It is proposed that PFOA degradation occurs via a decarboxylation reaction triggered by SO4−•, followed by a HF elimination process aided by •OH, which produces one-CF2-unit-shortened perfluoroalkyl carboxylic acids . The decarboxylation and HF elimination processes would repeat and eventually lead to the complete mineralization all PFCAs.Perfluorooctanoic acid (PFOA) is an emerging contaminant of concern due to its toxicity for human health and ecosystems. However, successful degradation of PFOA in aqueous solutions with a cost-effective method remains a challenge, especially for groundwater. In this study, the degradation of PFOA using activated persulfate under mild conditions was investigated. The impact of different factors on persulfate activity, including pH, temperature (25 °C–50 °C), persulfate dosage and reaction time, was evaluated under different experimental conditions. Contrary to the traditional alkaline-activated persulfate oxidation, it was found that PFOA can be effectively degraded using activated persulfate under acidic conditions, with the degradation kinetics following the pseudo-first-order decay model. Higher temperature, higher persulfate dosage and increased reaction time generally result in higher PFOA degradation efficiency. Experimental results show that a PFOA degradation efficiency of 89.9% can be achieved by activated persulfate at pH of 2.0, with the reaction temperature of 50 °C, molar ratio of PFOA to persulfate as 1:100, and a reaction time of 100 h. The corresponding defluorination ratio under these conditions was 23.9%, indicating that not all PFOA decomposed via fluorine removal. The electron paramagnetic resonance spectrometer analysis results indicate that both SO Becauair base . PFOA waair base . These cair base . Comparii.e., H2O2 and Fe2+ salts), alkaline ozonation and peroxone , utilizing the hydroxyl radical in AOPs, have been shown to be relatively ineffective for PFOA destruction [However, PFOA is chemically inert and recalcitrant to most chemical and microbiological treatment ,10,11,12truction ,23,24. 2O82−), a strong and relatively stable oxidant (E0 = 2.01 eV), can be activated to generate free radicals to achieve a higher oxidative potential under conditions of heat [4−• (E0 = 2.6 V), •OH (E0 = 2.7 V) and O2−• (E0 = −0.33 V) [2 [Persulfate  ,31,32, r−0.33 V) ,18,19, U−0.33 V) ,16,17,1833 V) [2 . Howeveret al. reported a low degradation efficiency of PFOA by persulfate using iron-modified diatomite under alkaline conditions [et al. observed some PFOA degradation by persulfate under acidic conditions [Little research has been reported on the degradation of PFOA by persulfate under mild conditions. Celyna nditions . Lee et nditions , showing7F15COOH, molecular weight 414.07, 96% purity) and the free radical spin trapping reagent 5,5-dimethyl-1-pyrrolidine N-oxide  were purchased from Aladdin . The rest of the chemical reagents used in this study, including sodium persulfate , sulfuric acid , hydrochloric acid , sodium hydroxide , sodium fluoride , potassium phosphate dibasic anhydrous , potassium dihydrogen phosphate , trisodium citrate dihydrate , methanol  and ethanol  were of analytical grade and purchased from Sinopharm Chemical Reagent Corporation Ltd. . The deionized water used in all experiments, with a resistivity of 18.0 MΩ cm, was purified by a Milli-Q system. PFOA . In subsequent experiments, pH of solutions was adjusted to 2.0 by H2SO4, the initial concentration of PFOA was 20 μM, and initial persulfate concentrations ranged from 0.5 mM to 4 mM. The containers were then placed into constant-temperature water baths without shaking . At the designated sampling times, an appropriate amount of ethanol (molar ratio of ethanol and persulfate was 100:1) was added to quench the reaction immediately. Samples were then taken and preserved at 4 °C in the refrigerator before analysis. All experiments were conducted in triplicate.Batch experiments to evaluate the influence of different experimental factors  on PFOA degradation efficiency were carried out in 40-mL borate glass bottles. Due to the significance of pH in the persulfate activation under mild conditions, experiments were conducted first to study the effect of pH. In these experiments, pH of solutions was adjusted by adding NaOH or H2SO4 to 2.0. In these experiments, PFOA (20 μM) and persulfate (2 mM) were mixed to react at different temperature: 30 °C, 40 °C and 50 °C. At the designated sampling times, an appropriate amount of ethanol was added and samples were taken and preserved at 4 °C in the refrigerator. For PFOA degradation byproducts analyses, PFOA (20 μM) and persulfate (2 mM) were mixed to react at a temperature of 50 °C, with pH adjusted by H2SO4 to 2.0. After a reaction time of 100 h, ethanol was added to quench the reaction immediately and samples were taken for analysis.Experiments for PFOA kinetics study were conducted under acidic conditions, with pH adjusted by H2SO4 to 2.0 and NaOH to 12, respectively. After a reaction time of 10 min, an appropriate amount of DMPO , dissolved in phosphate buffer at pH 7.4, was added immediately to react with free radicals produced by persulfate. 20 μL of the mixed solution were then loaded in the quartz capillary tube of EPR for analysis.Free radical analyses, which were used to identify the effective radicals contributing to the degradation of PFOA, were conducted under three different experimental conditions: (1) PFOA only in aqueous solution as the control system; (2) PFOA with persulfate under acidic conditions; and (3) PFOA with persulfate under alkaline conditions. In the control system, 25 mL of PFOA at a concentration of 20 μM was added to the 40-mL borate glass bottles at a temperature of 50 °C. The solution pH was not adjusted and the final pH was 4.2. For experiments under acidic conditions and alkaline conditions, 20 μM PFOA and 2 mM persulfate were mixed to react at a temperature of 50 °C, with their solution pH adjusted by H4OH in methanol, 4 mL of methanol, and 4 mL of Milli-Q water. The cartridges were washed with 4 mL of buffer solution , centrifuged for 3 min at 3000 revolutions/min to remove residual water, and then washed with 4 mL of methanol. Target compounds were then eluted with 4 mL of 0.1% NH4OH in methanol. The latter fraction was concentrated to 0.5 mL using a bath-typed nitrogen blowing instrument and then passed through a polypropylene-membrane syringe filter . 100 μL of filtrate and 100 μL of Milli-Q water were transferred into a polypropylene vial for concentration analysis. Further details of the analysis method, such as column installation, column temperature, mobile phase and its flow rate, can be found in the study by Yu et al. [Concentrations of PFOA and its degradation byproducts were analyzed by a high-performance liquid chromatograph (HPLC), using an ACQUITY UPLC system  equipped with a Waters ACQUITY TQD triple quadrupole mass spectrometer. All samples were extracted using solid phase extraction (SPE) without prefiltration. The Oasis WAX cartridges  were cleaned and conditioned with 4 mL of 0.1% NHu et al. . PFOA an−1. The retention time for fluoride ion was 3.12 min. In addition, a fluoride-ion-selective electrode was also used to analyze concentrations of fluoride ion, after a 1:1 dilution with an ionic strength adjustment buffer. The ionic strength adjustment buffer solutions were prepared by adding hydrochloric acid into trisodium citrate dehydrate aqueous solution (1 M) adjusting solution pH to 6.00. The quantitative detection limits were 1 μg/L and 20 μg/L, for the ion chromatography and ion selective electrode, respectively.Concentrations of fluoride ion were analyzed using a Dionex-500 ion chromatography system . The samples were filtered through Dionex OnGuard cartridges  to reduce the interference of organic compounds before analysis. The cartridges were cleaned and conditioned with 4 mL of methanol and 10 mL of Milli-Q water before use. Fluoride analysis was carried out using an AS14A (4 mm × 250 mm) analytical column and Ionpac AG14A 4-mm guard column (4 mm × 50 mm) with a degassed 8.0 mM sodium carbonate and 1.0 mM sodium bicarbonate mobile phase at a flow rate of 1 mL·min4.An electron paramagnetic resonance (EPR) spectrometer was used to analyze free radicals generated by activated persulfate. The EPR spectra were obtained using a Bruker-EMX-10/12 spectrometer  with a magnetic field modulation frequency of 9.77 GHz, microwave power of 19.97 mW, modulation frequency of 100 kHz, modulation amplitude of 2.0 G, sweep width of 100 G, time constant of 40.95 ms, sweep time of 83.85 s, and receiver gain of 2.0 × 10The defluorination ratio (DF ratio), expressed as (moles of fluorine released due to PFOA degradation)/, is an indicator of PFOA degradation and was used to evaluate the defluorination efficiency of PFOA by activated persulfate. PFOA degradation efficiency, expressed as (moles of PFOA degraded)/, was also used in the evaluation of PFOA removal efficiency by activated persulfate.2SO4 and NaOH  resulted in an increase in the DF ratio ; both SO4−• and •OH are present at neutral pH; and at pH > 12, •OH is the predominant free radical, and reductant radical O2−• may also be present. The higher DF ratio under acidic conditions (pH < 3) could be due to the production of predominant SO4−•, as explained through a proton-catalyzing process to form sulfate free radicals as shown in Equations (1) and (2) [− may act as a fast scavenger, converting SO4−• to •OH through the path demonstrated in Equation (3), considering the reaction rate of Equation (3) is much higher than that of Equation (4) [4−• free radical plays a major role in PFOA degradation, and •OH itself cannot oxidize PFOA directly [The effects of pH on PFOA degradation, with a molar ratio of 1:100 PFOA to persulfate, are illustrated in and NaOH a, HCl  . The meation (4) . The mucdirectly ,39.(1)S2SO4versus HCl) used to adjust the solution pH also affected the DF ratio. When solution pH was adjusted by HCl, the DF ratio was much lower –(7) [In addition, it was noted that the acid reagent (H (5)–(7) ,41. This (5)–(7) .(5)Cl−+i.e., to study the effect of persulfate dosage, temperature, and reaction time) was adjusted by H2SO4 to 2.0 in an effort to obtain optimal experimental conditions for PFOA removal. Effects of persulfate dosage, temperature and reaction time on PFOA degradation by persulfate under acidic conditions were further explored.Based on the results discussed above, pH for subsequent experiments  versus reaction time at temperature of 30 °C, 40 °C and 50 °C. The degradation of PFOA by activated persulfate under acidic conditions was found to follow the pseudo-first-order decay model, with the r2 correlation values of 0.894, 0.988 and 0.998, for temperature of 30 °C, 40 °C and 50 °C, respectively. The kinetics of PFOA degradation by activated persulfate under acidic conditions are shown in 1/2), which can be used to evaluate the degradation rate at different temperature. The k values of PFOA degradation by activated persulfate under acidic conditions increase with increasing temperature, further demonstrating the significant effect of temperature on PFOA degradation. r2 = 0.986), which is in good agreement with previous study by Lee et al. [e et al. . This ape et al. , indicat4) with hyperfine splitting constant of aN = 13.51, aHβ = 9.91, aHγ1 = 1.34 and aHγ2 = 0.88 in the EPR spectra for different solution systems in which pH was adjusted by H2SO4 and NaOH. The EPR spectra of radical adducts show the generation of •OH and SO4−• in solutions under both acidic and alkaline conditions. Relatively little degradation of PFOA was observed in persulfate solution systems with high pH values. According to Equation (3), SO4−• reacts rapidly with OH− to produce •OH under alkaline conditions. This suggests that •OH itself may not be able to degrade PFOA, consistent with the conclusion of early studies that showed •OH cannot oxidize PFOA directly [In order to further understand PFOA degradation mechanisms under acidic conditions, free radicals analysis experiments were carried out along with quantification of PFOA and its daughter products. directly ,44. 4−• produced by protons under acidic conditions (pH < 3), as discussed earlier, plays a significant role in the successful degradation of PFOA. It was speculated that the proton increases the reaction rate of sulfate radicals with PFOA, which leads to the efficient degradation of PFOA. Furthermore, consistent with the results of a previous study [4. A possible explanation is the radical conversion as described in Equations (10) and (11) [The predominant SOus study , the sigand (11) .(10)2S2Furthermore, concentrations of PFOA and its daughter products were quantified, after a reaction time of 100 h, and the results are summarized in It was observed that, in general, the total molarity of perfluoroalkyl carboxylic acid (PFCA) intermediates was equal to the initial concentration of PFOA. As indicated by the earlier conclusion that not all PFOA decomposes via fluorine removal (as seen from the discrepancy between DF ratio and degradation efficiency), it can be deduced that concentrations of shorter-chain PFCs increase with increased reaction time. The analysis results indicate a stepwise degradation process, consistent with those reported in early studies ,36. 4−• to initiate a decarboxylation reaction and perfluoroalkyl radicals are formed . The newly formed CnF2n+1• continues to react with •OH to form CnF2n+1OH, which undergoes an HF elimination to form Cn−1F2n−1COF [2-unit-shortened PFCA (Cn−1F2n−1COOH) via hydrolysis [2 and fluoride SO44−•, along with •OH, are the effective free radicals contributing to PFOA degradation. It was speculated that SO4−• initiated a decarboxylation reaction in PFOA, followed by HF elimination due to •OH, and formed one-CF2-unit-shortened PFCA (Cn−1F2n−1COOH). The discoveries of this study provide a basis for the cost-effective remediation approach of PFOA contamination in aqueous solutions, especially in groundwater. However, it is still technically challenging due to the fact that remediation at elevated temperature is difficult to implement in the field at large scale. Hence, further study to improve the efficiency of sulfate radical production to accelerate the degradation of PFOA and its associated intermediate products is needed. In addition, the influence of soil on the PFOA degradation by activated persulfate under acidic conditions was not investigated in this study. Further study to evaluate the impact of soil will prove to be valuable. This study demonstrates that advanced chemical oxidation of PFOA by activated persulfate under acidic conditions in aqueous solutions is effective, while its degradation by the traditional alkaline-activated persulfate shows rather low degradation efficiency. Experimental results showed that higher temperature, higher persulfate dosage and a longer reaction time resulted in higher PFOA degradation efficiency by activated persulfate under acidic conditions. The degradation kinetics of PFOA was found to follow the pseudo-first-order decay model. It was demonstrated that 89.9% of PFOA can be degraded after a reaction time of 100 h in a solution system with pH 2.0 at a temperature of 50 °C. The corresponding DF ratio of 23.9% was much less than the PFOA degradation efficiency, indicating that not all PFOA degradation is achieved via fluorine removal. EPR data showed that SO


Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound  that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of DOI:http://dx.doi.org/10.7554/eLife.07141.001 DNA contains the instructions required to make proteins and other molecules in cells. DNA is made of four building blocks called deoxyribonucleotides, which are in turn made from molecules called ribonucleotides by enzymes known as ribonucleotide reductases (RNRs for short). RNR enzymes are responsible for maintaining a good balance in the levels of the different deoxyribonucleotides in cells, which is essential for DNA to be made and repaired correctly.Previous work has shown that each RNR can act on all four ribonucleotides. However, these enzymes become more selective for certain ribonucleotides depending on which deoxyribonucleotide is most common within the cell. For example, when a deoxyribonucleotide called dGTP is plentiful, it binds to a so-called “specificity site” on the enzyme and alters the shape of the enzyme’s active site. This then means that a ribonucleotide called ADP will bind in preference to the other ribonucleotides. However, it was not clear how the binding of deoxyribonucleotides to the enzyme influences the shape of the active site.Zimanyi et al. used a technique called X-ray crystallography to determine the three-dimensional structures of a bacterial RNR enzyme when it is bound to all four different combinations of deoxyribonucleotides and ribonucleotides. In the absence of nucleotides, the active site adopts a shape that resembles an open barrel. However, when RNR is bound to a deoxyribonucleotide at the specificity site and a ribonucleotide at the active site, the barrel clamps down, bringing the specificity site and the active site closer together. Additionally, a loop of the protein interacts with each of the deoxyribonucleotides in a different way and communicates their identity directly to the active site, which rearranges itself to hold on to the corresponding preferred ribonucleotide.Zimanyi et al.’s findings provide an explanation for how RNRs can select between ribonucleotides so that they produce a good balance of deoxyribonucleotides in cells. This will inform future efforts to develop molecules that inhibit RNRs, which may have the potential to be used to treat bacterial infections or to kill cancer cells.DOI:http://dx.doi.org/10.7554/eLife.07141.002 Escherichia coli that employs a di-iron-tyrosyl-radical cofactor to initiate chemistry and requires two dimeric protein subunits for enzymatic activity. The α2 subunit contains two (β/α)10 barrels, which house the active sites at the barrel centers  . Three c centers , and thecofactor   substrates. Whereas high levels of dATP inhibit class Ia RNR, at lower levels, dATP promotes CDP or UDP reduction. Likewise, TTP promotes GDP reduction, and dGTP promotes ADP reduction (2 and β2 subunits for each other is weak (~0.4 μM) in the absence of effectors, whereas the binding of a complementary substrate/specificity effector pair increases the affinity of the class Ia RNR subunits fivefold (E. coli α2 (2 from Saccharomyces cerevisiae (2 from Thermotoga maritima (S. cerevisiae (E. coli numbering) and which regions of the structure are involved in the communication between the specificity site and the active site. A flexible loop, termed loop 2 (residues 292–301 in E. coli) NTP effectors to an allosteric specificity site in αeduction   , bridgesE. coli) . An addiE. coli) . Thus, p4β4 crystal form of the E. coli class Ia RNR 10 barrel around the bound substrate that is not observed in the absence of substrate. An initial superposition of one α chain from the previously reported free α2 soaked with AMP-PNP  found in the center of the (β/α)10 barrel. In this structural alignment, one half of the active site barrel overlays exactly, whereas the second half of the barrel and the N-terminal portion of the structure has undergone a clamping movement  onto thmovement . To bettE. coli RNR, that do not employ metal cations such as Mg2+ for this purpose. The positions of residues that contact the ribose O3' (Glu441 and Asn437) are virtually unchanged between the NDP-free and NDP-bound structures  reveals that the movements described above directly affect substrate binding . Residueructures . All fou2 barrel observed in our substrate/effector-bound α4β4 structures, no clamping motion of the barrel is observed between substrate-free α2 and a previously reported structure of α2 with GDP substrate and TTP effector bound  form almost identical contacts with RNR . Despiteolecules . There aFo–Fc composite omit electron density  in adenine and a hydrogen bond acceptor (O) in guanine; and the ‘backward’ position of the Gly299 carbonyl interacts with the 1-position and 2-position of the purine, which are both hydrogen bond donors (NH and NH2) in guanine but not in adenine   . Results(175 μM) . The neg(175 μM) . The effecreased . These r2 , the baID: 3R1R . HoweverID: 3R1R   or adenosylcobalamin (class II), and initiate catalysis  and the E. coli and the S. cerevisiae α subunits (29.3%), a structural comparison shows that all three enzymes interact with the phosphate groups and ribose moieties of their substrates and effectors in similar ways  shows direct contact. This GDP/TTP-bound structure is the only T. maritima RNR structure in which loop 2 is fully ordered and it is remarkably similar to our E. coli structures (E. coli numbering) and 208 (T. maritima numbering) to ‘fall back’ to contact N1 and N2 of GDP.The only noteworthy difference is that Arg298, critical for reduction of all four NDPs in ructures . All conructures , 4C. TheT. maritima class II RNR structures also reveal a movement of Gln294 in and out of the active site in response to effector binding. In particular, the GDP/TTP and CDP/dATP bound structures from T. maritima class II RNR, which have an ordered and semi-ordered loop 2, respectively, show that Gln203  interacts directly with CDP when dATP is bound, and moves away from the active site when GDP/TTP are bound , indicate that the mechanism of allosteric regulation is not conserved among RNRs. However, it is also possible that these observed structural differences are a result of crystal packing variations or other deviations in how crystals were prepared. Additional structures of high-affinity RNR-substrate complexes will help to clarify the degree to which the roles of Gln294 and Arg298 are conserved across RNR species. Regardless of their exact roles in S. cerevisiae RNR, there are a number of studies that support the importance of these residues  to Ala is lethal, and mutation of Gln288  to Ala yields a severe S phase defect ). The pH of each solution was slowly adjusted to 7–8 with NaOH, and the nucleotide concentrations were determined spectroscopically, using ε271 of 9.1 mM-1 cm-1 for CDP, ε259 of 15.4 mM-1 cm-1 for ADP, ε262 of 10.0 mM-1 cm-1 for UDP, ε253 of 13.7 mM-1 cm-1 for GDP, ε259 of 15.4 mM-1 cm-1 for ATP, ε259 of 15.2 mM-1 cm-1 for dATP, ε253 of 13.7 mM-1 cm-1 for dGTP, and ε262 of 9.6 mM-1 cm-1 for TTP. Preparation of nucleotides for crystallography was the same as described above except that 100 mM solutions of sodium salts of dATP, dGTP, and TTP were purchased from USB Corporation  or Invitrogen .For the enzyme assays, sodium salts of CDP, ADP, UDP, GDP, dATP, dGTP, and TTP were purchased from Sigma-Aldrich  and dissolved into assay buffer . For all structures, hydroxyurea-inactivated β2 (met-β2) was used in place of active β2. Met-β2 was prepared from purified active β2 as described  isopropyl-β-thiogalactopyranoside) with 1 μL of precipitant solution and equilibrating over a reservoir of 500 μL of precipitant at room temperature (~25°C). For the CDP/dATP structure, the precipitant solution was 9.5% (w/v) PEG 3350, 100 mM MOPS pH 7.5, 250 mM Mg(CH3COO)2, 25 mM MgCl2, and 5% (v/v) glycerol. For the UDP/dATP, ADP/dGTP, and GDP/TTP structures, the precipitant solution was 12% (w/v) PEG 3350, 100 mM MOPS pH 7.5, 300 mM Mg(CH3COO)2, 30 mM MgCl2, and 5% (v/v) glycerol. Freshly prepared drops were streak seeded with microcrystals grown under the same conditions with the exception of the MgCl2 concentration in the precipitant being 100 mM MgCl2. Streak seeding was used to obtain larger single crystals.Crystals were grown using the hanging drop vapor diffusion technique by mixing 1 μL of protein 2, 5% (v/v) glycerol, 5 mM DTT, and 10 mM CDP. For the GDP/TTP, UDP/dATP and ADP/dGTP structures, the soaking solutions were the same as for CDP except for having 13% (w/v) PEG 3350, and each nucleotide at 10 mM. Crystals were left in the soaking solution for 2 min and then cryoprotected. CDP-soaked crystals were looped through a solution of 12% (w/v) PEG 3350, 100 mM MOPS pH 7.5, 250 mM Mg(CH3COO)2, 60 mM MgCl2 and 10%, 15%, and 20% (v/v) glycerol in succession and then plunged directly into liquid N2. GDP/TTP, UDP/dATP, and ADP/dGTP soaked crystals were looped through a solution of 14% (w/v) PEG 3350, 100 mM MOPS pH 7.5, 300 mM Mg(CH3COO)2, 100 mM MgCl2, and 10%, 15%, and 20% (v/v) glycerol in succession and then plunged directly into liquid N2.After 2 days of growth, crystals were transferred to a drop of soaking solution. For the CDP/dATP structure, dATP was co-crystallized, and CDP was soaked into the structure using a solution containing 10.5% (w/v) PEG 3350, 100 mM MOPS pH 7.5, 25 mM MgClDiffraction data were collected at the Advanced Photon Source at Argonne National Laboratory. The CDP/dATP data set was collected on beamline 24ID-C at 100 K on an ADSC Q315 detector. The UDP/dATP, ADP/dGTP, and GDP/TTP data sets were collected on beamline 24ID-C at 100 K on a Pilatus 6M detector. All data were processed using HKL2000  Table 2Table 2.4β4 complex structures soaked with substrates and effectors were solved to the full extent of the data resolution using the previously published 3.95-Å resolution structure . Residues 4 to 737 of chain A from each structure were used for the analysis.2 and Arg298Ala-α2 were constructed from the previously published vector pET-nrdA  were carried out as previously described , and their concentrations were determined using ε280 of 189 mM-1cm-1. Hexahistidine tags were not removed since previous studies showed that these tags on E. coli α2 do not significantly alter activity  after drop-line subtraction of the diferric cluster absorbance  and E. coli thioredoxin (Trx) were prepared. The gene for TrxR was amplified from E. coli genomic DNA using primers with ends suitable for a second round of PCR to generate the plasmid borne gene in the pRham SUMO fusion vector . The primers, ordered from Integrated DNA Technologies, were: forward 5’-CGCGAACAGATTGGAGGTGGCACGACCAAACACAGTAAACTG-3’, and reverse 5’-GTGGCGGCCGCTCTATTATTTTGCGTCAGCTAAACCATCGAG-3’. DNA sequencing performed by Genewiz was used to confirm the sequence of the resulting construct. This construct contains a hexahistidine tag and SUMO protein fusion at the N-terminus under a rhamnose promoter. The resulting protein was expressed according to the plasmid manufacturer protocol (Lucigen) and purified as described above for hexahistidine-tagged α2, using the same buffers. The amount of flavin cofactor was quantified by absorption at 440 nm    . The fini strain . E. coliescribed .2 and the Gln294Ala and Arg298Ala mutants were performed using a continuous, coupled, spectrophotometric assay monitoring the consumption of NADPH by the Trx/TrxR system , 1 mM CDP and 175 μM dATP (control for inactivation by dATP), 1 mM ADP and 120 μM dGTP, 1 mM GDP and 250 μM TTP, and 1 mM CDP/UDP and 1 μM dATP. Substrate, effector, E. coli Trx (30 μM), E. coli TrxR (0.5 μM), and NADPH from Sigma-Aldrich (200 μM) were mixed in assay buffer, and the reaction was initiated by the addition of α2 (0.1 μM) and wild-type β2 (1 μM) to a final volume of 120 μL. The decrease in NADPH absorbance at 340 nm was monitored over 1 min. The basal level of NADPH oxidation was monitored over 30 s prior to the addition of enzyme.Activity assays for wild-type αR system . All exp


Trichuris muris. Classically activated Mφs predominate initially (as the inflammation develops) and then, following worm expulsion (as the inflammation resolves), both the resident and inflammatory populations of Mφs become alternatively activated. A small but significant increase in the proliferation of inflammatory Mφs is seen but only during the resolution phase of the inflammatory response following both worm expulsion and the peak in Mφ accumulation. In contrast to recent studies in the pleural and peritoneal cavities, the proliferation of resident and alternatively activated Mφs does not increase during the inflammatory response. Furthermore, in CCR2−/− mice, monocyte recruitment to the gut is impeded, and the accumulation of alternatively activated Mφs is greatly reduced. In conclusion, the recruitment of blood monocytes is the principle mechanism of Mφ accumulation in the large intestine. This study provides a novel insight into the phenotype and behavior of intestinal Mφ during infection-driven inflammation.Macrophages (Mφs) accumulate at sites of inflammation, and, because they can assume several functionally distinct states of activation, they can either drive or restrain inflammatory responses. Once believed to depend on the recruitment of blood monocytes, it is now clear that the accumulation of Mφs in some tissues can result from the proliferation of resident Mφs in situ. However, little is known about the proliferation and activation state of Mφ subsets in the gut during the development and resolution of intestinal inflammation. We show that inflammatory Mφs accumulate in the large intestine of mice during the local inflammatory response to infection with the gastrointestinal nematode parasite  Macrophages (Mφs) are mononuclear phagocytes of the innate immune system and are involved in host-defense, metabolism, and the homeostatic regulation of healthy tissues. Playing diverse and contrasting roles, Mφs can initiate, amplify, and regulate the adaptive immune system and both drive and resolve inflammatory responses. The gut is the largest reservoir of Mφs in the body , and intMφs can assume several functionally different states of activation that are regulated by the prevailing cytokine milieu and other factors that are present at sites of inflammation. Mφs respond to IFN-γ, with or without LPS, to become classically activated , 4. Clasgfp/+ transgenic mice, which express eGFP under the control of the CX3CR1 promoter  Act 1986 (as amended).Specific pathogen-free AKR, BALB/c, and C57BL/6 mice were purchased from Harlan. CX3CR1T. muris was maintained as described previously (T. muris excretory/secretory (E/S) Ags were prepared by culturing adult worms in vitro at 37°C for 4 h  or a high-level infection (200 eggs given).The E strain of eviously . T. muri for 4 h . T. muriT. muris E/S Ags for 48 h as previously described (Mesenteric lymph node (MLN) cells were cultured and stimulated with 50 μg/ml escribed . The culA Cytometric Bead Array kit  was used in accordance with the manufacturer’s instructions to assay cytokines using an LSR II flow cytometer (BD Biosciences).Lamina propria leukocytes (LPLs) were isolated from the proximal colon and cecum by enzymatic digestion as previously described , 15.1, S, G2, and M) but is absent from quiescent cells (G0).Two approaches were taken to measure proliferation. Firstly, mice were injected i.p. with BrdU, which is incorporated into the newly synthesized DNA of replicating cells during the S phase of the cell cycle. The mice were killed 4 h later, and an Ab was used to detect the BrdU in the DNA of Mφs by flow cytometry (as described next). Secondly, an Ab was used to measure Ki-67 in Mφs by flow cytometry. This nuclear protein regulates cell division and is present during all active phases of the cell cycle  and then incubated with rat, anti-mouse CD16/32 mAb  for 30 min on ice to block FcR. The cells were then stained with the following Abs to extracellular markers for 30 min on ice: PE rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 hamster, anti-mouse CD11c mAb (eBioscience), allophycocyanin–eFluor 780 rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), biotin rat, anti-mouse MHC class II (I-A/I-E) mAb (eBioscience) used in conjunction with PE-Vio770 mouse, anti-biotin mAb , and VioGreen rat, anti-mouse CD45 mAb; or PE rat, anti-mouse CD103 mAb (BD Biosciences), PE rat, anti-mouse Siglec-F mAb (BD Biosciences), PE rat, anti-mouse Ly6G (BD Biosciences), FITC rat, anti-mouse CD11b mAb (eBioscience), PerCP-Cy5.5 rat, anti-mouse F4/80 mAb (eBioscience), Alexa Fluor 700 rat, and anti-mouse CD45 mAb (eBioscience). To detect live and dead cells, a Live/Dead Fixable Dead Cell Stain Kit (the Blue Fluorescent Reactive Dye version) was used according to the manufacturer’s instructions (Life Technologies). An allophycocyanin BrdU Flow kit was then used according to the manufacturer’s instructions to detect BrdU that had been incorporated into the cells (BD Biosciences). As recommended, the staining of intracellular proteins was performed at the same time using the following Abs: eFluor450 rat, anti-mouse Ki-67 mAb (eBioscience) and rabbit, anti-mouse RELMα polyclonal Ab  used in conjunction with Qdot 605 donkey, anti-rabbit IgG (Life Technologies). To precisely control the gating for the staining of Ki-67, BrdU, and RELMα, the following control Abs were used in parallel for each mouse: rat IgG2a eFluor 450 isotype control (eBioscience), rat IgG1 allophycocyanin isotype control (eBioscience), and rabbit IgG control , respectively.The LPLs were washed in Flow Cytometry Buffer , and for iNOS and RELMα, we used biotin goat, anti-rabbit IgG F(ab′)2 . The appropriate isotype control mAbs or polyclonal control IgGs were used in parallel sections. The color development was monitored and was stopped, by washing in PBS, before any false-positive staining occurred in the isotype control sections. The sections were counterstained in Haematoxylin QS (Vector Laboratories). After randomization and blinding of the slides, the number of positively stained cells was determined in each section by light microscopy. The staining was performed in triplicate, and all of the positively stained cells in each section were counted .Immunohistochemistry was performed on frozen cross-sections of proximal colon using standard immunoperoxidase techniques as described previously . The folStatistical analysis was performed by the Kruskal–Wallis test with Dunn’s posttest .T. muris, leukocytes accumulated in the large intestine. There was a significant increase in the number of both Mφs and Th cells in the proximal colon 21 d postinfection, and ∼80% of the leukocytes were Mφs . Eosinophils are known to express F4/80 as well as Mφs. However, because Siglec-F+ cells were much less abundant than F4/80+ cells, only a small fraction of the F4/80+ cells were potentially eosinophils.The detection of CD45, F4/80, and CD4 by immunohistochemistry allowed the number of leukocytes, Mφs, and Th cells, respectively, to be quantified in the proximal colon of C57BL/6 mice. In uninfected mice, >90% of the leukocytes were Mφs . Followiwere Mφs . Similarwere Mφs . EosinopT. muris. MLN cells from C57BL/6 mice released high levels of all four cytokines after day 21 postinfection . In BALB/c and C57BL/6 mice, the number of M1s reached a peak 21 d postinfection and then subsequently decreased. In contrast, in AKR mice, the M1s emerged later and they persisted  eosinophil-like polymorphonuclear leukocytes were also observed  from the subsequent analysis. The F4/80analysis . ParadoxT. muris , infecteT. muris , the immThe marker RELMα was chosen for the analysis of M2s by flow cytometry because it exhibited similar staining profiles to Arginase-1 and Ym1, yet it revealed the greatest differences between uninfected and infected mice . In all + DCs also expressed RELMα in all three strains of mouse, and there was a small but significant increase in the relative percentage of these RELMα+ DCs 42 d postinfection in C57BL/6 and BALB/c mice . Approximately 5% of eosinophils also expressed RELMα, but there was no significant change postinfection .A minor fraction of CD103gfp/+ mice (on a C57BL/6 background), three distinct populations of CD11b+ leukocytes were identified by their differential expression of eGFP . Se. Segfp/+f CX3CR1 , which wf CX3CR1 . The vasf CX3CR1  was prevT. muris  and coulT. muris  . The relative percentage of monocytes (P1), immature inflammatory Mφs (P2), mature inflammatory Mφs (P3), and resident Mφs (P4) expressing RELMα was analyzed. Furthermore, to establish whether a Th2 response and worm expulsion was required for the accumulation of M2 in the large intestine, two disparate strategies of T. muris infection were employed: firstly, the familiar high-level infection protocol that resulted in a mixed Th1/Th2 response and worm expulsion [with the same kinetics that was observed for wild-type (WT) C57BL/6 mice  expressed the M2 marker RELMα , 5E. In + DCs (P5) expressed RELMα, but there was no significant difference postinfection .A minor fraction of CX3CR1+ Mφs also expressed Ki-67 . Therefore, a small number of Mφs proliferated in the large intestine in its resting state. Postinfection no significant increase in the relative percentage of BrdU+ or BrdU+Ki-67+ Mφs was detected , suggesting that proliferation may contribute to the accumulation of this subpopulation of Mφs in the large intestine postinfection . However, in contrast to peritoneal and pleural cavity  were resistant to a high-level infection with T. muris (not shown) as reported previously  and then predominate (after worm expulsion). Indeed, a similar transition from M1s to M2s has been observed following infections with parasites as diverse as ngolense , 37, andngolense . This rengolense . Howeverhigh resident Mφs are the predominant population in the uninfected large intestine, although CX3CR1int inflammatory Mφs are also encountered (T. muris, inflammatory Mφs accumulate in the large intestine and become more prevalent than resident Mφs. Importantly, we show for the first time, to our knowledge, that both inflammatory and resident Mφs become alternatively activated following infection with a gastrointestinal nematode. Furthermore, both inflammatory and resident Mφs remain alternatively activated for several weeks after the immunopathology appears to have subsided. That inflammatory Mφs can be alternatively activated reveals an interesting and far-reaching paradox because inflammatory Mφs are thought to amplify inflammation, whereas M2s are implicated in the resolution of inflammation (Using published approaches to define monocyte and Mφ subsets by multiparameter flow cytometry , 16, 26,ountered , 15. Posammation , 39.highCX3CR1low inflammatory monocytes (T. muris infection, the accumulation of Mφs in the large intestine does not coincide with an increase in the proliferation of resident Mφs. Furthermore, the inhibition of monocyte recruitment greatly impedes the accumulation of Mφs in the gut. Therefore, monocyte recruitment is the principle mechanism of Mφ accumulation during the development of the inflammatory response to T. muris. Nevertheless, following worm expulsion, there is a small but significant increase in the proliferation of inflammatory Mφs. Given that, in the large intestine, resident Mφs are derived from inflammatory Mφs (The proliferation of resident Mφs drives the accumulation of Mφs in the pleural and peritoneal cavities following infection with filarial nematode parasites , 19, andonocytes , 15, 17,tory Mφs , the incT. muris. Furthermore, we show that the accumulation of M2s in the large intestine is greatly reduced by disrupting monocyte recruitment to the gut. This is consistent with previous work showing a CCR2-dependent mechanism underlying the recruitment of Ly6ChiCCR2hiCX3CR1lo blood monocytes to the colon during an inflammatory response (Schistosoma mansoni. Taken together, it is becoming clear that the mechanisms that underlie the accumulation of M2s following infection with parasitic nematodes are either tissue specific or parasite species specific.Importantly, we reveal that the vast majority of M2s do not proliferate in the large intestine at any stage either before or postinfection with response , 15, 17.response , 19, in response  on the aIn summary, this study reveals the dynamic changes that take place to the phenotype of Mφ subsets during the initiation, amplification, and resolution of intestinal inflammation. We describe the emergence of M1s during worm infection and M2s following worm expulsion. However, in contrast to previous studies , 19, in


Purpose: Risk of aortic rupture is evaluated based on the vessel diameter; this parameter is probably insufficient. In vivo evaluation of biomechanical property of the aortic tissue might be of interest to discriminate between normal and altered aortic tissue . The purpose of this study was to describe a technique to measure regional aortic compliance (AC), aortic distensibility (AD) and aortic stiffness with 1.5 T MRI in young individuals under resting conditions and during supine bicycle exercise.Materials and Methods: Fiftheen young adults  yrs) with no risk factor for atherosclerosis were recruited. They all underwent 1.5T MRI  at rest and during supine exercise using an amagnetic ergometer . The following parameters were used for rest and exercise: cine = TR/TE = 30 ms/1.8 ms, thickness = 6 mm, α = 65°, matrix = 148 × 256, resolution = 30 ms, retrospective gating; Phase contrast = TR/TE = 25 ms/2 ms, thickness = 6 mm, matrix = 256 × 256, flow encoding = 200 cm/s, α = 25°, resolution = 25 ms, retrospective gating). For exercise the volunteer was instructed to cycle for 2 min at 25W, work load was increased each 2 min for a minimum of 10 min, to obtained twice the resting heart rate. The volunteer was instructed to stop cycling and immediately following which a breath held stress dataset of 2 acquisitions were acquired. The volunteer was instructed to cycle for at least 2 min at the last exercise level, and the images acquisition was repeated. AC and AD were determined from cine-MRI at four locations, ascending aorta (AA), proximal descending aorta (PDA), distal descending aorta (DDA), and aorta above renal artery (RA). Segmental aortic pulse wave velocity (PWV) was assessed by phase contrast.Results All volunteer complete the whole protocol. Stress induced a significant decrease of AC and AD at all sites p <10-3). At rest and during stress, AC was statistically higher in AA compared to the whole descending aorta (p ≤0.0007). We found a strong correlation between the rate pressure product and AC at all sites (Figure 0-3. At rConclusion This study demonstrates the feasibility to analyze regional aortic function during an exercise-induced stress MRI. Further studies are required to evaluate its interest to detect AA's dysfunction among patients with thoracic aortic diseases.Not applicable.


Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1–200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure–function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in  Hantaviruses may cause severe disease in humans, either hemorrhagic fever or cardiopulmonary syndrome. Both conditions are associated with high lethality. Vaccines and effective treatments approved for application in humans are not available. The L protein plays a central role in hantavirus replication and represents a key target for antiviral drug development. However, the protein is difficult to study, as it can hardly be expressed in cells, and therefore structure and function are largely unknown. We succeeded to express and purify the N-terminal domain of ANDV, a South American hantavirus, in mutant forms. The atomic structure of the domain was solved by X-ray crystallography. The structural data in conjunction with our enzymatic activity data demonstrate that the N-terminal part of ANDV L protein harbors an endonuclease that cleaves RNA, presumably to “steal” cap structures from cellular mRNA for virus transcription. The investigation of a range of mutant enzymes revealed the detailed role of amino acid residues within the endonuclease. Importantly, using a known inhibitor of viral endonucleases we demonstrate the potential of the ANDV enzyme as an antiviral target. The discovery of a highly active mutant form of the endonuclease, which can be expressed at high level, will facilitate drug discovery programs for hantaviruses. Hantavirus within the family Bunyaviridae. Hantaviruses can be pathogenic to humans and are distributed worldwide [Oligoryzomys longicaudatus) [Andes virus (ANDV) belongs to the genus orldwide –3. Theirorldwide . ANDV isaudatus) . The viraudatus) , 7.The hantavirus genome consists of three RNA segments: L (large), M (medium), and S  . The L sOrthomyxoviridae) [Orthobunyavirus, family Bunyaviridae), Lassa virus, and lymphocytic choriomeningitis virus (both family Arenaviridae) [The 5’ ends of hantavirus mRNAs contain a stretch of heterologous nucleotides suggesting that the virus uses a mechanism called ‘cap-snatching’ to initiate transcription of its mRNAs , 13. Capviridae) . Here, tviridae) –19. In oviridae) –23. An eviridae) –35.Investigation of L protein of ANDV and other hantaviruses has been challenging, because the protein is difficult to express in heterologous systems , 37. We The aim of the current study was to provide proof for the existence of a cap-snatching endonuclease in ANDV L protein using a structural and biochemical approach. We took advantage of the previously characterized mutations enhancing the L protein level in mammalian cells to facilitate expression of the endonuclease domain in bacteria.E. coli. To this end, plasmids for expression of the N-terminal 163, 179, 191, 194, 197, 200, 211, 214, and 228 amino acid residues of wild-type ANDV L protein were generated using E. coli cloning strain DH5α. To facilitate purification, the expression constructs contained an N-terminal His-tag or His-tag plus maltose-binding protein (MBP), respectively. However, we encountered difficulties in expressing the L protein fragments in bacteria, as observed previously in mammalian cells [E. coli with constructs containing 200 or more residues failed repeatedly, suggesting that background expression of soluble wild-type endonuclease is already toxic to the cells. To circumvent this problem, we took advantage of 15 amino acid exchanges that enhanced L protein expression in mammalian cells, presumably because they attenuate the enzymatic activity of the endonuclease [1–200) and the mutated constructs were tested for expression in E. coli BL21 cells. The results are summarized in 1–200 Y32V and D37A) showed a phenotype like the wild-type protein, i.e. no bacterial growth was observed. The remaining 13 mutants  were successfully expressed with varying yield and purified via nickel affinity and size exclusion chromatography . The mutant was expressed as His-tag fusion of variable lengths  and subjected to crystallization trials. We succeeded to crystallize ANDV L1–200 K127A in the presence of Mn2+ after cleavage of the His-tag. The crystal structure was solved in space group P4212 by molecular replacement using coordinates for residues 32–162 from the endonuclease of Hantaan virus  of ANDV endonuclease and LACV, LCMV and IAV endonucleases are 1.7 Å, 1.9 Å and 2.2 Å, respectively ..Fig 3). His36, Asp97, and Glu110 (group 1) are the central catalytic residues coordinating the manganese ion Figs  and 3. Am) as a measure of stability varies from 34.2°C for the least stable L1–200 I43A mutant to 49.2°C for the most stable L1–200 K124A mutant  abolishes this stabilizing effect. Interestingly, increasing the manganese concentration to 16 mM led to a substantial additional Tm increase of up to 8°C for some mutants, suggesting binding of another metal ion by the active site  RNA molecule was used as substrate, and the level of activity was measured by substrate degradation. None of the mutants was active in the absence of divalent ions. Addition of manganese rescued activity for some mutants , while manganese . Mutatioanganese . The mos1–200 N167A with high activity, L1–200 K44A with intermediate activity, and L1–200 D97E as a negative control. First, four different substrates were tested: a 27mer ssRNA that can form secondary structures (predicted hairpin Tm = 77°C), the same sequence as double stranded (ds) RNA, and the corresponding ssDNA and dsDNA molecules  did not show background degradation, demonstrating that our purification procedure removes potentially contaminating bacterial nucleases.To investigate substrate specificity of the ANDV endonuclease, we used three mutants with a range of enzymatic activity: Lolecules . ANDV L1d length . Compari1–200 mutants H36R, K44A, N50A, D97E, E110A, K125A, K127A, and N167A in the presence of 16 mM MnCl2. All mutants that showed a stabilization of > 8°C in the presence of manganese, also showed a further increase in the melting temperature upon addition of DPBA  seem to slightly destabilize the protein, which might affect the enzymatic activity of the enzyme. The corresponding wild-type residues are involved in hydrogen bonds with other side or main chain atoms or in hydrophobic interactions with neighboring secondary structure elements. Consistent with the structural data, most of these mutants are characterized by low temperature stability and high residual enzymatic activity. Residue Asn167 is exceptional as it is located distant to the active site. Via hydrogen bonds with β-sheet βc it positions a small helix (αe) that is not present in the otherwise similar LACV protein. Consistent with the peripheral location of Asn167, the corresponding mutant is the least attenuated. Its wild-type like features include the highest enzymatic activity and a reduced growth rate of the expressing The biochemical data also revealed a clear co-factor and substrate specificity of the ANDV endonuclease. Consistent with its presumed function as a cap-snatching enzyme, it preferred ssRNA over dsRNA substrates and was inactive on ssDNA or dsDNA. RNA length and secondary structure seem to influence substrate binding. Dependence of enzymatic activity from manganese rather than magnesium has also been observed for the endonucleases of the other segmented negative strand viruses , 20, 21.1–200 N167A mutant is the most promising enzyme candidate for this purpose.This study also has implications for development of antiviral drugs to treat hantavirus infections. The influenza virus endonuclease has been used as a target in antiviral drug discovery programs and several specific inhibitors have been found –35. LikeE. coli strain DH5α was used. The sequence of the inserts was confirmed by sequencing.The cDNA of fragments encoding residues 1–163, 1–179, 1–191, 1–194, 1–197, 1–200, 1–211, 1–214 and 1–228 from the L protein of ANDV  were amplified by PCR using the pCITE-ANDV-L plasmid  as a temE. coli strain BL21 Gold (DE3) (Novagen) at 17°C overnight using LB medium and 0.5 mM isopropyl-β-D-thiogalactopyranosid for induction. Cells were pelleted, resuspended in lysis buffer containing 50 mM Tris-HCl pH 7.3, 300 mM NaCl, 10 mM imidazole, 10% glycerol, 10 mM MnCl2, and 1 mM phenylmethylsulfonyl fluorid and disrupted by sonication. The soluble material was loaded onto a Ni-NTA affinity column, washed with 10 column volumes of lysis buffer containing 50 mM imidazole, and eluted with 5 volumes of lysis buffer containing 500 mM imidazole. The His-tag of eluted protein was cleaved off using glutathione S-transferase-tagged 3C protease at 4°C overnight during dialysis against lysis buffer. The untagged proteins were further purified by size exclusion chromatography using a Superdex 200 column in 50 mM sodium citrate pH 5.5, 1 M NaCl, 5% glycerol. Purified proteins were concentrated using centrifugal devices, flash frozen in liquid nitrogen, and stored in aliquots at –20°C.Proteins were expressed in 1–200 K127A (10 mg/ml) was crystallized in the presence of 2 mM MnCl2, 100 mM sodium acetate pH 5.0, 27.5% polyethylene glycol 3350, and 500 mM (NH4)2SO4 by sitting drop vapor diffusion. Crystals were flash frozen in liquid nitrogen with 8% butanediol as cryo protectant and diffraction data were collected to 2.4 Å at beamline ID23-1 at the European Synchrotron Radiation Facility, Grenoble, with a wavelength of 0.98 Å and 1.77 Å for respectively native and anomalous data. Datasets were processed with iMosflm [12 by molecular replacement using residues 32–162 from the endonuclease from Hantaan virus [ANDV L iMosflm . The cryan virus  and PHASan virus , and refan virus  and compan virus . TLS refan virus  with thean virus . Electroan virus , 50. The1–200 K127A in 2 mM MnCl2, 50 mM Tris-HCl pH 7.3, 250 mM NaCl, 5% glycerol were collected at the SAXS beamline P12 at the PETRA III storage ring of the Deutsches Elektronen-Synchrotron, Hamburg [–1 < s < 0.45 Å–1 was covered . Data were analyzed using the ATSAS 2.6 package [g were extracted from the Guinier approximation calculated with the AutoRG function within PRIMUS [max. Ab initio reconstructions were generated with the program DAMMIF [Small angle X-ray scattering (SAXS) data for 1, 2, and 5 mg/ml of ANDV L Hamburg . Using a package . The forn PRIMUS . GNOM [5n PRIMUS  providedm DAMMIF . Ten indm DAMMIF  and averm DAMMIF .1–200 protein with 0.1 μM 32P-labeled single stranded (ss) or double stranded (ds) 27mer RNA or DNA substrate (5’-GAU/TGAU/TGCU/TAU/TCACCGCGCU/TCGU/TCGU/TC-3’) or 40mer ssRNA substrate (5´-GAUGAUGCUAUCACCGCGCUCGUCGUCGAUGAUGCUAUCA-3’) in 50 mM Tris-HCl pH 7.3, 250 mM NaCl, 5% glycerol, 0.25 U/μl RNasin (Promega) in the absence or presence of 2 mM MnCl2 at 37°C for 1 or 2 h. RNA was annealed by heating to 98°C for 2 minutes, followed by a slow cooling over 2 hours to room temperature. A 27 or 40mer poly(A) ssRNA was used as unstructured substrate. In selected experiments, 2,4-dioxo-4-phenylbutanoic acid (DPBA) was added to the assay. The reaction was stopped by adding 2 × loading buffer , 0.025% sodium dodecyl sulfate, xylene cyanol, and bromophenol blue) and heating the samples to 98°C for 5 min. The reaction products were separated by 8 M urea, 20% polyacrylamide, Tris-borate-EDTA gel electrophoresis and visualized by phosphor screen autoradiography using a Typhoon scanner . Intensity of the signals was quantified by ImageJ software [Nuclease activity was measured by incubating 1 μM ANDV Lsoftware .1–200 protein was measured by thermofluor assay at a protein concentration of 10 μM in 100 mM Tris pH 7.0, 250 mM NaCl, 5% glycerol complemented with either 10 mM EDTA, 4 mM MnCl2, 16 mM MnCl2, 16 mM MgCl2 or 16 mM MnCl2 plus 100 μM DPBA as described [The stability of ANDV Lescribed .S1 Fig1–200 K127A had been cleaved off for crystallization experiments. All other proteins feature an N-terminal His-tag.Following purification, 1 μg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with Coomassie. The His-tag of ANDV L(PDF)Click here for additional data file.S2 FigThe stability of the purified protein was tested in thermofluor assay under various conditions. The protein is most stable at lower pH and higher salt concentrations.(PDF)Click here for additional data file.S3 Fig2 –values of fits are given for each structure, curves were calculated with CRYSOL [Experimental scattering curves (black dots) are compared with theoretical scattering curves for the ANDV crystal structure (black line), the LACV crystal structure (blue line), the LCMV crystal structure (green line) and the IAV crystal structure (orange line). χh CRYSOL .(PDF)Click here for additional data file.S4 Fig1-200 N167A shows no accumulation of larger intermediate RNA products with specific lengths.Degradation of unstructured polyA RNA by ANDV L(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Yarrowia lipolytica has six JEN genes: YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D20108g, YALI0D24607g, and YALI0E32901g. Through phylogenetic analyses, we found that these genes represent a new subfamily, Jen3 and that these three Jen subfamilies derivate from three putative ancestral genes. Reverse transcription-PCR. revealed that only four YLJEN genes are expressed and they are upregulated in the presence of lactate, pyruvate, fumarate, malate, and/or succinate, suggesting that they are able to transport these substrates. Analysis of deletion mutant strains revealed that Jen3 subfamily proteins transport fumarate, malate, and succinate. We found evidence that YALI0C15488 encodes the main transporter because its deletion was sufficient to strongly reduce or suppress growth in media containing fumarate, malate, or succinate. It appears that the other YLJEN genes play a minor role, with the exception of YALI0E32901g, which is important for malate uptake. However, the overexpression of each YLJEN gene in the sextuple-deletion mutant strain ΔYLjen1-6 revealed that all six genes are functional and have evolved to transport different substrates with varying degrees of efficacy. In addition, we found that YALI0E32901p transported succinate more efficiently in the presence of lactate or fumarate.Jen proteins in yeast are involved in the uptake of mono/dicarboxylic acids. The Jen1 subfamily transports lactate and pyruvate, while the Jen2 subfamily transports fumarate, malate, and succinate.  Saccharomyces cerevisiae  are (1) regulated through a complex pathway which involves different stages of expression ; (2) strain-dependent; and (3) strongly affected by the carbon source present, especially in the case of glucose , YALI0C15488p (YLJen1p), YALI0C21406p (YL-Jen2p), YALI0D20108p (YLJen3p), YALI0D24607p (YLJen4p), and YALI0E32901p (YLJen6p). Phylogenetic analyses by Lodi et al.  and JEN1.The genome of i et al.  revealedY. lipolytica share a conserved motif in the seventh TM, NXX(S/T)HX(S/T)QDXXXT, which is involved in transport ability and substrate affinity  strain (Table− Ura−) has been previously described by Barth and Gaillardin . During the experiment, this minimal medium was supplemented with uracil (0.1 g/L) and/or leucine (0.1 g/L) if necessary. To test growth on different carbon sources, we used the following medium: 0.17% (wt/vol) yeast nitrogen base , 0.5% (wt/vol) NH4Cl, (Difco), and 50 mmol/L phosphate buffer (pH 6.8). For growth and consumption tests, a carbon source was added at 0.15–0.3% (wt/vol). Solid media were obtained by the addition of 1.6% agar. Y. lipolytica precultures were grown in minimal medium to avoid stress on strains that would be used in phenotype tests.The ain Table. The auxillardin . The proo et al. . All of k et al. , and thoillardin . Rich meillardin . MinimalStandard molecular genetic techniques were used throughout this study . The auxotrophic marker (either URA3ex or LEU2ex) was then inserted into the vector IsceI site to generate the corresponding JMP (UpDnURA3ex/LEU2ex) vectors  were then introduced at the I-sceI site, yielding the constructs JMP1667 and JME1680, which respectively contained the YALI0B19470g::URA3ex and YALI0B19470g::LEU2ex cassettes. The deletion cassettes used in this study were obtained by digestion of these plasmids by NotI.For sed Table. The Up YALI0C15488g disruption, primer pairs C15488UpNotI/C15488UpIsceI and C15488DnNotI/C15488DnIsceIIceuI were used (TableURA3ex and LEU2ex markers (from JMP802 and JMP803) were then introduced at the I-sceI site, yielding the constructs JMP1671 and JME1682, which respectively contained the YALI0C15488g::URA3ex and YALI0C15488g::LEU2ex cassettes. Deletion cassettes were obtained by digestion of these plasmids by NotI.For sed Table. The Up YALI0C21406g disruption, primer pairs C21406UpNotI/C21406UpIsceI and C21406DnNotI/C21406Dn2IsceIIceuI were used (TableURA3ex marker (from JMP803) was then introduced at the I-sceI site, yielding the construct JMP1698, which contained the YALI0C21406g::URA3ex cassette. Deletion cassettes were obtained by digestion of these plasmids by NotI.For YALI0D20108g disruption, primer pairs D20108UpNotI/D20108UpIsceI and D20108DnNotI/D20108Dn2IsceIIceuI were used (TableURA3ex marker (from JMP803) was then introduced at the I-sceI site, yielding the construct JMP1702, which contained the YALI0D20108g::URA3ex cassette. Deletion cassettes were obtained by digestion of these plasmids by NotI.For YALI0D24607g disruption, primer pairs D24607UpNotI/D24607UpIsceI and D24607DnNotI/D24607Dn2IsceIIceuI were used (TableLEU2ex marker (from JMP802) was then introduced at the I-sceI site, yielding the construct JMP1813, which contained the YALI0D24607g::LEU2ex cassette. Deletion cassettes were obtained by digestion of these plasmids by NotI.For YALI0E32901g disruption, primer pairs E32901UpNotI/E32901UpIsceI and E32901DnNotI/E32901Dn2IsceIIceuI were used (TableURA3ex and LEU2ex markers (from JMP802 and JMP803) were then introduced at the I-sceI site, yielding the constructs JMP1713 and JME1715, which respectively contained the YALI0E32901g::URA3ex and YALI0E32901g::LEU2ex cassettes. Deletion cassettes were obtained by digestion of these plasmids by NotI.For YLJEN1-6 genes was performed using the respective primer pairs StartB19470/EndB19470, StartC15488/EndC15488, StartC21406/EndC21406, StartD20108/EndD20108, StartD24607/EndD24607, and StartE32901/EndE32901. All complementation genes were cloned into pCR4Blunt-TOPO and then digested by AvrII and BamHI. They were then cloned into JMP1392 that had been previously digested by BamHI and AvrII; this generated JMP1792, JMP1794, JMP1821, JMP1809, JMP1796, and JMP1798, respectively.Complementation of the sextuple mutant with individual Construction of deletion strains was controlled by PCR using Ver1 and Ver2 primers . They were then resuspended into 1 mL of YNB N and P. Washed cells were then sown into 96-well microplates with adapted medium at OD600 = 0.1 in 200 μL of final volume. Growth was observed using a microtiter plate reader , following the manufacturer's instructions.YLJEN genes, precultures of the reference strain JMY2900 were established in liquid YNB supplemented with 1% glucose and 0.5% yeast extract; cultures grew for 15 h at 28°C. Cells were then washed with distilled water and transferred into fresh liquid YNB media supplemented with one of the following: 1% glucose, 0.3% lactate, 0.3% fumarate, 0.3% malate, 0.3% pyruvate, or 0.3% succinate. Cultures were incubated in baffled Erlenmeyer flasks at 28°C and 160 rpm, then harvested at 2 and 6 h postinoculation, frozen in liquid nitrogen, and stored at −80°C. RNA was extracted from cells using the RNeasy Mini kit (Qiagen), and 2 μg were treated with DNAse . cDNA was synthesized using the Maxima First Strand cDNA synthesis kit . PCR was then performed using the GoTaq DNA polymerase kit (Promega) with specific primers designed by the Primer3 program and listed in TableμL of cDNA.To determine if carbon sources induced the expression of Y. lipolytica strain precultures were grown overnight, then centrifuged and washed two times with YNB supplemented with NH4Cl and phosphate buffer (YNB N and P). They were then resuspended in 1 mL of YNB N and P. Cultures were inoculated at OD600 nm = 0.1 in 50 mL of YNB medium supplemented with 50, 100, or 200 mmol/L of phosphate buffer (pH = 6.8) depending of the quantity of substrate  and 170 mmol/L each of fumarate, lactate, malate, and succinate, then incubated at 28°C with agitation (160 rpm). To analyze the consumption of carbon sources, a sample of each culture was centrifuged for 1 min at 13 000 rpm; the resulting supernatant was analyzed with HPLC  using a HyperREZ XP carbohydrate H+ 8-μm column  coupled to a UV (210 nm) detector. The column was eluted with 0.01 N H2SO4 at room temperature and a flow rate of 0.6 mL min−1. Standard samples were used to determine the quantity of each compound. Prior to HPLC analysis, samples were filtered on 0.45-μm pore-size membranes.S. cerevisiae – SGD (http://www.yeastgenome.org/); C. albicans – CGD (http://www.candidagenome.org/); Schizosaccharomyces pombe – Pombase (http://www.pombase.org/); Komagataella (Pichia) pastoris – Gent University (https://bioinformatics.psb.ugent.be/gdb/pichia/); Wickerhamomyces ciferrii, Kazachstania naganishii, and Naumovozyma castellii – the NCBI database (http://www.ncbi.nlm.nih.gov/); Candida tropicalis – the Broad Institute (http://www.broadinstitute.org/); Candida glabrata, Zygosaccharomyces rouxii, Eremothecium (Ashbya) gossypii, K. lactis, Lachancea kluyveri, Lachancea thermotolerans, and Debaryomyces hansenii – Génolevures (http://www.genolevures.org/); Blastobotrys (Arxula) adeninivorans, Cyberlindnera fabianii, and Y. lipolytica – GRYC (http://gryc.inra.fr). Species closely related to Y. lipolytica, that is, Y. yakushimensis CBS10253, Yarrowia galli CBS9722, Y. phangngensis CBS10407, Y. alimentaria CBS10151, and C. hispaniensis CBS9996, were sequenced and annotated in our laboratory at INRA Thiverval-Grignon (data not shown). Homologues of YLJEN genes were found via a homology search based on a two-step reciprocal approach using BLASTp  and a Jenp tree built as described above were reconciled with synteny data. A parsimonious approach was used to minimize the number of duplications and losses of JEN genes. For the phylogeny of JEN genes within the Yarrowia clade, KLJen2p was used as an outgroup. Protein sequences were aligned with MAFFT HX(S/T)QDXXXT. One exception to this was found, however, in YALI0B19470p, in which the first S/T residue was replaced by an alanine . We also note that the F270 residue was replaced by a leucine in CAJen1p, but as both F and L are nonpolar amino acids, this change is unlikely to have serious consequences for protein specificity.The six YLJen proteins were much more similar to each other  and five that emerged prior), two species of Wickerhamomycetaceae, and three CTG species, in addition to Komagataella (Pichia) pastoris, Blastobotrys (Arxula) adeninivorans, six species closely related to Y. lipolytica, and Schizosaccharomyces pombe, which served as an outgroup species . We then retrieved the genomes of these yeasts from genetic databases and attempted to identify the JEN genes by BLAST searches; this effort was successful in 18 of the 23 species. With the exception of S. cerevisiae, none of the species that diverged after the whole genome duplication event has a JEN gene, nor S. pombe and Zygosaccharomyces rouxii. In total, 61 genes representing 18 species were included in the phylogenetic analysis  occurred in the three remaining subgroups. In the group containing the YLJEN3 and YLJEN4 genes, an additional duplication occurred, but this duplication could not be dated, as all other species, with the exception of Y. galli, have lost these two genes. In total, 12 duplications and losses were predicted in the Yarrowia clade, indicating that the Jen3 family has a highly dynamic history.To better understand the evolution of ysis Fig.. The treKurtzman . This dunsis Fig.. Three sSCJEN1 is tightly regulated by various carbon sources; for example, it is repressed by glucose but activated by lactate , a pseudogene that has been lost in all other species.ated Fig., suggestYLJEN5 was slightly upregulated in the presence of lactate and pyruvate, repressed by fumarate and, to a lesser extent, malate, and expressed in succinate as in glucose , suggesting one of three potential scenarios: (1) this yeast does not possess a transporter for this compound, (2) the glutarate concentration was too low to support observable growth, or (3) Y. lipolytica is not able to metabolize glutarate. The single-deletion strains grew in the presence of all other carbon sources tested, with the exception of ΔYLjen1 when fumarate, malate, or succinate are added into the medium . This result confirms that YLJEN2, YLJEN3, YLJEN4, and YLJEN5 are not involved, or are only weakly involved, in the transport of fumarate and malate. This finding was not surprising with regard to YLJEN3 and YLJEN4 because they were not expressed even in the wild-type strain, but it was unexpected for YLJEN2 and YLJEN5 because both are expressed in the wild-type under the conditions tested here. Nevertheless, the deletion of YLJEN6 together with YLJEN1 was sufficient to prevent growth on malate medium and strongly reduce growth on fumarate medium . This shows that YLJen5p is also able, at least weakly, to transport fumarate. Analysis of the quintuple mutant ΔYLjen1,2,3,5,6 and sextuple mutant ΔYLjen1-6 also reveal an absence of growth in media containing fumarate, malate, or succinate , a result which lent support to this hypothesis.Taken together, these results indicate that (1) YLJen1p and YLJen2p are generalist transporters with no specific substrate affinity; (2) YLJen3p, YLJen4p, and YLJen5p are more specialized for the transport of fumarate and succinate; and (3) YLJen6p displays a high degree of affinity for fumarate. The best transporters, both in terms of transport efficiency and the diversity of substrates transported, are therefore YLJen1p and YLJen2p, which derivate from a common ancestor in the Jen3 phylogenetic tree Fig.. It is pKurtzman . This spYLJEN genes participate in fumarate transport, our results provide an explanation for the diversity of phenotypes found in the different deletion mutants grown with this substrate. However, it is very curious that, in vivo, the deletion of YLJEN1 completely inhibited growth in succinate-based medium, because our overexpression analysis revealed that YLJen2p and YLJen5p can also transport this compound . It is therefore possible that the transport of succinate is highly regulated. The same observation can be made about malate transport, as the deletion of YLJEN1 with YLJEN6 prevented growth, even though YLJEN5 and more specially YLJEN2 are also able to transport malate. One explanation could be that YLJen1p and/or YLJen6p control expression of the other YLJEN genes. However, RT-PCR performed in ΔYLjen1 and ΔYLjen1ΔYLjen6 deletion mutants revealed that the remaining JEN genes had the same expression profile (data not shown) as seen in the wild-type , suggesting that these strains consumed lactate solely for the purpose of cell maintenance. Based on the results of our previous experiments, we expected that ΔYLjen1,6 and ΔYLjen1-6 would not grow in this medium; however, it was surprising to see that the ΔYLjen1 mutant did not grow either. One potential explanation for this could be that the culture conditions in this experiment were different from those used to generate the results presented in FigureInterestingly, three general patterns of strain behavior were observed. In the first, strains did not grow and did not consume fumarate, malate, and succinate; this group included Δn1,6 Fig.. HoweverYLjen6 and ΔYLjen1-6 strains in which YLJEN1 or YLJEN2 were overexpressed , YLJEN3 , and YLJEN4   grew similarly to JMY2900 in the different substrates while members of group 3  grew more slowly in malate.The second pattern of strain behavior involved growth that began at approximately 20 h of culture and substrate consumption that was as efficient as or better than that found in the JMY2900 wild-type, at 86–129% of the WT rate of consumption. This group was composed of Δssed Fig. and C4. ely) Fig. and D4. YLJEN6-overexpression strain of ΔYLjen1-6 which displayed characteristics of both groups 2 and 3. It began to grow after approximately 20 h in culture (more slowly than the other strains in group 2) and it consumed fumarate and malate more slowly as well . Growth and substrate consumption were quantified by spectroscopy and HPLC, respectively , while malate and succinate were consumed slowly  or malate , an observation which suggests that the transport of lactate is not coupled with that of either fumarate or malate , providing evidence that they are co-transported . Instead, malate consumption was quite similar to that observed in other malate-containing media. Together, these results suggest that malate somehow inhibited fumarate consumption. We observed better growth on the fumarate and succinate medium. Under these conditions, fumarate was consumed 15-fold more slowly than succinate  and after 42 h of culture, there was still approximately 0.6 g L−1 fumarate remaining in the medium , and demonstrate similar expression patterns, even if YLJEN1 is up-regulated under more conditions than YLJEN2 is. We were unable to determine if the YLJenp can transport pyruvate or lactate. However, some YLJEN genes, for example, YLJEN6, are upregulated by lactate. YLJen6p is also unusual in that exposure to lactate, but also to fumarate, increased succinate uptake in a mutant strain in which YLJen6p was the only functioning Jen protein, an observation which could explain the divergent results we obtained in experiments that involved YLJEN6 (Figs.Lodi et al.  showed tEN6 Figs..Y. lipolytica is a an important organism in the biotechnology sector for its ability to produce both lipids (Beopoulos et al. YLJEN genes could be a promising avenue for future research since the absence of succinate consumption by the mutant strain could increase its production.Since


Perna viridis, an aquatic species of commercial value, promoted the production of Hsp70 and enhanced its resistance to stresses. Initially, the LT50 and LHT for P. viridis were determined to be 42°C and 44°C, respectively, with no heat shock induced death of mussels at 40°C or less. Immunoprobing of western blots revealed augmentation of constitutive (PvHsp70-1) and inducible (PvHsp70-2) Hsp70 in tissue from adductor muscle, foot, gill and mantel of P. viridis exposed to 38°C for 30 min followed by 6 h recovery, NLHS conditions for this organism. Characterization by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that PvHsp70-1 and PvHsp70-2 respectively corresponded most closely to Hsp70 from P. viridis and Mytilus galloprovincialis. Priming of adult mussels with NLHS promoted thermotolerance and increased resistance to V. alginolyticus. The induction of Hsp70 in parallel with enhanced thermotolerance and improved protection against V. alginolyticus, suggests Hsp70 functions in P. viridis as a molecular chaperone and as a stimulator of the immune system.Mild heat stress promotes thermotolerance and protection against several different stresses in aquatic animals, consequences correlated with the accumulation of heat shock protein 70 (Hsp70). The purpose of this study was to determine if non-lethal heat shock (NLHS) of the Asian green mussel,  Aquatic organisms experience environmental stresses including temperature fluctuation, salinity shift, oxygen deprivation and pollution –3 as welCyprinus carpio, allowing it to survive a normally lethal temperature [Artemia franciscana promotes Hsp70 build-up, induces thermotolerance and guards Artemia larvae against V. campbellii and V. proteolyticus, two pathogens of this branchiopod crustacean [Penaeus monodon to a short hyperthermic stress enhances Hsp70 accumulation and resistance against gill associated virus (GAV) [Non-lethal heat shock (NLHS) is an effective method to protect aquatic organisms against stress, an outcome often associated with increased Hsp accumulation , 5. NLHSperature . NLHS ofustacean , 10. Expus (GAV) . The conus (GAV) , 1, 4.Perna viridis [P. viridis and other bivalves in cage culture systems [V. alginolyticus, a common pathogen of bivalves and crustaceans, causing mortalities of 95–98% in cultured P. viridis. Oysters cultivated in the same rafting area exhibit clinical signs similar to those of P. viridis. Sessile organisms like P. viridis depend on physiological responses such as the increased synthesis of Hsps to accommodate stresses because they cannot escape by swimming [P. viridis upon NLHS was investigated in this study, revealing a potential role for this protein in tolerance to heat and resistance to bacterial infection.Several issues impede sustainable production of the Asian green mussel  viridis , a major viridis –17. Addi systems –20. Thesswimming . Thus, tP. viridis measuring 70–80 mm in length were purchased from various long-line culture farms in Masai, Johor (1°29′36.5″N 103°52′40.94″E). Animals were acclimatized in the Universiti Malaysia Terengganu Marine Hatchery under constant aeration (>6 ppt) at 28°C and salinity of 30 ppt for two weeks prior to use. During acclimation, mussels were fed daily with the microalgae Chaetoceros sp. at 6 x 107 cells/ml, the final number of algae in the tank. The rearing water was replaced every 2 days.Adult L. viridis acclimatized at 28°C were exposed to abrupt 30 min heat shocks ranging in temperature from 34°C to 44°C in a water bath accurate to ±0.5°C. Mussels were then transferred to 28°C and mortality was determined 24 h later by counting live animals; gaping mussels that failed to respond to gentle tapping on the shell were considered dead. The percent mortality was calculated as (N0 - Nt)/ N0 × 100 where N0 and Nt are initial and final numbers of living mussels [To determine the minimum temperatures that caused 50% mortality (LT50) and 100% mortality (LHT) groups of 20 2, 5 mM NaH2PO4, 40 mM HEPES, pH 7.4) [2O2 in 0.1 M Tris-HCl, pH 7.6, for detection of antibody reactive proteins [PvHsp70-1 and PvHsp70-2 with Quantity One software . The amounts of Hsp70 in tissues of P. viridis exposed to NLHS were interpreted as reflective density/mm2, the density value generated by Quantity One software. Human Hsp70 served as the control for antibody specificity.For protein extraction approximately 100 mg of tissue prepared individually from the adductor muscle, foot, gill and mantle was rinsed with sterile, cold, distilled water several times, and homogenized in 500 μl cold buffer K   containi pH 7.4)  was addeproteins , 10. HumP. viridis were 30 min heat shock at 38°C followed by 6 h recovery at 28°C, a value used in subsequent stress experiments.Adult mussels (n = 5) acclimated at 28°C were abruptly heat shocked at temperatures ranging from 30 to 40°C for 30 min and then transferred to 28°C for 6 h recovery prior to protein extraction. Mussels held at 28°C served as controls. As determined by SDS polyacrylamide gel electrophoresis and immunoprobing of western blots the temperature that induced maximum Hsp70 accumulation in mussel tissues and did not result in mussel mortality was 38°C. In subsequent experiments, adult mussels (n = 30) acclimated to 28°C were heated at 38°C and allowed to recover at 28°C for 0, 6, 12, 24 and 48 h prior to protein extraction. After 48 h, protein was extracted every 2 days until 10 days post-heat shock. Determination of Hsp70 band density on Western blots, heat shock parameters and recovery conditions established that the optimal conditions for NLHS of PvHsp70-1 and PvHsp70-2 were excised and freeze-dried overnight. Proteins were then digested with trypsin and the extracted peptides [In preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) , protein samples from mussels receiving NLHS were resolved in 5% SDS polyacrylamide gels by electrophoresis, stained with Biosafe Coomassie (BioRad Laboratories) and destained. Gel slices containing peptides  were loaThermotolerance induction was determined by challenging mussels exposed to NLHS at their LT50 and LHT with survival ascertained 24 h later as described above. Ten mussels were used for each treatment and experiments were done in triplicate. Control animals were held at 28°C.V. alginolyticus causing 50% mortality of mussels, bacteria were grown overnight at 28°C with constant shaking in marine nutrient broth to stationary phase, harvested and suspended in sterile seawater prior to determining density at 600 nm. The number of bacteria was calculated from a standard curve obtained according to the equation, y = (2 x 108)x – (3 x 107), where y is the number of bacteria/ml and x is the OD600 value. Mussels were incubated by immersion with 1 x 106, 1 x 107, 1 x 108 and 1 x 109V. alginolyticus/ml for various times. Survival was determined daily by counting live animals. Gaping mussels that failed to respond to gentle tapping on the shell were considered dead. The experiment was done in triplicate.To determine LC50, the concentration of 8V. alginolyticus/ml, for 72 h, the LC50, after which survival was determined as described in the previous section. Mussels challenged with V. alginolyticus without NLHS served as controls. The experiment was done in triplicate.Mussels (n = 10) subjected to NLHS were challenged with 1 x 10Survival percentages were ArcSin-transformed to satisfy normality. The significance of differences between survival of groups of challenged mussels either exposed or not exposed to NLHS and the amounts of Hsp70 were evaluated by using one way ANOVA with SPSS version 20.0 for Windows.P. viridis but mortality occurred above 40°C, with LT50 and LHT at 42°C and 44°C respectively , an effect accompanied by reduction of viral replication [A. franciscana larvae against pathogenic V. campbellii and V. proteolyticus . Clearly, the accumulation of Hsp70s induced by NLHS correlates with survival against subsequent infection, suggesting Hsp70 influences the immune response. Hsps may stabilize cells against injury in response to pathogen proliferation, mediate folding of cell proteins synthesized due to bacterial pathogens, store and re-fold partially denatured protein and stimulate the innate immune response, possibly by sending danger signals to the innate immune system [Cross-protection or cross-tolerance, an enhanced tolerance to a particular stress, acquired by an initial transient, but different, stress , 7, 4, w marinus . The acclication . Additioe system –44, 4, 7C. farreri [C. virginica [A. irradians [M. mercenaria [P. viridis.Although bivalves rely solely on an innate, non-lymphoid system of immune responses , 45, som farreri , C. virgirginica , A. irrarradians  and M. mrcenaria  immune aP. viridis by NLHS and that Hsp70 plays a role in increasing the thermotolerance of P. viridis and enhancing survival against V. alginolyticus challenge. Further work is required to elucidate the role of Hsps in induced thermotolerance and the immune response of mussels, perhaps with the application of molecular tools such as RNA interference (RNAi) an option. Such studies are of fundamental interest and have applied significance through formulation of strategies to protect aquatic organisms against stress and disease, of particular importance in aquaculture.The data presented herein demonstrate that Hsp70 is induced in S1 TablePvHsp70-1 in adductor muscle, foot, gill and mantle of P. viridis exposed to heat shock at 30, 32, 34, 36, 38 and 40°C were determined by densitometry analysis of antibody-stained Western blots as described in Materials and Methods. Data are presented as mean ± standard deviation. Asterisk (*) represents statistical difference against the control treatment (P<0.05). 28, mussels not receiving NLHS (control) (S1A Table). Amounts of PvHsp70-2 interpreted as reflective density/mm2 in tissues of P. viridis exposed to NLHS. The amounts of PvHsp70-2 in adductor muscle, foot, gill and mantle of P. viridis exposed to heat shock at 30, 32, 34, 36, 38 and 40°C were determined by densitometry analysis of antibody-stained Western blots as described in Materials and Methods. Data are presented as mean ± standard deviation. Asterisk (*) represents statistical difference against the control treatment (P<0.05). 28, mussels not receiving NLHS (control) (S1B Table). Amounts of PvHsp70-1 interpreted as reflective density/mm2 in tissues of P. viridis upon NLHS at 38°C with different recovery length. The amounts of PvHsp70-1 in the adductor muscle, foot, gill and mantle of P. viridis upon NLHS at 38°C with different recovery length. Data are presented as mean ± standard deviation. Asterisk (*) represents statistical difference against the control treatment (P<0.05). c and 28, mussels not receiving NLHS (control) (S1C Table). Amounts of PvHsp70-2 interpreted as reflective density/mm2 in tissues of P. viridis upon NLHS at 38°C with different recovery length. The amounts of PvHsp70-1 in the adductor muscle, foot, gill and mantle of P. viridis upon NLHS at 38°C with different recovery length. Data are presented as mean ± standard deviation. Asterisk (*) represents statistical difference against the control treatment (P<0.05). c and 28, mussels not receiving NLHS (control) (S1D Table).The amounts of (DOCX)Click here for additional data file.