During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming. Cryopreservation remains the most common method for the long-term storage of various cells. However, during the cryopreservation process a significant number of cells experience irreparable damage1234Ice recrystallization is the process that occurs during freezing and thawing whereby large ice crystals increase in size at the expense of smaller ones. This process occurs primarily during warming/thawing of a frozen sample and the subsequent cellular damage it causes is the primary source of cell injury during cryopreservationThe most significant short fall of current cryopreservation protocols making them suboptimal is the failure to control ice recrystallization. RBC transfusions are lifesaving for patients suffering from leukemias, hemolytic anemias, and from traumas resulting in severe blood loss. Cryopreservation is the only technology allowing access to large quantities of RBC units necessary when high numbers of RBC transfusions are required. However, the cryopreservation of RBCs for routine transfusion is not a common practice because current protocols do not permit direct transfusion immediately after thawing. Clinical cryopreservation protocols utilize high concentrations of glycerol (40% v/v) as a cryoprotectant however, after thawing time-consuming deglycerolization procedures are necessary to prevent intravascular hemolysis.C-linked) antifreeze glycoprotein (AFGP) analogues . TWEs have been recognized as another significant contributor to reduced post-thaw viabilities in sperm181921In this paper, we demonstrate that these IRIs are capable of mitigating ice growth 3,4), aryl-aldonamide 5 and lysine-based non-ionic surfactants with the general structure of 616We have identified several different classes of small molecules that inhibit ice recrystallization . These i3 and 4 to inhibit ice recrystallization has been previously quantified and the IRI activity of 5 is shown in 5 is the least active of the three compounds and substitution of the para-methoxy substituent in 3 with a bromine atom results in a potent inhibitor of ice recrystallizationThe ability of 3–5, optimal in vitro concentrations for each IRI were determined using the two-step rate-controlled freezing experiments3–5 to preserve RBCs using only 15% glycerol during slow cooling. It is interesting to note that the required “dosage” of each IRI is very different . As shown in 4 is effective at only 30 mM, while the optimal concentration of 3 is 110 mM. This is not surprising as 4 is approximately twice as active as 3 with respect to IRI activity. However, 5 is equally effective (p > 0.05) as 4 at only 5 mM but is less IRI active than both 3 and 4 suggesting that factors other than IRI activity may be important for the cryoprotective activity. The ability to reduce the amount of glycerol in the presence of an IRI without compromising the number of RBCs recovered is significant because cryopreservation using less glycerol will reduce post-thaw processing time in the clinical setting.Each of these compounds was investigated for the ability to prevent cryo-injury during freezing and thawing. For these studies, human RBCs were utilized because they are annucleate and assays to reliably assess levels of post-thaw hemolysis are well established. Prior to performing cryomicroscopy with RBCs in the presence of IRIs 3–5 was re-assessed at their effective in vitro concentrations reported in 4 does not change dramatically, however ice crystal size in the presence of 110 mM 3 is dramatically smaller in size. Interestingly, 5 appeared to be less sensitive to concentration effects as there is little difference in ice crystal size despite the fact the concentration is approximately four-fold less.Given that the optimal concentrations for the freezing of RBCs were not the same as those utilized for the assessment of IRI activity (22 mM), the IRI activity of 4 (30 mM) was cooled at a rate of 25 °C/min to a temperature of −40 °C. The sample was then warmed to −10 °C at a rate of 10 °C/min and held for 10 minutes prior to taking a picture. 4 samples stays constant even in the presence of RBCs. In other words, the presence of RBCs does not appear to influence the amount of ice in the sample. However, less ice is observed in samples (with and without RBCs) when 4 (30 mM) is present compared to the 15% glycerol control. In each of these images, the percentage of frozen fraction is small. This is because the holding temperature of −10 °C is close to the colligative freezing point depression of the 15% glycerol solution (−4 °C) and therefore a large fraction of the sample is unfrozenAnalysis of ice crystal size is a key aspect of the splat IRI assay that our laboratory has developed410163–5 was assessed in conditions with high amounts of ice present, the experiment was repeated at a lower holding temperature to ensure higher ice volume. 4 and 5 relative to the 15% glycerol control. Analysis using domain recognition software4 and 5. This is interesting as it has been hypothesized that cellular injury in slowly frozen red cells is a result of solution effects  and the reduction of unfrozen fraction in the extracellular space.As the IRI activity of 3–5 resulted in significantly smaller ice crystals in vitro, we sought to increase the extent of ice recrystallization in the frozen sample controls. To do this, an experiment was performed in which a sample was frozen by placing it directly in dry ice (dump freeze) at −80 °C (cooling rate of 90 °C/min) and then warmed to −20 °C. After stabilization at −20 °C, the sample was cooled again to −80 °C. This process could be repeated for several cycles  before being thawed . The sample was held for 15 minutes and then quickly warmed to −20 °C (90 °C/min) and held for an additional 15 minutes before being cooled to −80 °C again and then warmed to −20 °C prior to thawing. Images were acquired throughout the temperature cycling. The data from this experiment are shown in 4, the ice crystal sizes throughout the experiment remained constant after warming to −20 °C and repeated cycling (181921A remarkable aspect of this experiment is that in the presence of 30 mM 3–5 at the optimized concentrations during freezing of RBCs with only 15% glycerol using slow freezing rates will yield a higher number of intact RBCs post-thaw compared to only 15% glycerol. RBCs were frozen with 3–5 at the optimized concentration in 15% glycerol. Samples were cooled rapidly using “dump freeze” conditions (fast freezing rates). After freezing to −80 °C, the sample was then warmed to −20 °C and after stabilization at −20 °C, the sample was cooled again to −80 °C. This constitutes one “cycle”. This cycle was repeated 2 and 4 more times prior to thawing and percent intact RBCs post-thaw was measured corresponding to 1, 3 and 5 freezing-warming-freezing cycles  the percentage of intact RBCs decreases dramatically for the 15% glycerol control (55% intact RBCs) and for compound 3 (40% intact RBCs). The percentage of intact RBCs frozen using 15% glycerol with 30 mM 4 stays constant (92% intact RBCs). When the freezing-warming-freezing cycles are repeated five times, the percent intact RBCs frozen with 15% glycerol and 110 mM 3 is only 36% and 39% respectively. 15% glycerol with 5 mM 5 decreases by 10% to 65% intact RBCs but with IRI 4 (30 mM) the percentage of intact RBCs holds at approximately 90%. With each successive freezing-warming-freezing cycle, the amount of ice recrystallization is increased and the amount of cellular damage is also increased. This is the reason why we observe a decrease in the number of percent intact RBCs with the 15% glycerol control. Interestingly, the 40% glycerol control protects the RBCs against this transient warming injury. Small molecule IRI 4 at 30 mM is a very effective inhibitor of ice recrystallization, in fact it is the most potent inhibitor examined in this study and is also very effective at preventing the cellular injury resulting from ice recrystallization during TWEs with reduced amounts of glycerol.Based upon the observations that IRIs can greatly reduce the ice crystal size and that ice recrystallization results in significant decreases in post-thaw cell viability, we hypothesize that inhibiting ice recrystallization during freezing and thawing/warming will result in increased post-thaw cell viabilities. Consequently, we predict that the use of compounds g cycles . Percent3–5 are effective inhibitors of ice recrystallization in the presence of human RBCs and result in 70–80% intact RBCs post-thaw with reduced amounts of glycerol. The 15% glycerol control furnishes only 40% intact RBCs post-thaw. The ability of 3–5 to reduce the mean grain size of extracellular ice was verified by cryomicroscopy and validates the positive correlation between inhibiting the process of ice recrystallization and increased post-thaw recovery of RBCs. Finally, compound 4, the most effective inhibitor of ice recrystallization in this study was shown to prevent cellular injury due to ice recrystallization during TWEs further demonstrating the utility of these novel small molecule ice recrystallization inhibitors as cryoprotectants.Ice recrystallization is a major cause of cellular damage during freezing. Conventional cryoprotectants such as DMSO and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization. Novel small molecule IRIs control the growth of ice and recrystallization during freezing and unlike the many polymers that are reported to inhibit ice recrystallization, have low molecular masses and are readily amenable to cellular systems. Small molecule IRIs All methods were carried out in accordance with approved guidelines.3 and 4 were prepared as described previously by our laboratoryN-(4-chlorophenyl)-D-gluconamide 5 was synthesized as follows. To a solution of D-gluconic acid-d-lactone  in acetic acid (5 mL) was added 4- chloroaniline . The mixture was stirred under reflux for 2 hours. The crude product was precipitated with hexanes, filtered and the crude solid was recrystallized in EtOH to afford 5 as white crystals ; 1H NMR : δ 9.7 , 7.8 , 7.35 , 5.71 , 4.59 , 4.55–4.53 , 4.36 , 4.18 , 4.02–3.99 , 3.61–3.57 , 3.52–3.51 , 3.42–3.36 ; 13C NMR : δ 171.77, 137.56, 128.40, 126.89, 121.17, 74.20, 72.19, 71.53, 70.31, 63.27; LRMS (ESI) (m/z): [M + Na]+ calcd. for C12H16ClNaNO6, 328.70; found, 327.95.Aryl-glycosides Sample analysis for IRI activity was performed using the “splat cooling” method as previously describedAll RBC units were obtained from NetCAD (Canadian Blood Services’ Network Centre for Applied Development). Whole blood was collected from healthy volunteers using standardized phlebotomy guidelines approved by Canadian Blood Services (CBS). Informed consent was obtained from all donors. All experimental protocols were approved by NetCAD and CBS. Ethics approvals were obtained from Research Ethics Board (REB) at CBS and the University of Alberta. For cryovial experiments, whole blood units were collected and processed by NetCAD . The whole blood was processed using the buffy coat (BC) method to produce leukocyte reduced SAGM RBC units, which has been previously describedThe freezing solution consists of a 30% glycerol solution prepared from a commercially available glycerol solution  by diluting it with 0.2%/0.9% dextrose/saline (SD). An equal volume of freezing solution was added to 150 μL of RBCs for a final volume of 300 μL. The final concentrations of all freezing solutions were as indicated in the results and discussion. RBC suspensions were transferred to cryotubes and incubated at room temperature for 10 minutes prior to immersion in a methanol bath cooled to −5 °C. A thermocouple was inserted into a RBC/15% glycerol sample (temperature probe) to monitor temperature at 1 second intervals. Once the internal solution from the temperature probe reached −5 °C, ice nucleation was induced by touching the outside of the glass cryotubes with pre-cooled (in liquid nitrogen) forceps. Controlled nucleation is performed to ensure that ice nucleation occurs at the same sub-zero temperature of −5 °C in each vial. RBC samples were then held at −5 °C for 5 minutes. Samples were then cooled at a rate of 1 °C/min to −40 °C, then thawed immediately by plunging in a 37 °C water bath. Post-thaw hematocrits (Hcts) and percent hemolysis was determined for all freezing experiments by comparing the supernatant hemoglobin concentration to total hemoglobin concentration using the cyanmethemoglobin Drabkin’s methodThe freezing solution consists of a 30% glycerol solution prepared from a commercially available glycerol solution  by diluting it with 0.2%/0.9% dextrose/saline (SD). An equal volume of freezing solution was added to 150 μL of RBCs for a final volume of 300 μL. The final concentrations of all freezing solutions were as indicated in the results and discussion. RBC suspensions were transferred to cryotubes and incubated at room temperature for 10 minutes prior to immersion in dry ice (−80 °C). A temperature probe was used for temperature measurements at 1 second intervals. Once the internal solution reached −80 ± 2 °C, the samples were immersed in a methanol bath cooled to −20 °C. Once the internal solution reached −20 °C, the samples were plunged into dry ice again. RBC samples were held in dry ice until the internal solution from the temperature probe reached −80 ± 2 °C, after which the samples were either thawed (representing one cycle of transient warming) or immersed in a methanol bath cooled to −20 °C. One, three and five cycles of immersion in a −20 °C methanol bath and dry ice were performed. Samples were thawed quickly by plunging in a 37 °C water bath. Post-thaw Hcts and percent hemolysis was determined for all freezing experiments by comparing the supernatant hemoglobin concentration to total hemoglobin concentration using the cyanmethemoglobin Drabkin’s methodPercent post-thaw RBC integrity was calculated using the measured percent hemolysis values according to the following equation: % post-thaw RBC integrity = −100% hemolysis. Data is represented as the mean percentage of post-thaw RBC integrity for each condition. Error bars are reported as the standard error of the mean (SEM). Statistical significance for all data was determined by unpaired Student’s t-test with a 95% confidence level.The nucleation and growth of extracellular ice in solutions containing the IRI compounds were documented using a cryomicroscope that consists of a Nikon 80i fluorescent microscope with a long working distance condenser and objectives, CCD cameras (Hammamatsu ORCA) interfaced to a personal computer and a convection cryomicroscope stage (Linkam FDCS196).How to cite this article: Briard, J. G. et al. Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing. Sci. Rep. 6, 23619; doi: 10.1038/srep23619 (2016).


In this study, we found that TRAP150 interacted with the cleavage and polyadenylation specificity factor (CPSF) and co-fractionated with CPSF and RNA polymerase II. Moreover, TRAP150 preferentially associated with the U1 small ribonucleoprotein (snRNP). However, our data do not support a role for TRAP150 in alternative 5′ splice site or exon selection or in alternative polyadenylation. Because U1 snRNP participates in premature cleavage and polyadenylation (PCPA), we tested whether TRAP150 is a cofactor in the control of PCPA. Although TRAP150 depletion had no significant effect on PCPA, overexpression of TRAP150 forced activation of a cryptic 3′ splice site, yielding spliced PCPA transcripts. Mechanistic studies showed that TRAP150-activated splicing occurred in composite but not authentic terminal exons, and such an activity was enhanced by debilitation of U1 snRNP or interference with transcription elongation or termination. Together, these results indicate that TRAP150 provides an additional layer of PCPA regulation, through which it may increase the diversity of abortive RNA transcripts under conditions of compromised gene expression.The spliceosomal factor TRAP150 is essential for pre-mRNA splicing  Eukaryotic RNA polymerase II (RNA pol II) produces mRNAs as well as a variety of non-coding RNAs. The majority of RNA pol II products necessarily undergo several interconnected processing steps to become functionally mature ,2. A myrin vivo and its overexpression promotes the splicing efficiency  is an integral component of the spliceosome . Identifficiency . We haveficiency . Most inficiency . This obficiency . NeverthPhysical interactions between splicing factors and cleavage/polyadenylation factors may cooperatively facilitate terminal exon definition and recognition of proper cleavage/polyadenylation signals in the 3′ ends of transcripts . HoweveOur observation that TRAP150 associated with U1 snRNP and the cleavage and polyadenylation specificity factor (CPSF) prompted us to evaluate its function in PCPA. We found that TRAP150, although not essential for PCPA, activates cryptic 3′ splice sites in PCPA transcripts. Our finding provides a molecular mechanism for splicing following PCPA.d-ribofuranosyl-benzimidazole  or 10 μM camptothecin (Sigma) alone or with both at 37°C for 4 h. To block splicing, transfected cells were treated with 1 μM pladienolide B  at 37°C for 4 h.HEK293 and HeLa cell culture and transfection were performed as described . To inhiThe NR3C1 minigenes including the wild-type, 5′ splice site mutant and poly(A) signal mutant were constructed into the pcDNA3.1 vector as described . To geneAntibodies against the FLAG epitope, hRrp6 and U2AF65 were from Sigma-Aldrich, antibodies against BCLAF1 (A300-608A), CPSF73 (A301-091A), CPSF160 (A301-580A), CstF64 (A301-092A), TRAP150 (A300-956A), Xrn1 (A300-443A) and Xrn2 (A301-102A) were from Bethyl Laboratories, anti-pol II C-terminal domain (4H8) was from Abcam, anti-poly(A) polymerase (H-300) was from Santa Cruz Biotechnology, anti-HA epitope was from Bioman Scientific and anti-α-tubulin (DM-1A) was from Thermo Scientific. The antibody against the U1-70K protein was a gift of R. Luhrmann (University of Göttingen).6) were transiently transfected with 10 μg of the FLAG-TRAP150 expression vector by calcium phosphate. At 48 h post-transfection, cells were lysed in 1 ml of hypotonic buffer  Triton X-100) at 4°C for 15 min followed by addition of 37.5 μl of 4 M NaCl to the final concentration of 150 mM. After centrifugation at 13 400 × g for 15 min, the soluble fraction was subjected to immunoprecipitation using anti-FLAG (7 FLAG-TRAP150-expressing HEK293 cells were used. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and stained with SYPRO Ruby (Bio-Rad). The bands of interest were excised, trypsinized and subjected to liquid chromatography coupled with tandem mass spectrometry .HEK293 cells (∼3 × 10The 15–40% glycerol gradient was prepared in buffer containing 20 mM HEPES (pH 7.9), 0.2 mM EDTA and 150 mM KCl. HeLa cell lysate  was layered onto a 12-ml gradient and centrifuged in a Beckman SW41 rotor at 36 000 rpm at 4°C for 36 h. Gradient fractions (500 μl each) were manually collected from top to bottom, and proteins were precipitated with 10% trichloroacetic acid. To determine the phosphorylation status of TRAP150, 250 μl of fraction 21 was treated with 80 units of alkaline phosphatase (Roche) at 37°C for 30 min and precipitated with 10% trichloroacetic acid.32P-labeled antisense riboprobe to detect spliceosomal snRNAs . One-fifth the amount of the RNAs was separated by a 6% denaturing polyacrylamide gel followed by northern blotting analysis using l snRNAs .6 of HEK293 cells. We used pCEP4-based human influenza hemagglutinin epitope (HA)-tagged TRAP150 expression vectors for the NR3C1 minigene assay, because the minigene was derived from pcDNA3.1. In brief, 0.5 μg of the NR3C1 minigene or a CAT(In)-derived splicing reporter was co-transfected with 2.5 μg of the HA-tagged TRAP150 expression vector into ∼1 × 106 HeLa cells. For U1 blockage, the U1 decoy plasmid  was included. After 24 h, cells were harvested and RNAs were recovered using TRIzol reagent. Reverse transcription was performed as described (® No-ROX kit (Biolone). PCR was performed on the LightCycler 480 (Roche) using the default run mode with SYBR Green I. Actin served as reference. For the 3′ RACE analysis, the first-round PCR, >35 amplification cycles were performed using the gene-specific forward primer and the reverse XbaKpnBam adaptor primer. All possible poly(A) containing transcripts of each gene could be selectively amplified. Subsequently, 10 and 15 cycles of nested PCR were performed for mRNA and PCPA, respectively, using the gene-specific chimeric reverse primers (Supplementary Table S1), each of which contained a string of 12 Ts followed by several residues complementary to the sequence immediately upstream of the putative cleavage site. To detect the cryptically spliced products of PCPA (hereafter termed CSPP) product, 20 amplification cycles of PCR were performed. The annealing temperature used for the first-round PCR reaction was 55°C whereas it was lowered to 50°C for the nested PCR, because a low GC content primer was used. For the knockdown experiments, cells were transfected with 100 nM short interfering RNA (siRNA) and incubated for 48 h. Cells were further transfected with the NR3C1 minigene and an effector expression vector and then incubated for another 24 h. Total RNA was isolated using Trizol reagent, followed by 3′ RACE analysis. All siRNAs used in this study were purchased from Life Technologies, and their sense-strand sequences were as follows: si-BCLAF1: 5′-gguuauagaccugucuggaauagaa-3′; si-CPSF73: 5′-aggugcaguacagaagguuucuaaa; si-hRrp6: 5′-ucuucuauaggucuguauaacuggg; si-Luc.: 5′-ggatttcgagtcgtcttaatgtata; si-TRAP150: 5′-ccaaaagguauaagcuccgagaugauu; si-Xrn1: 5′-gggaucuggaaagaugcaauacuuu; si-Xrn2: 5′-gagaggagcauugaugacuggguuu.To evaluate the effect of TRAP150 in PCPA of endogenous genes, 10 μg of pcDNA3.1-based expression vector encoding FLAG-tagged TRAP150 or deletion mutant was transfected into ∼5 × 10escribed , except escribed  was usedhttp://genome.ucsc.edu/). We searched for the intronic promoter-proximal poly(A) signals using the following criteria: poly(A) signals located  of CPSF160 co-precipitated with TRAP150 regardless of the presence of RNase Figure , suggestTo test whether TRAP150 mediates the interaction between spliceosomal snRNPs and CPSF, we immunoprecipitated FLAG-TRAP150 and recovered co-precipitated RNA for northern blot analysis using probes complementary to snRNAs. The result showed that TRAP150 preferentially associated with U1 snRNA in comparison to other RNA processing factors Figure , lane 7.The interactions between TRAP150 and U1 snRNP as well as components of CPSF initially prompted us to examine whether TRAP150 could modulate alternative splicing or polyadenylation. Using minigene-based splicing assays, we observed that overexpression or knockdown of TRAP150 had no significant effect on either 5′ splice site usage of the adenovirus E1a transcript or exon selection of α-tropomyosin . To our surprise, TRAP150 could modulate terminal exon selection of the calcitonin/calcitonin gene-related peptide (CT/CGRP) minigene  Supplem. At presCDC42, GABPB1 and UBAP2L mRNAs in TRAP150 siRNA transfected HEK293 cells. While ∼70% of TRAP150 was depleted, no obvious change was detected in the alternative polyadenylation isoform expression of these three genes . Nevertheless, the role of U1 snRNP in PCPA  in TRAPe Figure , lane 4.e Figure ,9, we deC Figure  and C. Ad Figure , lane 6.A Figure , Ex2–Ex3cedented , but itsNR3C1, i.e. the polyadenylation site of PCPA is located <500 nt downstream of the 5′ splice site, and potential 3′ splice sites exist between these two sites. Finally, we selected seven candidates, ACN9, CMTM8, GPR126, GSK3B, NF1A, PLCE1 and PLCH1  to examine their propensity to undergo PCPA. PCPA transcripts were detected in all seven genes, even without blocking U1 snRNA function   signals that are located within a <5-kb region from the transcription start site. We then focused on those with features similar to the PCPA property of NR3C1 PCPA transcripts. Unlike TRAP150, overexpression of SRm160 did not elicit NR3C1 CSPP expression  signal disrupted the expression of both PCPA and CSPP  signal and the major cryptic 3′ splice site could function in heterologous genes. We inserted an NR3C1 fragment spanning nucleotides −156 to +51 relative to the poly(A) signal  into the intron (derived from the human β-globin gene) of a previously used splicing reporter . Overexpression of TRAP150 dramatically enhanced the use of the authentic 3′ splice site . Together, our results indicated that PCPA is not functional in the exon context and that TRAP150 is unable to activate 3′ cryptic sites downstream of the authentic/strong one, and its splicing activity is likely independent of the polyadenylation signal.To further examine whether the poly(A) signal dictates TRAP150-induced cryptic 3′ splice site usage in the authentic terminal exon, we placed the PCPA We previously reported that TRAP150 co-transcriptionally promotes pre-mRNA splicing . The preper se, but our data indicate that TRAP150 could neither modulate terminal/3′-most alternative polyadenylation selection nor facilitate intronic polyadenylation  site-bearing intron into a composite terminal exon. Although TRAP150 knockdown had no significant effect on the PCPA process, its overexpression activated the splicing of PCPA transcripts Figure . This efOur data reveal that TRAP150 overexpression activates the authentic 5′ and cryptic 3′ splice sites in PCPA. We gathered that the CPSF complex, which binds to the intronic poly(A) site, might dictate TRAP150 in 3′ splice site selection in composite exons so that the activated 3′ splice sites are close to (≤100 nt upstream) the cleavage/polyadenylation site Figures . We noteTreatment of cells with DRB or camptothecin or knockdown of Xrn2 or CPSF73 enhanced CSPP expression Figure . PerhapsThe biased distribution of the 5′ splice site-poly(A) site axis toward the sense strand of genes ensures the expression of long/functional transcripts ,18,36. NSupplementary Data are available at NAR Online.


Dear Editor,End-binding protein 1 (EB1), a member of microtubule plus-end tracking proteins (+TIPs), plays an important role in the regulation of microtubule dynamics and has been implicated in cancer development  domain, T154, S155, S156, S157, S165, and T166 in the linker region, and T206 and Y217 in the end binding homolog (EBH) domain . It is noteworthy that for the consecutive linker-region residues T154, S155, S156, and S157 (TSSS), and S165 and T166 (ST), the phosphorylation sites could not be unambiguously assigned based on the mass spectrometry profiles , yet they were all likely to be phosphorylated with certain stoichiometry. Therefore, we treated TSSS or ST as one phosphorylation motif and mutated all the residues in the motif in subsequent functional assays. According to the crystal structures of the CH and EBH domains available in the protein data bank, we analyzed the localization of five identified phosphorylation sites in the three-dimensional structures. By molecular modeling, we found that all the five phosphosites present in the CH and EBH domains were exposed to the surface of EB1 .To explore the functional roles of EB1 phosphorylation, we generated a panel of phospho-deficient  and phospho-mimic  mutants. Immunoblot analysis with antibodies against phosphorylated serine and threonine did not show any dramatic changes in EB1 phosphorylation level for the S27A , T33A , TSSSAAAA , STAA , and T206A mutants  . Considering that in total five serines and four threonines were identified to be phosphorylated, it was conceivable that the overall phosphorylation level of EB1 was not significantly altered by single-site or single-motif mutations. By contrast, the phosphorylation of Y71 and Y217 contributed significantly to the overall tyrosine phosphorylation of EB1, as both of the phospho-deficient mutants showed dramatically reduced signals in the immunoblot, with antibodies against phosphorylated tyrosine . We then overexpressed wild-type or mutant EB1 to investigate whether the phosphorylation at specific sites affects EB1 interaction with microtubules/tubulin. It turned out that none of the EB1 mutants changed its interaction with α-tubulin in the GST pulldown assays . Immunofluorescence microscopy further revealed that all the mutants of EB1 were located at the plus end of microtubules in a pattern similar to wild-type EB1 .To analyze the effect of EB1 phosphorylation on microtubule dynamics, we overexpressed GFP-EB1 wild-type and mutants and took serial images by time-lapse microscopy. We then used the PlusTipTracker software to analyze the dynamics of microtubules  and mitotic centromere-associated kinesin (MCAK) as examples for SxIP motif-containing proteins  and p150Glued are known to interact with EB1 through cytoskeleton-associated protein glycine-rich (CAP-Gly) domains . Further experiments are warranted to investigate this possibility.Considering that the concentration of EB1 is hundreds of nanomoles in cells and that the concentration of EB1 needed for dimerization is lower than 1 nanomole, the newly translated EB1 monomers are obligatory to form dimers immediately and it is quite hard for EB1 dimers to dissociate into monomers Below is the link to the electronic supplementary material.


CaMKII inhibition led to enlargement of TJ-areas and up-regulation of the barrier function. CaMKII inhibition induced excess TJ formation in part by the activation of AMP-activated protein kinase (AMPK) and subsequent phosphorylation of claudin-1. As up-regulation of epithelial barriers is essential for the prevention of chronic inflammatory diseases, the identification of CaMKII as a modulator of TJ function paves the way for the development of new drugs to treat these diseases.Epithelial cells define the boundary between the outside and the inside of our body by constructing the diffusion barrier. Tight junctions (TJs) of epithelial cells function as barriers against invasion of harmful microorganisms into the human body and free diffusion of water or ions from the body. Therefore, formation of TJs has to be strictly controlled in epithelial cells. However, the molecular mechanisms governing this regulation are largely unknown. In this study, we identified Ca Although the molecular components of tight junctions (TJs) were discovered12678It is now well-established that claudins, proteins with four transmembrane domains, are essential for TJ formation. A previous study revealed that over-expression of claudin is enough to reconstitute ectopic TJ formation in L fibroblastsPolymerization of claudins into TJ strands is likely strictly regulated. One important regulator of TJ formation is the ZO family of proteins. We and others have revealed that the ZO family of proteins is essential for TJ formation11121313In addition to being regulated by the ZO family of proteins, excess claudin-1 should be removed from the plasma membrane to prevent spontaneous polymerization of claudins into extra TJs in epithelial cells. Although unpolymerized claudins are retained at the basolateral membrane in cultured epithelial cells and in tissues, extra TJs are not formed at the basolateral membrane domains. To prevent spontaneous polymerization of claudins and unnecessary TJ formation, the concentration of unpolymerized claudins should be kept under the threshold of spontaneous polymerization. Thus, molecular mechanisms to control the concentration of unpolymerized claudins at the plasma membrane exist, but have yet to be clarified.In the present study, we revealed that CaMKII inhibition led to enlargement of TJ areas and up-regulation of epithelial barrier function. These effects were owed to increased junctional contractility and decreased mono-ubiquitination of claudin-1.We first established several clones of L fibroblasts expressing GFP-tagged mouse claudin-1 (GFPmCL1L) at various levels. We isolated the GFPmCL1L cell clone, which had a low expression level at which GFP-claudin-1 was unable to form TJs, but localized entirely to the plasma membrane . UnexpecWe examined the effects of approximately 400 bioactive chemical regents on the polymerization of GFP-claudin-1 in GFPmCL1L cells. We identified KN-93, a specific CaMKII inhibitorWe next examined whether KN-93 induces enlargement of TJs in epithelial cells. When compared with the staining pattern of endogenous claudin-1, treatment with KN-93 induced the formation of laterally enlarged TJ networks in cultured epithelial cell lines, such as CSG1 cells  and S1 oTo distinguish laterally enlarged TJ areas from the normal TJ strands, we used anti-claudin-1 mAb (clone 2H10D10). The 2H10D10 antibody was able to detect excess TJs formed by treatment of CSG1 cells with KN-93 . The epiTaking advantage of this unique character of the mAb, we quantified the effect of KN-93 treatment on the formation of enlarged TJs . ImmunosThese enlarged TJ networks contained other TJ membrane proteins such as claudin-3 . KN-93 tΩ/cm2) compared with that of control cells (469.6 ± 23.5 Ω/cm2). We therefore concluded that KN-93 is a potent TJ-formation promoting agent. In the following experiments, we further investigated how treatment with KN-93 up-regulated the epithelial barrier.We next investigated the effects of KN-93 on the epithelial barrier function by measuring trans-epithelial resistance (TER) . After tWe looked at changes in the distribution of cell adhesion molecules other than claudin-1 after treatment with KN-93. Among the cell adhesion molecules tested, the intensity of staining with the α18 antibody, which recognizes a tension-dependent conformation of α-cateninWe next reasoned that up-regulation of junctional contractility may be involved in the increase of barrier function induced by treatment with KN-93. Inhibition of rho-kinase by treatment with Y-27632 abrogated the increase of TER by KN-93-induced CaMKII inhibition, indicating that up-regulation of junctional contractility is necessary for the enhanced epithelial barrier function we saw with KN-93 treatment alone .Taken together, these results demonstrated that CaMKII inhibition leads to up-regulation of junctional contractility, which in turn contributes to the strengthening of epithelial barrier function.In addition to the increased junctional contractility, CaMKII inhibition by KN-93 caused enlargement of the TJ-containing areas. We previously demonstrated that ZO family proteins determined where claudin-1 should be polymerized as forced dimerization of ZO-1 was sufficient to induce ectopic TJ formation at the lateral membrane1718Owing to our observation that TJs were formed in serum starved GFPmCL1L cells at the initial stage of this study, we wondered if CaMKII activity is down-regulated by serum starvation. As the activated CaMKII further autophosphorylates at Thr-286 to be activeA relationship between the inhibition of CaMKII and the activation of AMPK had not been shown previously, so we examined whether crosstalk existed between these two kinases in an attempt to narrow down the signaling pathway(s) that control TJ formation. Although the molecular mechanisms are not clear at present, we found that CaMKII inhibition strongly activated AMPK, but AMPK activation by AICAR treatment did not induce CaMKII inhibition . When GF2+ -induced TJ assembly22In previous studies, two groups reported that AMPK is activated during CaIn GFPmCL1L cells, N-terminally GFP-tagged claudin1 migrated as a doublet by SDS-PAGE. We noticed that the upper band of GFP-claudin-1 decreased when treated with KN-93 or AICAR . This fiNext, we tested the possibility that the slower-migrating form of claudin-1 was owed to ubiquitination. Treatment with MG-132, an inhibitor of proteasome, increased the intensity of the upper band of GFP-claudin-1, supporting the idea that this upper band corresponds to mono-ubiquitinated claudin-1 . Similarin vitro phosphorylation assay using recombinant AMPK proteins and GST fusion proteins containing the cytoplasmic tail of claudin-1 confirmed this hypothesis  of Japan. The following primary antibodies were used for the immunofluorescence microscopy and immunoblotting: rabbit anti-claudin-3 pAb (Zymed), rabbit anti-claudin-1 pAb, mouse anti-α tubulin mAb (Sigma), rabbit anti-AMPK antibody, rabbit anti-phospho-AMPK mAb, rabbit anti-CaMK II antibody, rabbit anti-phospho-CaMK II mAb, mouse anti-phospho MLC2 mAb , mouse anti-claudin-1 mAb clone 2H10D10 (Life Technologies), rabbit anti-Par3 pAb (Millipore), mouse anti-ZO-1 mAb (T8-754) and α18 antibody (a generous gift from Dr. A. Nagafuchi).The following chemical reagents were used for this study: KN-93 (Wako), AICAR (Sigma) and MG-132 (Peptide Institute. Inc.).Escherichia coli and purified using Glutathione Sepharose Beads . Kinase reactions contained recombinant AMPK and GST-COOH-terminal tail of mouse claudin-1 substrates in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 25 μM ATP, and 15 μCi [γ-32P] ATP for 30 min at 37 °C. Proteins were separated by SDS-PAGE and visualized by autoradiography (Typhoon9500).Recombinant active AMPK was purchased from Millipore. Glutathione S-transferase (GST)-tagged cytoplasmic tail of mouse claudin-1 (183 aa- 211 aa) were expressed in 2, 1% Triton X-100, 2 mM DTT, 1 mM PMSF, 2 μg/mL leupeptin, and 0.25% aprotinin; lysates were cleared by centrifugation. Each supernatant was incubated with anti-Myc mAb (9E10)-conjugated protein G beads. After extensive washing with the lysis buffer, bound proteins were eluted with SDS-sample buffer. Samples were resolved by SDS/PAGE and electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell). This membrane was incubated successively with anti-GFP mAb or anti-claudin-1 mAb which were visualized using Super Signal West Dura (Pierce).HEK293 cells were transfected with GFP-tagged claudin-1 or claudin-1 K189R and Myc-tagged ubiquitin. CSG1 cells were transfected with Myc-tagged ubiquitin alone. Cells were lysed with lysis buffer containing 20 mM Hepes (pH 7.4), 50 mM NaCl, 2 mM MgClFreeze fracture electron microscopy was performed as described previouslyImmunofluorescence microscopy was performed as described previouslySpecimens were observed at RT with a confocal microscope  equipped with a Plan-APO (63/1.40 N.A. oil-immersion) objective with appropriate binning of pixels and exposure time. The images were analyzed with ZEN 2012 .How to cite this article: Shiomi, R. et al. CaMKII regulates the strength of the epithelial barrier. Sci. Rep. 5, 13262; doi: 10.1038/srep13262 (2015).


Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C.The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and 50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode.The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 of ASB against JBU and HPU was 3.28 ± 0.13 mM and 3.17 ± 0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki = 2.86 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki* = 1.33 × 10−4 mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap.The ICASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases. Helicobacter mustelae, a gastric pathogen of ferrets, was recently found to synthesize a distinct iron-dependent oxygen-labile urease with dinuclear Fe and less activity [2+) and the sulfhydryl group, especially the multiple cysteinyl residues in the active site of the enzyme, are essential for the catalytic activity of all ureases. As the reaction results in the increase of pH values, urease is responsible for negative effects of urease activity in human health and agriculture.Urease  which catalyzes the hydrolysis of urea to produce ammonia and carbon dioxide has been found in plants, algae, fungi, bacteria and soil . In addiHelicobacter pylori (H. pylori) is a well-established etiologic agent of gastritis, gastric and duodenal ulceration, even gastric carcinoma [H. pylori urease (HPU), a highly active urease produced by H. pylori, is a virulence factor in infections of gastrointestinal tracts. HPU could initiate the hydrolysis of urea generating ammonia, which allows the bacterium to survive and colonize the low pH environment of the gastric mucosa [arcinoma . H. pyloc mucosa . TherefoIn past decades, varieties of urease inhibitors have been investigated including phosphoramidates , hydroxa20H30O5), the major diterpenoid lactone and the primary effective constituent of Andrographis paniculata , has multiple pharmacological properties, including antimicrobial [H. pylori activity [H. pylori associated gastritis in clinical practice [20H29O7S · Na, shown in Fig. Andrographolide , urea (molecular biology reagent), D,L-dithiothreitol (DTT) , L-cysteine (L-cys), boric acid and sodium fluoride (NaF) were purchased from Sigma Aldrich. JBU  was also from Sigma Aldrich, of which one unit of urease activity is defined as the amount of enzyme needed to liberate 1.0 μmol of NH3 from urea per min at pH 7.0 at 25 °C. Brucella broth was purchased from Becton–Dickinson. . Other chemicals were obtained from Guangzhou Chemical Reagent Factory (China). All reagents were of analytical grade. Phosphate buffer  was prepared by adjusting pH of phosphoric acid with NaOH. 2 mM EDTA was added to all enzyme-containing solutions.Andrographolide sodium bisulfite  was grown in brucella broth supplemented with 10 % heat-inactivated horse serum for 24 h at 37 °C under microaerobic conditions  [8 CFU/mL) were centrifuged  to collect the bacteria, and after washing twice with phosphate-buffered saline (pH 7.4), the H. pylori precipitation was stored at -80 °C. H. pylori was returned to room temperature, and after addition of 3 mL of distilled water and protease inhibitors, sonication was performed for 60 s. Following centrifugation , the supernatant was desalted through Sephadex G-25 column . The resultant crude urease solution was added to an equal volume of glycerol and stored at 4 °C until use in the experiment.85 % N2) , 31. ForThe standard urease assay mixture contained 50 mM urea in 20 mM phosphate buffer (pH 7.0) containing 2 mM EDTA. After addition of the enzyme-containing solution of 0.25 mg/mL JBU, the assay ran for 20 min, and the enzyme activity was determined by measuring the concentration of the ammonia released in the reaction mixture. For ammonia measurement, aliquots were withdrawn from the reaction mixtures, and the ammonia was determined at 595 nm spectrophotometrically according to the modified Berthelot (phenol-hypochlorite) method  at ambieUrease solutions mixed with serial concentrations of ASB ( 0–6 mM) were incubated at 37 °C for 20 min, which contained 0.25 mg/mL JBU, 20 mM phosphate buffer (pH 7.0), and 2 mM EDTA. The initial time of incubation was defined as the moment once the enzyme and inhibitor were mixed. After appropriate period of time, aliquots from the incubation mixture were transferred into the standard assay mixtures for urease residual activity determination. The activity of uninhibited urease was defined as the control activity of 100 %.KM and the maximum velocity vmax in the absence of the inhibitor were determined by measuring the initial reaction velocities at different urea concentrations ranging from 0.4 to 10 mM. The values were obtained by applying nonlinear regression to the Michaelis-Menten equation.The Michaelis constant Unpreincubated System. The progress curves were determined by the reactions directly initiated by the addition of the JBU into the reaction mixtures containing different concentrations of ASB .Preincubated System. The JBU was preincubated with ASB for 20 min first, and the reaction was then initiated by addition of urea solution into the reaction preincubation mixtures containing different concentrations of ASB .The reaction progress was studied in the absence or presence of ASB using the following two procedures.JBU activities in both procedures were determined as described in standard urease activity assay. A curve-fitting computer program was employed to fit the experimental points to the integrated equation describing slow-binding inhibition progress curves :1\documep is the amount of product accumulated at time t after initiation of the reaction. v0 and vs are the reaction initial and steady-state velocities, respectively, and kapp denotes the apparent first-order velocity constant for interconversion between v0 and vs.where This part of the study was carried out as follows. JBU was first preincubated with different protectors for 20 min. Then, samples of the protected JBU were incubated with 4 mM ASB for additional 20 min. The urease activity was assayed upon incubation of the mixture. For protection by boric acid and NaF, JBU was preincubated with 4 mM boric acid and 4 mM NaF, respectively. For protection by thiols, the applied thiol-containing compounds (L-cys and DTT) were of a series of concentrations.JBU was added to the mixture after a 20 min contact of ASB with the thiol.ASB was added to the mixture after a 20 min contact of JBU with thiol.Thiol was added to the mixture after a 20 min contact of JBU with ASB.The incubation mixtures contained JBU solution, ASB, and mono thiols (L-cys) or dithiol (DTT). The components of the incubation mixture were mixed according to the following three procedures:The complete mixture was mixed thoroughly and incubated for additional 5, 10, 20, and 40 min. Then, JBU activity assay was determined as described in the inactivation of JBU by ASB.After a 20 min preincubation of JBU with ASB (4 mM), the mixture was further incubated with DTT or L-cys for 120 min. The activity of JBU was determined before and after the addition of DTT.ASB (4 mM) was preincubated for 10 and 20 min, respectively, with the enzyme to establish the equilibrium: The reactivation of inactivated JBU was studied in two ways: by using DTT, L-cys and by multidilution in the reaction mixture containing urea.x, y, z ) with a spacing of 0.5 Å and grid maps were built. The center of the grid was set to the average coordinates of the two Ni2+ ions. The Lamarckian genetic algorithm (LGA) was selected as the search algorithm. The Lamarckian job consisted of 100 runs. Default settings were used with an initial population of 150 randomly placed individuals, a maximum number of 2.5 × 106 energy evaluations, and a maximum number of 2.7 × 104 generations. A mutation rate of 0.02 and a crossover rate of 0.8 were chosen. Van der Waals and hydrogen bonding were included in the calculated non-bonded energy. Results differing by less than 0.5 Å in positional root-mean-square deviation (RMSD) were clustered together, and the results of the most favorable free energy of binding were chosen as the resultant complex structures.The automated docking studies were carried out using Auto-Dock version 4.0 as implemented through the graphical user interface AutoDock Tools (ADT 1.5.2). The three-dimensional crystal structure of JBU (PDB code: 3LA4) was obtained from the RCSB Protein Data Bank,whose resolution was 2.05 Å . The required actions were to remove water molecules from the protein, add all hydrogen atoms, calculate Gasteiger charges, and merge nonpolar hydrogen atoms to carbon atoms. The standard 3D structure (PDB format) of ASB was obtained with chem3D Ultra 8.0 software. The PDB files were further transferred to PDBQT files with AutoDock Tools. The three-dimensional results were created by the PyMol molecular graphics system . The cubH. pylori and other bacteria producing urease. Data from Fig. 50 indicated the ASB concentration that could descend the activity of urease to 50 %. The obtained IC50 value of ASB against HPU and JBU was 3.17 ± 0.34 mM (R2 = 0.976) and 3.28 ± 0.13 mM (R2 = 0.997), respectively. The linear function for this relation was a good-enough approximation. Based on the above mentioned, a conclusion could be drawn that ASB performed similar effectiveness on the inhibition of these two ureases.The system of jack bean urease-ASB-urea, can be treated as a model system in the studies on the utility of ASB in the therapy of diseases caused by KM and vmax of ureolytic reaction by applying nonlinear regression to the Michaelis-Menten equation were 2.001 ± 0.08 mM and 0.125 ± 0.07 mM/min, respectively. As the Lineweaver-Burk plots for ASB shown in Fig. KM value did not significantly change in the presence of ASB, while the Vmax value decreased as the ASB concentration increased, indicating that the effect of ASB on JBU might be a noncompetitive mechanism of inhibition. Additionally, our data indicated a slow-binding inhibition relationship of enzyme activity versus preincubation time [EI and EI*. It was clear in Fig. EI) and (EI*) (E + I ⇔ EI ⇔ EI* ) was achieved, which was characterized by the constant urease activity, since the slow-binding effect would not be revealed unless the enzyme interacted with the inhibitor for sufficient time. Otherwise, it would lead to a misinterpretation as a noncompetitive type if determined by the initial reaction rates method. Hence, the progress curves analysis was employed to confirm the slow-binding model of JBU inactivation by ASB.Enzyme kinetics was determined in the absence and presence of various concentrations of ASB. ion time , 34, whiv0) to a much slower steady-state velocity (vs) according to the apparent first-order velocity constant kapp. Such a behavior is characteristic of slow-binding inhibition elaborated by the theory of Morrison and Walsh. The obtained results also showed that the v0 and vs were inhibitor concentration-dependent. In terms of the preincubation system , being different from each studied inhibitor concentration.The progress curves for urea hydrolysis under ASB-inhibited JBU catalyzation were shown in Fig. v0; vs) versus the inhibitor concentration is characteristic of a two-step enzyme inhibitor interaction, mechanism B described as follows,The obtained relationship of the reaction velocities  respectively.where v0 and vs on the inhibitor concentration according to formula [Ki = (2.86 ± 0.09) × 10−3 mM, followed by a slow isomerization into the final ASB-urease complex with the overall inhibition constant of Ki* = (1.33 ± 0.11) × 10−4 mM. The rate constant of the ASB-JBU isomerization indicated that forward process was rapid in contrast with slow reverse reactions. The overall inhibition constant obtained by the steady-state analysis was (1.18 ± 0.13) × 10−3 mM. Furthermore, the shape of the curves in that case corresponded to the competitive slow-binding type of inhibition, as represented by formula [v0. Then, in the later period, the inhibition became stronger, characterized by lower reaction rates vs. This indicated a competitive inhibition in both initial and steady-state stages of the inhibition reaction.By calculating from reciprocal dependence of  formula , it was Taken together, the progress curves analysis and preincubation studies proved that the ASB inhibition on urease was indeed in a slow-binding and competitive manner.Up to now, it has been found that there were two well-defined urease protectors. One is the inorganic compounds (NaF and boric acid) reacting with active-site nickel ions, and the other is thiol-containing compounds such as DTT and L-cys, which interact with sulfhydryl groups of urease , 36. WheThe role of thiols in ASB inactivation was studied by comparing urease activities in thiol-free system at three time points of incubation. It was found that monothiol (L-cys) and dithiol (DTT) could alleviate the inactivation by ASB, and urease regained activity in spite of ASB presenting in the incubation mixture. When the thiol-containing compounds provided thiol groups, concentration was equal or higher than that of ASB. However, as Fig. The presence of the thiol-protector in the incubation system allowed ASB to react with thiols from the JBU and those in the “free” thiol-protector. The thiols presenting in the protein were much less reactive than those presenting externally in the form of L-cys or DTT. The decreases of urease activity in the thiol-free system and system with the thiols were compared, suggesting that the general losses of urease activity in both systems remained, but it was slowed down in the presence of thiols, especially in the presence of L-cys. These data suggested that ASB-thiol interaction was strategic for the inactivation rate decrease.To investigate whether the inactivation of JBU by ASB was reversible, the reactivation of ASB-inactivated JBU was carried out in two ways. In the first way, by addition of L-cys or DTT after the 20 min incubation of JBU with ASB, urease activity recovered in a time-dependent manner: after 1.5 h, JBU had restored 90.77 % and 70.98 % of its initial activity, respectively . Taken together, there would be a supposed reversibility between urease and ASB, in which the chemical approach but not multidilution could recover the enzyme activity that had been inactivated by ASB.In order to elucidate the inhibition mechanism revealed by the kinetics study, molecular docking of ASB into the crystal structure of JBU (3LA4 in the Protein Data bank) was performed by the AutoDock program, and the best possible binding modes were shown in Fig. 3H of ASB, as the hydrogen bond donor ,was found between the OH and the backbone S atom of CME 592 (H∙ ∙ ∙S distance = 2.7 Å), which was located on the mobile flap closing the active site of the enzyme. In addition, the 12- SO3H of ASB was bound via two hydrogen bonds to NH2 of ARG 439 with O∙∙∙H distance of 2.7 Å and 2.8 Å, respectively. The Cys-592 (marked as CME592) is a key residue located at the mobile flap covering the active site, one per each of the six sites in the hexameric molecule [In the best possible binding mode, ASB tightly anchored the helix-turn-helix motif over the active-site cavity through O − H∙ ∙ ∙S, N − H∙ ∙ ∙O, and O − H∙ ∙ ∙O hydrogen bonding interactions. This mode made ASB engage a cleft beside the active-site cavity, using 3 typical hydrogen bonds to anchor the flap tightly with the backbone of the enzyme, thereby preventing the flap from backing to the close position. The 12- SOmolecule . Besidesmolecule , 39. Themolecule . And modmolecule .As the results described, ASB possibly made hydrogen bonding interactions with the side chains of the above-mentioned residues, especially the active-site flap Cys-592, hence preserving the flap in an open conformation and resulting in an inactivation. The observation was soundly supportive of the earlier conclusion drawn from the urease protection experiments performed with the active-site binding inhibitors, which supported the observation that inhibition by ASB involved the participation of sulfhydryl group of the active-site cysteine. Taking into account the peculiarities of the active-site flap cysteine in the urease catalysis and sulfhydryl group in urease activity, it could be inferred that ASB made contacts with the side chains of cysteine residues, especially sulfhydryl group, which was reflected in their enhanced affinity to the Cys-592. As a result, the mobility of flap was restricted, and the enzymatic activity significantly declined finally.Based on the research on the mechanism and kinetics of urease inhibition by ASB in the present study, it could be concluded that ASB was a competitive inhibitor targeting thiol groups in the active site of urease in a slow-binding manner, both concentration dependent and reversible. Hence, ASB deserves to be further exploited as a promising urease inhibitor for treatments of urease-related diseases.


This study was aimed to investigate the roles of serum macrophage inflammatory protein-3α (MIP-3α) and cystatin A in nasopharyngeal carcinoma (NPC) prognosis.The serum levels of MIP-3α and cystatin A in 140 primary NPC patients without distant metastasis were detected by enzyme-linked immunosorbent assay before and after treatment. The results were compared with those in 100 healthy controls. The log-rank test was used to compare survival curves of the 2 groups. Multivariate analysis of prognostic factors used Cox proportional hazards regression model.Serum levels of MIP-3α and cystatin A in pretreatment patients with NPC were higher than those in healthy controls. Concentrations of these 2 factors in the majority of patients after the therapy decreased to control level. Patients with high serum level of MIP-3α and cystatin A before treatment had poorer overall survival (OS), local recurrence-free survival, and distant metastasis-free survival than the ones with low level. In addition, serum pretreatment MIP-3α and cystatin A levels were independent prognostic factors for OS and distant metastasis-free survival of NPC patients; serum posttreatment MIP-3α and cystatin A levels were independent prognostic factors of local recurrence-free survival.Our results revealed that serum MIP-3α and cystatin A may be promising candidate prognostic factors for NPC, and higher serum levels of MIP-3α and cystatin A correlate with shorter probability of OS, local recurrence, and distant metastasis. However, the fact that patients with the same clinical stage have various therapeutic effects indicates that TNM stage does not accurately predict the prognosis for NPC. So far, it is mostly dependent on imaging examination for checking NPC residual lesion, relapse, and distant metastasis, evaluating the sensitivity of radiotherapy and chemotherapy, and predicting prognosis. Identification of serum tumor markers of NPC is urgently needed and may be helpful for convenient and noninvasive early disease screening, prognosis prediction, treatment planning, and residual or recurrent tumor monitoring.Nasopharyngeal carcinoma (NPC) constitutes a serious health problem in southern China as it is one of the most common cancers there. Wuzhou City of Guangxi province in southern China is one of the endemic areas with high NPC incidence.6 Chemokines are classified into 4 families  based on the number and spacing of cysteine residues and exert their biological effects by interacting with specific cell surface receptors that belong to transmembrane G protein-coupled receptor family. These chemokines can activate the genes associated with cell movement, invasion, and metastasis through different transmembrane signal pathways. Macrophage inflammatory protein-3α (MIP-3α), encoded by the CCL20 gene, is a chemokine that induces leukocyte migration into inflammation sites and regulates leukocyte trafficking through lymphoid tissues; and its expression has effects on tumor cell proliferation, angiogenesis, inflammatory cell infiltration, invasion, and metastasis.7 Lysosomal cysteine proteases (cathepsins) may degrade extracellular matrix, which is a key step for tumor cells breaking through the basement membrane and then causing invasion and metastasis. So far, more and more reports suggest that cystatin families, as the endogenous inhibitors of cathepsins, are closely associated with tumorigenesis, development, invasion, and metastasis, and it may be a novel tumor marker for clinical diagnosis and prognostic prediction.8An important pathological feature of NPC is a wide range of lymphocytic infiltration. Although the characteristics of tumor infiltrating lymphocytes have not yet been elucidated, interaction between tumor cells and tumor-infiltrating lymphocytes in NPC microenvironment may play an important role in tumor formation, and cytokines may mediate the interaction as well. Chemokines are recently reported to be involved in tumor proliferation, progression, and metastasis.10 The clinical significance of serum MIP-3α and cystatin A levels at different time points (pretreatment vs posttreatment) has not been fully explored. In this prospective study, we evaluated the serum MIP-3α and cystatin A levels before and after treatment in NPC and correlated the results with treatment outcome.MIP-3α and cystatin A have been reported as novel biomarkers and prognosis predictors for NPC.11 The cancer stage was defined according to the Chinese 2008 staging system12  type I, nonkeratinizing differentiated carcinoma as type II, and nonkeratinizing undifferentiated carcinoma as type III.2 was delivered over 3 days for 3 cycles on d1, d22, and d43 during the course of radiotherapy as concurrent chemotherapy. Subsequent adjuvant chemotherapy consisted of 25 mg/m2 and 5-fluorouracil 450 mg/m2 injected on days 1 to 3 for 2 courses. Peripheral blood was taken before the commencement of treatment and after the completion of treatment.All patients underwent radical radiotherapy for NPC. For patients with stages I–II, the treatment was radiotherapy alone, whereas for those with stages III–IVa, chemotherapy was added to radiotherapy. Two radiotherapy technologies were used, including conventional 2-dimensional radiotherapy and 3-dimensional conformal radiotherapy. The accumulated doses to gross primary tumor and involved neck lymph nodes were 68 to 74 Gy and 64 to 70 Gy, respectively; the uninvolved areas received 50 to 56 Gy. Chemotherapy consisted of cisplatin and 5-fluorouracil. The chemotherapy regimen of cisplatin 25 mg/mBlood samples without any evidence of disease were obtained from 100 healthy individuals as controls. There were 71 male participants and 29 female participants; their median age was 49 years, with a range of 21 to 75 years. The clinical research ethics committee of Wuzhou Red Cross Hospital approved this study.Sera from peripheral blood samples were obtained by venipuncture at study enrollment and stored at −70°C until use. Serum levels of MIP-3α (expressed in picogram/milliliter) and cystatin A (expressed in nanogram/milliliter) were determined using a commercially available human MIP-3α or cystatin A quantitative Enzyme-Linked Immunosorbent Assay kit  according to the manufacturer’s instructions. All standards and samples were prepared and tested in duplicate and the mean value taken. The levels of MIP-3α or cystatin A were separately calculated from the standard curves.The OS was calculated from the first day of chemoradiotherapy until the date of death or until the date of the last follow-up. The local recurrence-free survival (LRFS) was calculated from the first day of chemoradiotherapy until the date of either primary lesions in the nasopharynx or regional lymph node recurrence or until the date of the last follow-up. The distant metastasis-free survival (DMFS) was calculated from the first day of chemoradiotherapy until the date of distant metastasis or until the date of the last follow-up.P values were 2-sided and values <0.05 were considered as statistically significant.The levels of serum MIP-3α and cystatin A in different groups were compared using the Mann–Whitney rank sum test. Survival curves were plotted by the Kaplan–Meier method and compared by the log-rank test. The significance of various variables for survival was analyzed by the Cox proportional hazards regression model. Variables that were included in the model were age, sex, pathological type, tumor (T)-classification, lymph node (N)-classification, and chemotherapy along with MIP-3α and cystatin A levels before and after treatment. All statistical analyses were performed using SPSS version 13.0 , and all The end of follow-up was January 26, 2014. After a median follow-up of 48 months , 9 patients were lost to follow-up, 11 patients were reported with local recurrence, 16 with distant metastasis, 5 with local recurrence plus distant metastasis, and 25 deaths. The 4-year follow-up rate was 93.6% (131/140). The 4-year OS, LRFS, and DMFS rates for all patients were 84.9%, 88.6%, and 84.7%, respectively.P = 0.252 and 0.850) . Elevated cystatin A levels were more likely to appear in the older group (age ≥ 45 years) than in the younger group (P < 0.001).The potential relationships between clinical characteristics and serum levels of MIP-3α and cystatin A are shown in Table r = 0.217, P = 0.010).Spearman rank correlation analysis showed that pretreatment serum level of MIP-3α correlated weakly with cystatin A (P < 0.001), 4-year LRFS , and 4-year DMFS  than those with low pretreatment MIP-3α level group. Further, 59 (42.1%) had higher posttreatment MIP-3α level (MIP-3α ≥ 18 pg/mL). Patients with high posttreatment MIP-3α level had a poorer 4-year OS  and 4-year LRFS  than those with low posttreatment MIP-3α level; there was no statistical difference in 4-year DMFS curves between the 2 groups   was selected to categorize patients as high pretreatment MIP-3α and low pretreatment MIP-3α. Among all patients, 29 (20.7%) had higher pretreatment MIP-3α level (MIP-3α ≥ 110 pg/mL). The OS, LRFS, and DMFS curves for the high pretreatment MIP-3α level group and low MIP-3α level group are shown in Figure P < 0.001), 4-year LRFS , and 4-year DMFS  of high pretreatment cystatin A level group were significantly lower than those of low pretreatment cystatin A level group , 4-year LRFS , and 4-year DMFS  of high posttreatment cystatin A level group were also significantly lower than those of low posttreatment cystatin A level group  was selected to categorize patients as high pretreatment cystatin A and low pretreatment cystatin A. There were 45 (32.1%) patients with higher pretreatment cystatin A level (cystatin A ≥ 20 ng/mL). All the 4-year OS , cystatin B, and cystatin C.Our study suggests that serum MIP-3α and cystatin A can be used as new markers to define patients with a high risk of metastasis who warrant more aggressive systemic therapy to improve long-term outcome. Combining T and N stage with pretreatment and posttreatment MIP-3α and cystatin A levels may provide new clinical criteria of patient selection for combined modality treatment.We found statistically significant correlation between MIP-3α and cystatin A. They may be intrinsically linked together to affect the pathogenesis and development of NPC. The specific mechanism still requires further research.Our results indicate that MIP-3α and cystatin A could serve as promising candidate markers in NPC prognosis. Higher serum levels of MIP-3α and cystatin A correlated with shorter probability of overall survival, local recurrence, and distant metastasis in univariate and multivariate analysis. However, a study of a large population is needed to evaluate the long-time prognostic importance and clinical relevance of these 2 factors in NPC.


In mammals, the ability to sense and respond to both intracellular and extracellular nutrient levels requires the integration and cooperation of multiple complex metabolic regulatory networks. Key among these are the mTOR and AMPK signaling pathways, which are activated in response to increased or decreased cellular energy levels, respectively. These pathways control cell growth, proliferation, metabolism and autophagy, and their dysregulation has been associated with both cancer and metabolic disease. The anti-diabetes drug metformin has been shown to function, at least in part, through activation of AMPK, however it also inhibits mTORC1 signaling, suggesting a potential role for mTORC1 inhibition in the beneficial effects of metformin. AMPK can suppress mTORC1 through activation of the TSC complex, an inhibitor of mTORC1, or inhibition of Raptor, an essential component of mTORC1. The liver plays a critical role in the maintenance of systemic metabolic homeostasis, and is thought to be the major target organ for the physiological effects of metformin, thus we are investigating the hepatic regulation of mTORC1 by metformin.The effects of metformin on mTORC1 were assessed in livers and primary hepatocytes from LTSC1KO mice that have conditional loss of TSC1 in the liver. mTORC1 activity was determined by analysis of the phosphorylation state of its targets, and the rate of protein synthesis using 35S-methionine incorporation. Physiological effects of metformin were determined using glucose and metformin tolerance tests.Dose-response and time-course assessments of mTORC1 activity in response to metformin show that inhibition of mTORC1 signaling is resistant to metformin in primary hepatocytes of LTSC1KO mice under conditions that fully suppress mTORC1 signaling in control hepatocytes. The rate of protein synthesis is also maintained at a higher level in these hepatocytes. Furthermore, while metformin results in decreased phosphorylation of mTORC1 targets in the livers of control mice, its activation is not as severely compromised in livers of LTSC1KO mice. However, conditional loss of TSC1 in the liver seems to have no effect on the systemic glucose lowering ability of metformin.Our findings demonstrate that conditional disruption of the TSC1-TSC2 complex desensitizes hepatocytes to the inhibition of mTORC1 by metformin, suggesting a dominant role for this tumor suppressor complex in the hepatic control of mTORC1 by energy stress. We are continuing to use this genetic model to determine the contribution of mTORC1 inhibition by metformin in the liver to the cellular and physiological effects of metformin.


ENO) levels over a one‐year period affects IgE concentrations. Altogether, 158 relatively well‐controlled but multi‐sensitized asthmatics (age 18–65 years), with ongoing ICS treatment at baseline, were included in this post hoc analysis of data from a randomized, controlled trial on FENO‐guided asthma therapy. Asthma control and quality of life (Juniper ACQ and mAQLQ), FENO, and serum IgE were measured at baseline and after one year. Concentrations of IgE antibodies to six common perennial aeroallergens were summed up . We found that perennial and total IgE decreased by 10.2% and 16.0% (P < .001 both comparisons). This was not related to allergen exposure, whereas the total use of ICS and LTRA during the year correlated with the reduction in perennial IgE (P = .030 and P = .013). The decrease in perennial and total IgE correlated significantly with the reduction in FENO (P < .003 and P < .001), and with improvements in ACQ and mAQLQ scores . We conclude that one year of optimization of treatment with ICS and LTRA in patients with persistent atopic asthma resulted in significant decreases in total IgE and IgE antibodies; these decreases correlated with a reduction in FENO and improvements in asthma control and quality of life. Thus, IgE is reduced by ordinary asthma controller medications and the effect on IgE seems to be clinically important.Immunoglobulin E (IgE) is crucial for the development of airway inflammation in atopic asthma, and inhibition of IgE using monoclonal antibodies is now part of asthma therapy. However, the impact of ordinary anti‐inflammatory treatment on IgE is unclear. The aim of this study was to investigate if optimization of treatment with inhaled corticosteroid (ICS) and leukotriene‐receptor antagonist (LTRA) according to symptoms or exhaled nitric oxide (F ENO), and recruitment and activation of eosinophilic granulocytes and mast cells Asthma is by definition an inflammatory disorder of the airways. Immunoglobulin E (IgE) is often involved in the inflammation, as the majority of asthmatics have atopic asthma, which is most commonly initiated in childhood by sensitization to airborne allergens Reports on the effect of corticosteroid treatment on IgE concentrations in patients with atopic asthma have been equivocal and few long‐term studies have been done. There are unanimous reports of an initial transient increase of IgE during the first 1–2 weeks of treatment with systemic corticosteroids ENO‐guided asthma therapy (the NOAK study), we merged data from two study groups (FENO‐guided or symptom‐guided asthma therapy) for analysis of one‐year follow‐up data In this post hoc analysis of a randomized controlled trial of FOptimisation of anti‐inflammatory asthma treatment using exhaled NO to improve asthma‐related quality of life within primary health care (the NOAK study), which was an open‐label, parallel‐group, randomized, controlled study, conducted at 17 primary health care centers in central and southern Sweden from November 2006 to March 2010 ENO‐guided treatment or control group  and were followed up at four visits over a period of one year. Those who had donated blood samples both at baseline and at the last visit of the study (n = 158) were included in this analysis. Eligible participants had a doctor's diagnosis of asthma, had been on prescribed ICS treatment for at least the past six months, and had confirmed IgE sensitization to at least one major airborne perennial allergen . Participants were 18–65 years old, non‐smokers for at least the past year, and with a smoking history of <10 pack‐years. For the FENO‐guided group, the anti‐inflammatory treatment was adjusted using an algorithm based on FENO ENO measurement was double‐blind and treatment was adjusted according to routine clinical practice . ICS and LTRA were used as anti‐inflammatory treatment and participants in both groups were placed in one of six fixed treatment steps, where LTRA was added at the two highest steps (Table S1). At every visit, the following data were registered: FENO, self‐reported exposure to pets, and dampness in the home (window pane condensation), asthma control measured with Elizabeth Juniper's Asthma Control Questionnaire (ACQ), and LTRA and ICS use  4‐antibody concentration, and serum eosinophilic cationic protein (S‐ECP) at the start and end of the study (See Table S2 for a study design summary). IgE, IgG4, and ECP were analyzed in a Phadia® 100 system with ImmunoCAP® reagents . IgE antibodies measured = Dermatophagoides pteronyssinus, Dermatophagoides farina, cat, dog, horse, timothy, birch, Cladosporium herbarum, and mugwort. IgG4 antibodies measured = cat and timothy. Serum samples were initially stored at −20°C (<6 months) and then at −80°C. Samples from each patient from the start and end of the study were analyzed side‐by‐side in the instrument for all serum measurements. Pollen count data were collected during 2006–2009 from three stations, Stockholm, Forshaga, and Malmö, which were as close to the participating primary health care centers as possible (see online repository for more information).All data in this study were collected from the study tudy n = 8 were inThe study was approved as a multi‐center study by the regional ethics committee in Stockholm (Dnr: 2006/185‐31). All participants provided written informed consent.ENO (≥24 ppb in women and ≥26 ppb in men)—or with more symptoms, depending on which group they belonged to—received more treatment, and participants with low FENO or few symptoms received less treatment ENO in both study groups, probably due to some degree of covariation of symptoms and FENO ). This resulted in a significant decrease in FENO in subjects with elevated FENO at baseline in both groups, with no significant difference between the groups . When results from the subjects with elevated baseline FENO in the two study groups were analyzed together, this showed an increase in the proportion of participants with LTRA from 2.5% to 22.2% (P < .001), mean ICS use increased by 180 μg/day (P < .001), and mean FENO decreased by 13.5 ppb (P < .001).IgE was a secondary endpoint in the original protocol. However, when analyzing the change in IgE, it was found that most IgE was reduced in both treatment groups, with no significant difference between groups (Table S3). Since a reduction in IgE has been rarely reported, we wanted to examine what variables associated with reduced IgE. For this post hoc analysis, the two groups were merged to increase the number of patients. Even though the treatment was adjusted on different grounds in the original study groups, both groups had to use the same treatment steps (Table S1). Mean ICS use did not change significantly within or between study groups during the study, but there was a significant redistribution of treatment steps in both groups perennial allergens , seasonal allergens , food allergens , and all allergens . Two‐sided tests with P‐values less than .05 were considered significant and indicated in bold in figures. Results were analyzed with Mann–Whitney U‐test for comparison between groups and Wilcoxon signed‐rank test for analysis of change within groups between first and last visit, or the corresponding Student's t‐tests. The effect of inclusion in different quarters of the calendar year was analyzed with Kruskal–Wallis test and corrected for multiple comparisons. Categorical data were analyzed with χ2 test, or Fisher's exact test when applicable. Spearman's rank test or Pearson's test were used for correlation tests. For IgE antibody measurements, values down to zero were used in the calculations. IgG4 and IgE data were converted to the log base 10 scale and analyzed using a paired t‐test when applicable. IgE values of zero were replaced by .001 before conversion to log base 10 scale. Statistical analyses were performed with Stata/IC11 version 11.2 . The study is registered with ClinicalTrials.gov (identifier NCT00421018).The concentrations of individual IgE antibodies were grouped and summed up. The groups were: Baseline characteristics and one‐year follow‐up data for included patients are presented in Table 4‐antibody concentrations against cat and timothy did not show any significant change during the study . These relations were not consistent at last visit except for perennial IgE. There was a positive correlation between the reduction in FENO and relative reduction in IgE concentrations for perennial IgE, all IgE and total IgE, and a trend for seasonal IgE (Table ENO (<20 ppb) between baseline and last visit, we observed a significant difference in the change in seasonal and all IgE as well as total IgE, and a trend for perennial IgE (Table S8). The change in S‐ECP showed a positive correlation with the change in perennial IgE only . Concentrations of IgE antibodies against pets were significantly higher both at the beginning and end of the study in those who had daily contact with pets than in those who never had contact. However, both groups significantly lowered their IgE concentrations for pets during the study, with no significant difference between the groups (Table S11). Further, there was no difference in the proportion of patients reporting dampness in the home between baseline and last visit (P = .27).Thirty‐one participants were exposed daily to relevant pets (cat and dog) and 84 reported that they never had contact with pets during the study (Table S10). At the study start, 28 participants reported a pet at home and six of them removed their pet during the study. Two participants got a pet during the study. The proportion of participants with pets at home compared with those who reported no contact with pets did not change significantly between baseline and last visit (P < .01 both comparisons).There was no major difference in reported pollen levels between the years of the study, except that birch pollen were high in 2006 . Fifteen participants were included during the year after the heavy birch pollen season 2006. These participants did not have higher IgE concentration for birch at inclusion than those who were included after the birch pollen season 2007 and onwards, but did have a significantly larger reduction of IgE against birch during the study, although both groups showed a significant reduction in birch IgE concentration (Table S11). However, there was no significant difference between the changes in other types of IgE between these two groups (Table S13). Analysis of the influence of the time of year when a patient was included in the study revealed a significant difference for seasonal and food IgE but not for the other IgE groups or total IgE. Those included during quarter two (April–June) had a significantly smaller reduction in seasonal IgE compared to those included during quarter four (October–December) and a smaller reduction in food IgE compared to those included during quarter three (July–September)  in asthmatics n = 2300) in adults 30–40 years of age showed unchanged prevalence of IgE sensitization to basically all aeroallergens and increased total IgE concentrations over a 9‐year follow‐up period The major finding of our study was a substantial decrease in IgE concentrations, with relative reductions of 10–20% for most IgE antibodies measured, as well as for total IgE. IgE to mold showed the largest relative reduction, but the mean baseline IgE‐antibody concentration was low for this allergen. Interestingly, not only IgE to aeroallergens but also to food allergens was significantly reduced in this study. We chose to include IgE to food in the analysis since a large proportion of adult asthmatics seem generally to be sensitized to food ENO or symptoms.Very few data on changes in IgE concentrations due to treatment with anti‐inflammatory drugs are available, and the results of earlier studies have been contradictory. In the 1970s, a transient increase of IgE concentrations, followed by a decrease to baseline or below, was described in children and adults with mainly steroid‐naïve atopic asthma who were treated with a short course of oral corticosteroids ENO. This may be due to the fact that reductions in IgE, but not FENO, related to improvements in the environmental (trend) and emotional domains of the mAQLQ instrument.Interestingly, the reduction in relevant groups of IgE antibodies and total IgE correlated with improvement in asthma symptom control, measured with ACQ and the symptom domain of mAQLQ. More importantly, the reduction in IgE was significantly larger in subjects showing a clinically important change in ACQ score. Thus, control of IgE formation may be related to asthma symptom control. Furthermore, improvement in asthma‐related quality of life correlated with reductions in relevant IgE‐antibody groups and total IgE, but not with changes in FENO and the relative change of important groups of IgE antibodies and total IgE. Serum ECP, which signals systemic eosinophilic inflammation, correlated only with the relative change of perennial IgE antibodies. However, blood eosinophil count may be a better marker of (IgE‐mediated) systemic Th2‐driven inflammation ENO seemed stronger than that between anti‐inflammatory drug use and changes in IgE. This is reasonable since the confirmed anti‐inflammatory effect (reduction in FENO) should be more important for effects on IgE levels than the amount of drug given per se. From a mechanistic point of view, it is reasonable to suggest that anti‐inflammatory treatment with ICS and LTRA can reduce IgE synthesis since both agents inhibit the Th2 lymphocyte, resulting in reduced secretion of IL‐4, IL‐13, and IL‐5 ENO through reduction of IL‐4 and IL‐13 release ENO, leads to reduced formation of IgE We could for the first time show a significant correlation between the change in F4 antibodies to the two common aeroallergens cat and timothy did not change at all during the study. The concentration of IgG4 antibodies has been shown to correlate with the degree of cat allergen exposure in the home regardless of the degree of IgE sensitization 4‐antibody concentrations ENO or symptoms. Thus, those with low FENO or fewer/milder asthma symptoms received less treatment and those with high FENO or more/stronger symptoms increased their use of ICS and LTRA. As a result, participants in both study groups with a high baseline FENO increased their LTRA and ICS use, and FENO decreased significantly in these patients without any significant difference between the two groups. A relationship between a change in FENO and a change in IgE concentrations was seen at the individual level in this study. However, whereas IgE concentrations were significantly reduced at the group level, FENO was not. This may seem contradictory but could be explained by the fact that the overall ICS use did not change during the study while LTRA use increased significantly. It is well known that FENO is strongly affected by ICS treatment but much less so by LTRA treatment A strength of the present study is that all paired serum measurements were analyzed side‐by‐side in the instrument. Another strength is the thorough collection of data on both outdoor (grass and tree pollen) and indoor  allergen exposure. These analyses showed very consistent exposure levels over the study years, with the exception of high birch pollen counts during the spring of 2006. This extreme season slightly strengthened the effect on IgE to birch pollen over one year but did not affect the reduction of IgE to the other allergens or total IgE. Furthermore, IgGENO as well as with improvements in asthma control and asthma‐related quality of life. While FENO signals short‐term (days and weeks) changes in Th2‐driven airway inflammation, the IgE concentration may reflect more long‐term positive effects (months and years) of anti‐inflammatory treatment that are clinically important.In conclusion, this one‐year follow‐up study on relatively well‐controlled adults with atopic asthma and ongoing inhaled corticosteroid treatment at baseline, using merged data from a randomized, controlled trial, showed that optimized treatment with inhaled corticosteroids and a leukotriene‐receptor antagonist resulted in a marked decrease in both total IgE and IgE‐antibody concentrations. The decrease in total IgE and perennial IgE‐antibody concentration correlated with reductions in FKA has received research support from Aerocrine AB and Thermo Fisher Scientific. MB is an associate of Thermo Fisher Scientific. JS has received research support from Aerocrine AB. AM, ALU, AA, and ML declare no conflict of interest.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Table S1. Treatment steps allowed in the study.Table S2. Study design.Table S3. Change in IgE in the two original study groups.Table S4. Correlation analysis between age and IgE concentrations at baseline and last visit.Table S5. Correlation analysis between age and relative change in IgE concentrations over one year.Table S6. Comparison of gender and relative change of IgE concentrations over one year.Table S7. Correlation analysis between IgE concentrations and FENO at baseline and last visit.Table S8. Median (IQR) change in IgE concentrations (kUA/L) in subgroups moving up or down between the normal and elevated range of FENO during the study (cut‐off 20 ppb).Table S9. Correlation analysis between relative change in IgE concentrations and change in mAQLQ domain scores over one year.Table S10. Exposure to pets during the study.Table S11. Comparison of change in IgE concentrations after different exposures to pets and birch pollen.Table S12. Sum of AUC for different pollens in Stockholm, Forshaga, and Malmö.Table S13. Median relative change in IgE concentrations over one year.Figure S1. Birch pollen count in Stockholm during the spring, 2006 and 2007. AUC, area under curve.Click here for additional data file.


Two polar vessels, Akademik Shokalskiy and Xuelong, were trapped by thick sea ice in the Antarctic coastal region just to the west of 144°E and between 66.5°S and 67°S in late December 2013. This event demonstrated the rapid establishment of extreme Antarctic sea ice conditions on synoptic time scales. The event was associated with cyclones that developed at lower latitudes. Near the event site, cyclone-enhanced strong southeasterly katabatic winds drove large westward drifts of ice floes. In addition, the cyclones also gave southward ice drift. The arrival and grounding of Iceberg B9B in Commonwealth Bay in March 2011 led to the growth of fast ice around it, forming a northward protruding barrier. This barrier blocked the westward ice drift and hence aided sea ice consolidation on its eastern side. Similar cyclone-induced events have occurred at this site in the past after the grounding of Iceberg B9B. Future events may be predictable on synoptic time scales, if cyclone-induced strong wind events can be predicted. Sea ice movement is strongly affected by the atmospheric circulation  and Xuelong (an ice breaker), were trapped by thick sea ice in the Dumont d'Urville Sea , just to the west of 144°E and between 66.5°S and 67°S. This extreme event, which has attracted wide press and public attention across the world, demonstrated that thick sea ice can be created on synoptic time scales at this specific site. The ice was too thick  for Xuelong to navigate, as Xuelong can only break sea ice with a thickness of less than 1.2 m. This event offers an important opportunity to investigate how strong winds associated with cyclones force Antarctic sea ice change and even lead to the rapid creation of extreme sea ice conditions in the Antarctic coastal region.https://earthdata.nasa.gov/labs/worldview/) around the site where the two vessels were trapped in late December 2013 (hereafter termed “the site”) was characterized by ice fixed to the coast (fast ice) connected to Iceberg B9B to the west (http://www.scp.byu.edu/data/iceberg/ascat/b09b.ascat). The grounding of Iceberg B9B provided favorable conditions for fast ice to grow around it, making Commonwealth Bay a permanent ice-covered region after March 2011.Sea ice distribution from high resolution MODIS (The Moderate Resolution Imaging Spectroradiometer) imagery  . Output from the Antarctic Mesoscale Prediction System that employs a meso-scale weather prediction model for the Antarctic region shows that the strongest wind is found around , with the annual mean wind speed being approximately 20 m/shttp://www.ecmwf.int/products/data/operational_system/) for four cases were analyzed  were analyzed to identify peak wind speeds and corresponding cyclone intensities in Decembers over the period of 1979 to 2012. The strong southeasterly winds at  around 23 December 2013 were not exceptional (but in the highest 29%), as stronger wind events occurred in Decembers of other 8 years derived from ERA-Interim reanalysis , along with wind stress derived using 10-m zonal and meridional wind components and the method in ref. Sea ice concentration from the OSTIA  datasetIn addition to fast ice connected with the continent at several places, there is a large amount of mobile sea ice in a broad region extending northward to about 63°S–64°S between 135°E–150°E. There were katabatic winds on the Adélie Land, with weaker winds to the east of this land. In the coastal region and around the site, katabatic winds are predominantly from the southeast, turned from the downslope direction by the Coriolis force. To the north of the site, the winds generally come from the north and are turned westward.For ice floes that have weak internal ice stress, sea ice velocity can be estimated by the terms of air-ice and ice-water stress, and the sea surface tiltTo further illustrate how sea ice was moved by the wind during this event, potential sea ice drift velocity is also overlaid on sea ice concentration. Here, potential sea ice drift velocity is simply obtained by assuming a constant wind factor (the ratio of ice drift speed and overlying 10-m wind speed) of 2% and the turning angle is 30°, as ice-water stress and sea surface tilt data are not available for a quantitative estimation. The wind factor has been reported to increase with wind speed1213Just to the east of the site, sea ice drifted mainly westward under the forcing of southeasterly winds, with the drift speeds, for example, of 0.15 m/s at 00, 06 and 12 UT and 0.25 m/s at 18 UT at 66.5°S, 146°E. Further to the north of Mertz polynya, there was some southward drift of sea ice. This wind-driven sea ice drift made the sea ice retreat towards the regions covered by very compact fast ice, and become much more consolidated .The large open water area to the east of the site on 3 December 2013  occurredCyclone-induced strong wind events around the site where two polar vessels were trapped were characterized by dominant southeasterly winds, as the Coriolis force turned the katabatic winds to the left when they approached the coastal region. Towards the event site, these strong southeasterly winds drove a large westward drift of ice floes, and the cyclones also drove southward ice drifts. The grounding of Iceberg B9B in Commonwealth Bay in March 2011 and the subsequent growth of fast ice around it formed a barrier for mobile ice, leading to the sea ice packing and hence extreme sea ice conditions on the eastern side of this barrier. Thus, the processes in the polar climate system on both long and short time scales caused this polar vessel beset event.https://earthdata.nasa.gov/labs/worldview/) after a cyclone-induced strong southeasterly wind event around 1 December 2012.The Adélie Land coastal region of Antarctica has been identified as one of the most prominent cyclogenesis regions in the Southern HemisphereThere is no doubt that similar events will happen again at this specific site with northward protruding fast ice. Future navigation should, if at all possible, avoid going to those regions where frequent strong katabatic wind events occur and with northward protruding barriers, such as fast ice, glacier tongues, or land, when large amounts of sea ice are present. Cyclone-induced strong southeasterly winds can result in extensive westward drift of sea ice, and hence have great potential to generate extreme sea ice conditions on the eastern side of these northward protruding barriers.These results demonstrate the important role of polar cyclones or storms in the rapid creation of extreme sea ice conditions. Poleward shifts of extratropical storm tracks and intensifications of polar storm activities in a warming climate have been detected  were used to show sea ice changes around the location where the two polar vessels were trapped. Mean sea level pressure (MSLP) fields and 6-hourly 10-m wind fields from the ECMWF operational analysis , which has a horizontal resolution of 14 km, were analyzed to examine changes in atmospheric circulation. The peak wind speed and the associated cyclone intensity in December 2013 were compared with those in Decembers over 1979 to 2012 derived from the ERA-interim reanalysis 6-hourly datahttp://www.ecmwf.int/products/data/archive/descriptions/ei/index.html). The OSTIA  daily sea ice data (with a resolution of 5 km)http://ghrsst-pp.metoffice.com/pages/latest_analysis/ostia.html) was employed for the purpose of illustrating cyclone-induced sea ice movements. Wind stress fields are derived using 10-m zonal and meridional wind components and the method in ref. High resolution MODIS (The Moderate Resolution Imaging Spectroradiometer) daily images (Z.W. and J.T. conceived the study. Z.W. conducted the analysis, C.L. prepared Fig. 1, and J.T., B.S., B.L., C.L. offered useful insights during this processes. Z.W. prepared the first draft, and J.T., B.S., B.L., C.L. joined in the writing of the paper.