G Sepharose beads overnight at 4°C and beads then were washed three times with cold immunoprecipitation buffer. Sodium dodecyl sulfate (SDS) buffer containing 62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 3% 2 beta-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue [Control or Hx-XO treated HUVECs in 100 mm petri dishes were lysed by incubation for 30 minutes in 500 nol blue  was addeImmunoprecipitated proteins or whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 9% polyacrylamide gels using standard techniques , 22. TheAll experiments were performed in triplicate and repeated at least three times. Results are expressed as mean ± standard deviation. Data were compared using analysis of variance (ANOVA) followed by a Fischer test.To determine the consequences of oxidative stress on HUVECs adhesion to the matrix, HUVECs were incubated for 10, 20, 30, 60, or 90 minutes with Hx-XO, a ROS generating system, and adhesion was measured using a Calcein-AM probe. Results show that HUVECs adhesion was significantly increased within the first 20 min of treatment with Hx-XO-derived ROS . FollowiAs FAK plays a pivotal role in adhesion mechanisms  and ERK1μM), another PI3-kinase inhibitor, induced similar effects (data not shown). We then analysed the effect of wortmannin on cell adhesion after ROS treatment. Using Calcein-AM probe, we observed that, after 15 minutes of Hx-XO treatment, adhesion decreased significantly in cells treated with wortmannin (100 nM) compared to cells treated with Hx-XO alone . Orthovanadate also reversed the ROS-inhibitory effect on cell adhesion mainly at 60 min .To verify that HUVECs alterations were due to oxidative injury caused by oxidative stress induced by the Hx-XO system, HUVECs were pretreated with the antioxidant molecule, trans-3,4′,5-trihydroxystilbene (Resveratrol) , 26. Res Figures . QuantifIn this study, we have demonstrated that oxidative stress, produced by the hypoxanthine-xanthine oxidase (Hx-XO) system, modulated both HUVECs adhesion and FAK phosphorylation. During the first twenty minutes of ROS exposure, HUVECs adhesion was significantly increased in parallel with FAK phosphorylation. This early ROS-induced adhesion and FAK phosphorylation were dependent on PI3-kinase. Later, we observed a progressive decrease in cell adhesion, concomitant with a decreased in FAK and ERK1/2 phosphorylation. These latter dephosphorylation events could be related to the ROS-induced activation of one or several tyrosine phosphatases. These events are prevented by the antioxidant agent Resveratrol. These results demonstrated that ROS can have dual contrasting effects on HUVECs adhesion to the matrix depending on the length of exposure. This phenomenon is critical for HUVEC functioning and survival.ROS induced a biphasic effect on cell adhesion and FAK phosphorylation, following a transient increase during the first 20 minutes, a dephosphorylation of FAK and a decrease of cell adhesion occurred in a time-dependent manner. The early ROS-induced increase of FAK phosphorylation was regulated by PI3-kinase as shown by its inhibition by wortmannin. These results suggest that ROS can modulate PI3-kinase directly or indirectly as previously described , 28. TheWe also showed the involvement of tyrosine phosphatases in the oxidative stress regulation of these kinases. The very quick-acting dephosphorylation of ERK1/2 suggests the activation of constitutive phosphatases by ROS –36. DephThe protective effect of polyphenolic antioxidant agent such as Resveratrol , 26, on In our model of time-dependent oxidative stress we propose that initially the ROS-induced increase of FAK tyrosine phosphorylation is dependent on PI3-kinase. This leads to continued HUVECs adhesion in spite of a rapid dephosphorylation of ERK1/2. In the late phase of this process, ROS-induced activation of different phosphatases may be involved in ERK1/2 as well as FAK dephosphorylation, leading to a decrease in HUVECs adhesion. Kinetic studies should be performed to further investigate the effect of ROS on cell function.In conclusion, our data has demonstrated a time-dependent biphasic effect of ROS on FAK phosphorylation and endothelial cells adhesion. Under these conditions, FAK phosphorylation plays a critical role in regulating cell adhesion to the matrix and was dependent on equilibrium between PI3-kinase and protein tyrosine phosphatases. Such mechanisms could occur during different pathological inflammatory conditions as a result of the release of ROS in proximity to endothelial cells.