Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the DOI:http://dx.doi.org/10.7554/eLife.11620.001 The element nitrogen is required for all forms of life, and is an essential component of important biological molecules such as DNA and proteins. The most abundant form of nitrogen is dinitrogen, which comprises 78% of the Earth’s atmosphere. However, dinitrogen is highly unreactive, and so the nitrogen must be converted into a more reactive form before it can be used biologically. The only known enzyme capable of carrying out this reaction is called nitrogenase, but how this enzyme performs this difficult task is still not understood.Enzymes contain a region known as the active site, to which substrates – the molecules that the enzyme acts upon – bind. The active site of nitrogenase contains a region called the FeMo-cofactor, which must transform from an inactive to an active state to catalyze the conversion of dinitrogen to ammonia.Azotobacter vinelandii while the enzyme transformed selenocyanate. This revealed unexpected structural changes of the FeMo-cofactor that significantly challenge previous assumptions about how the active site works. For example, a single selenium atom from selenocyanate can be incorporated into a specific position of the FeMo-cofactor, which highlights the importance of this position for the enzyme’s initial interaction with substrates.Another substrate of the nitrogenase enzyme is a molecule called selenocyanate, which is made up of atoms of selenium, carbon and nitrogen. Spatzal, Perez et al. examined the structure of the active site of nitrogenase taken from the bacteria species Spatzal, Perez et al. then used the inserted selenium atom as a probe to investigate the changes in the active site structure that occur when either reacting with a substrate called acetylene or being inhibited by carbon monoxide. This revealed that selenium can migrate into the positions taken up by three of the FeMo-cofactor’s nine sulfur atoms (the three “belt-sulfurs”) during these interactions. The active site was not previously thought to be active in this way: this will need to be taken into account in all future models that describe how dinitrogen is converted into a biologically useful form.In the future, Spatzal, Perez et al. will investigate in detail how these “belt-sulfur” atoms exchange with atoms from the substrate, where the removed sulfur is stored, and the pathway by which it returns. Further experiments will also characterize the active site during the transformation of dinitrogen.DOI:http://dx.doi.org/10.7554/eLife.11620.002 We found SeCN-  = 410 ± 30 μM versus Ki (SCN-) = 12.7 ± 1.2 mM  to be a± 1.2 mM . In cont± 1.2 mM .10.7554/- could be incorporated into the FeMo-cofactor by incubating nitrogenase under assay (turnover) conditions with concentrations of SeCN- sufficient to inhibit acetylene reduction. The 1.60 Å resolution crystal structure of Av1 (Av1-Se2B), isolated from assays containing SeCN- (1Se:Mo:C]-R-homocitrate cluster (FeMoSe-cofactor) form of the MoFe-protein. The essentially exclusive substitution of S2B by Se was validated by anomalous difference Fourier maps calculated from data measured at the Se-K edge f’’-peak position of 12,662 eV  followed by sample workup for crystallographic investigation. Crystal structures of the enzyme were determined at seven time points after initiation of substrate turnover corresponding to 2 to 5360 acetylene reduced per active site. These structures (designated Av1-Se-fq-2 to Av1-Se-fq-5360), at resolutions of 1.32–1.66 Å, represent the first example of time-dependent structural snapshots of the nitrogenase active site during turnover . . Consequently all belt-S atoms of FeMo-cofactor, separated by 5.7 Å in the resting state, can interchange during substrate (acetylene) reduction . The Se Our previous study of CO inhibited Av1 found replacement of S2B by CO . The CO -, with different chemical properties, are incorporated at this site. Significantly, the side-chain residues flanking this position were previously identified to be catalytically important as demonstrated by mutagenesis studies  buffer (Nitrogenase activity was determined by monitoring acetylene and ethylene in the headspace (9 mL) of reaction mixtures (1 mL) that consisted of 20 mM creatine phosphate, 5 mM ATP, 5 mM MgCl) buffer . All rea2 reduction was monitored by determining ammonia formation, based on a modification of the previously described fluorescence method . The headspace (9 mL) of the assay vial was flushed with N2 before incubation for 5 min at 30°C. Reactions were initiated by addition of the nitrogenase component proteins , and terminated at specific time points by the addition of 300 μL 0.5 M EDTA (pH=8.0). The liquid chromatography step in the previously described method was replaced by filtering the assay mixture  and collecting the filtrate, subsequently used for fluorescence measurement using a Flexstation 3 plate reader .Ne method . The 1.02  as a standard.Dihydrogen from proton reduction was measured by gas chromatography (molecular sieve 5A-80/100 column) equipped with a thermal-conductivity detector. The assay was identical to the acetylene reduction assay with the acetylene omitted. Ar was used as the reference/carrier gas. Calibration curves were prepared using 10% HMethane, one product of the KSeCN and KSCN reduction, was determined by gas chromatography in the headspace of the assay as described above for the acetylene reduction assay. The assay mixture was identical to the acetylene reduction assay except that the acetylene was omitted and KSeCN or KSCN  were added. Calibration curves were determined with pure methane gas.2S2O4 to remove excess KSeCN. The protein was further purified by size-exclusion chromatography . The final protein concentration was adjusted to 30 mg/mL. Significantly, incorporation of Se required full nitrogenase turnover as SeCN- incubation with Av1 alone was ineffective; likewise, the non-substrate Na2Se failed to serve as a Se donor even when incubated under turnover conditions.Av1-Se2B was prepared based on the above described proton reduction activity assay protocol in the presence of 25 mM KSeCN, providing conditions commensurate with full inhibition of acetylene to ethylene reduction. Av1-Se2B was isolated from the assay mixtures by ultrafiltration using a 100 kDa cut-off membrane and washed two times (dilution ratio of 1:100 each) with 200 mM NaCl, 50 mM Tris/Cl buffer pH = 7.5 containing 5 mM NaoC. Av1 was isolated by ultrafiltration using 100 kDa cut-off membranes and twice was washed with 200 mM NaCl, 50 mM Tris/Cl pH = 7.5, 5 mM Na2S2O4 buffer to remove other assay components. The final protein concentration was adjusted to 30 mg/mL and 21°C for crystallization.Av1-Se freeze quenched samples were obtained by applying the above described acetylene reduction activity assays, with the replacement of wild-type Av1 with Av1-Se2B. Additionally, an alteration of the protein concentration per assay as well as a variation of the active site ratio of Av2:Av1 = 1:2 to 4:1 was required to either slow down acetylene reduction for the isolation of samples corresponding to low numbers of turnover, or to allow for the isolation of high turnover samples and to ensure non-limiting assay conditions. Termination of protein activity at distinct time points was achieved by rapid freezing the activity assay mixtures in liquid nitrogen, simultaneously measured by the formation of ethylene from acetylene. Time points and corresponding numbers of turnover per active site (#) at given Av2:Av1 active site ratios for the prepared samples were: #2 , #46 , #341 , #921 , #1785 , #2141  and #5361 . The samples were subsequently processed at 3For the preparation of Av1-Se-CO, CO-inhibited activity assays were prepared as described earlier  with the2 atmosphere. The reservoir solution contained 24–28% PEG 8000 (v/v), 0.75–0.85 M NaCl, 0.1 M imidazole/malate (pH 7.5), 1% glycerol (v/v), 0.5% 2,2,2-trifluoroethanol (v/v) and 2.5 mM Na2S2O4. Additionally, a seeding strategy was applied to accelerate the crystallization process and to optimize crystal shape. The cystals of the respective proteins  were obtained between 6 and 24 hr after setting up the crystallization experiment. Cryo-protection was achieved by transferring crystals into a 5 uL drop of reservoir solution containing 8–12% MPD (v/v). Diffraction data were collected at 12,662 eV  at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12–2 equipped with a Dectris Pilatus 6M detector.Av1-Se2B and all Av1-Se freeze-quenched samples were crystallized based on the sitting drop vapor diffusion method at 21°C in an anaerobic chamber containing a 95% Ar / 5% HThe data were indexed, integrated, and scaled using iMosflm, XDS and Scala . Phase i2 for isotropically refined B-factors in the Av1-Se2B structure). For Fe and S in the FeMo-cofactor, the corresponding B values are 9.0 ± 0.4 Å2, with the B for Se2B = 9.9 ± 0.5 Å2 and the average B for the entire protein structure = 13.8 Å2. Quantification of Se-occupancies was further improved by including the contribution of the residual anomalous density of sulfur, with the anomalous scattering of S being 6.3% that of Se (f’’(S)/f’’(Se)) at an energy of 12,662 eV. The largest deviation between Se-anomalous densities observed for the two crystallographically independent copies of FeMo-cofactor was found to be ~5%, which provides an estimate of the uncertainty in the occupancy values. The root mean square value of the anomalous electron density map is ~2% of the Se peak value, which sets a lower threshold on the minimum occupancy that can be detected at this site.Anomalous electron density maps were calculated based on the data collected at 12,662 eV using a combination of CAD and FFT embedded in the CCP4 program suite . Quantif review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , Wilfred van der Donk (Reviewing editor), and three reviewers, one of whom, Catherine Drennan, has agreed to share her identity.Thank you for submitting your work entitled "Catalysis-dependent Se incorporation and migration in the nitrogenase active site FeMo-cofactor" for consideration by The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:The MoFe cluster of nitrogenase is one of Nature's most impressive and most enigmatic metallocenters, and its reaction mechanism has been notoriously refractory to experimental attempts to capture reaction intermediates. This study shows that the MoFe cluster is far less rigid than generally anticipated. The authors solved a series of structures of the nitrogenase MoFe protein at high resolution and use a creative application of anomalous scattering to demonstrate the migration of sulfur ligands among three sites that bridge the Fe and Mo-Fe ends of the iron-molybdenum cofactor (FeMo-cofactor). The structures show that multiple sulfur positions on the MoFe cluster can exchange with selenium in a turn-over dependent manner, with the Se first entering into the S2B site, the same position that CO was previously shown to occupy. The high resolution data show that Se can rotate around the cluster, occupying different S belt positions, before it is finally displaced by S after multiple turnovers with acetylene. Other substrates do not cause Se migration. For a long time, scientists have investigated the potential positions on the MoFe cluster that represent the binding site(s) for substrates, and this work suggests that the 'resting state' of the cluster, with its sulfur belt intact, may not be the form of the cluster to which substrates bind. As such, this study should cause the re-examination of all mechanistic proposals for nitrogenase, and complex metalloclusters in general.Essential revisions:1) Based on the partial occupancy of Se in the S2B site, a large number of turnovers was required to chase the Se from the site (between 1000 and 2000). What does this mean in terms of the "average" reaction? Is the protecting ligand at S2B displaced by the substrate in every turnover, as the authors hint? If so where does it go? Is the migration of the S2B ligand a rare or common event?2) The comment in the fourth paragraph of the Main text about Se occupancy of a "low occupancy site (ranging from 0-20% under different conditions) adjacent to the previously identified potential sulfur-binding site (SBS)" needs an explanation and perhaps also a citation. Where is this site?3) The paper needs an improved statement of error estimates for the partial occupancies at the Se2B, Se3A and Se5A sites. What were the limits of detection of Se partial occupancy in the anomalous difference maps? Are the 1-2% occupancy values at sites Se3A and Se5A in the "Av1-Se2B" sample really different from the 0.3-0.8% values for "Av1-Se-fq-5361" ? The sta 1) Based on the partial occupancy of Se in the S2B site, a large number of turnovers was required to chase the Se from the site (between 1000 and 2000). What does this mean in terms of the "average" reaction? Is the protecting ligand at S2B displaced by the substrate in every turnover, as the authors hint? If so where does it go? Is the migration of the S2B ligand a rare or common event?These are critical questions for understanding the mechanistic underpinnings of our observations and we do not know the answers. We are presenting a study detailing our unexpected findings that serve as the basis for future work, but not a final report on all the new directions implied by the results to date. We do not pretend to know the answers to the many intriguing possibilities but are presenting the unexpected observation that provides these new directions.Although we feel this is too speculative to include in the manuscript, in the spirit of responding to the issues raised by these important questions, we present for the reviewers some musings about the mechanistic implications of this work. Substrate binding (excluding protons) appears to be accompanied by rearrangements of the FeMo-cofactor . It is important to emphasize that during turnover, we have no evidence that the S2B ligand completely dissociates from the cofactor (as it is in the CO-inhibited structure), but speculate it most likely remains bonded to at least one Fe. At the completion of a catalytic cycle, the FeMo-cofactor returns to the original arrangement with a high probability; however, with a small, but non-zero probability, this hypothetical intermediate can rearrange effectively resulting in net migration of the belt ligands. We have tried to develop kinetic models for the migration based on the data 2) The comment in the fourth paragraph of the Main text about Se occupancy of a "low occupancy site (ranging from 0-20% under different conditions) adjacent to the previously identified potential sulfur-binding site (SBS)" needs an explanation and perhaps also a citation. Where is this site?We inadvertently omitted the reference to our 2014 CO paper defining the potential SBS, which has now been added, along with a brief description .3) The paper needs an improved statement of error estimates for the partial occupancies at the Se2B, Se3A and Se5A sites. What were the limits of detection of Se partial occupancy in the anomalous difference maps? Are the 1-2% occupancy values at sites Se3A and Se5A in the "Av1-Se2B" sample really different from the 0.3-0.8% values for "Av1-Se-fq-5361" ? The staWe have added a section to the Methods on “Quantification of Se occupancies” clarifying this analysis. The 5% error is based on the largest deviation between Se-anomalous densities observed between the two crystallographically independent copies of the FeMo- cofactor, and is likely the most realistic estimate of the occupancy errors. The limit of detection (~2% ) reflects the root mean square density of the anomalous Fourier map relative to the peak height of the fully occupied Se2B site. Occupancies of 0.3-0.8% and 1-2% are not statistically different. We have rounded the occupancies in


Sphaerospora dykovae (formerly S. renicola). S. dykovae was reported to proliferate in the blood and in the swim bladder prior to the invasion of the kidney, where sporogony takes place. Due to the presence of emerging numbers of proliferative myxozoan blood stages at different carp culture sites in recent years we analysed cases of SBI, for the first time, using molecular diagnostics, to identify the myxozoan parasites present in diseased swim bladders.Swim bladder inflammation (SBI) is an important disease of common carp fingerlings in Central Europe. In the 1980s, its etiology was ascribed to multicellular proliferative stages of the myxozoan parasite in situ hybridisation, thereby determining which myxozoans are involved in SBI.We amplified myxozoan SSU rDNA in a non-specific approach and compared the species composition in swim bladders at culture sites where carp demonstrated 1. No signs of SBI, 2. Minor pathological changes, and 3. Heavy SBI. Based on DNA sequences, we determined the localisation and distribution of the most frequent species by S. dykovae, however, blood stages were predominantly represented by Sphaerospora molnari, whose numbers were greatly increased in carp with mild and heavy SBI, compared with SBI-free fish. S. molnari was found to invade different organs and cause inflammatory changes also in the absence of S. dykovae. One site with mild SBI cases was characterised by Buddenbrockia sp. infection in different organs and a general granulomatous response.Large multicellular myxozoan swim bladder stages characterised heavy SBI cases and were identified as S. molnari in the blood represent an important co-factor or precondition for SBI.We provide evidence that the etiology of SBI can vary in relation to culture site and disease severity and that emerging numbers of  Cyprinus carpio carpio L. Swim Bladder Inflammation (SBI) of fingerlings is an acute and severe disease of economic impact in Central Europe [In common carp, l Europe . The firl Europe ). Mortall Europe .Sphaerospora dykovae  (previously S. renicola) was identified as a key player in the etiology of the disease [Research into SBI first intensified in the 1980s, when the myxozoan  disease –7. MyxozS. dykovae were described in common carp: 1. Multicellular, proliferative stages floating in the blood , 2. Mulby Csaba  and Kováby Csaba ), and 3.by Csaba . K-stageby Csaba . The linby Csaba , 13 or sby Csaba , thus lein situ hybridisation (ISH) to determine parasite identity, number per species and exact location in the swim bladder and in other organs, thus identifying the etiological agents of SBI in common carp based on molecular diagnostics. Furthermore, we monitored the fish in an SBI-enzooic pond continuously throughout the year to better understand the seasonality and temperature dependence of parasite invasion and proliferation of the species involved in SBI in common carp.Using molecular methods, we recently demonstrated that myxozoan blood stages in asymptomatic common carp may belong to at least 7 different myxozoan species . To clarS. dykovae-enzooic sites in the Czech Republic and Hungary, between 2011 and 2013 , 50 clones from each site were sequenced unidirectionally , using the reverse nested PCR primer, to get an approximation of abundant myxozoans. Thereafter, sequence alignments were performed using Geneious 7.0  and specific primers were designed/applied for the most abundant myxozoan species  and used for in situ hybridisation (ISH) employing a DIG-antiDIG-alkaline phosphatase protocol [S. dykovae and Sphaerospora molnari Lom et al., 1983 [From each fish, first, a blood sample was taken with a sterile syringe, thereafter a drop of blood, several gill arches as well as parts of the swim bladder and kidney were examined microscopically in fresh smears, for semiquantitative estimation of infection intensity of protocol , 18. PCRl., 1983 , 18 wereAll animal procedures were performed in accordance with Czech legislation . We declare that animal handling complied with the relevant European and international guidelines on animal welfare, namely Directive 2010/63/EU on the protection of animals used for scientific purposes and the guidelines and recommendations of the Federation of Laboratory Animal Science Associations.S. dykovae infection and showed that prevalences of infection ranged between 8.3% and 81.8% (average 57%) and intensities of infection between 1.3 and 5.0 on a 0–5 scale (Table S. dykovae spore-forming stages in the kidney tubules was high (>4) at sites 2, 4, 5 and 6. At 3 of these sites (4–6), fish showed moderate to strong SBI pathology with mortalities on site whereas carp from site 2 were free from macro- or microscopic pathological changes, despite large parasite loads in the renal tubules. At one further site (site 3) SBI pathology was detected in fish, however, S. dykovae prevalence and intensity of infection was relatively low (29.4% and 2.7) when compared with other SBI sites . Due to the results from DNA sequencing all sites were later also screened for infection with S. molnari. Fish at all but one site were infected; average prevalence of infection was 73.4%. Prevalences of S. molnari were highest (82.4-100%) at the four SBI sites and at one other site without SBI symptoms , Tetracapsuloides sp. , Hoferellus cyprini Doflein, 1898 and Chloromyxum cyprini Fujita, 1927  and S. molnari (average 28%). While S. molnari was most prominent at sites with moderate SBI pathology (48-50%), S. dykovae clearly dominated the sites with heavy SBI pathology (46-94%). Buddenbrockia sp. was common at all sites (average 17%) with most of the clones sequenced from non-SBI sites and from one of the moderate SBI sites (site 3). Myxobolus encephalicus was sequenced from an average of 17% of all clones and was most common at non-SBI sites as well as at site 4 (moderate SBI). Tetracapsuloides sp., H. cyprini and C. cyprini were not detected at all sites and occurred in a low number of clones . T. hovorkai was present only at one site.Cloning of myxozoan PCR amplicons from swim bladders of carp showed that eight species were abundant, i.e. S. dykovae, S. molnari, Buddenbrockia sp., M. encephalicus and T. hovorkai. ISH showed that only three myxozoans occurred in large numbers within different swim bladder tissue layers and can thus potentially be related to the typical SBI pathology, i.e. S. dykovae, S. molnari and Buddenbrockia sp.In order to determine the exact location and distribution of infectious parasite stages in the tissues of the swim bladders and their relation to host tissue responses we performed ISH on histological sections of swim bladders of carp from the six sites, targeting all species with an important number of clones present in SBI cases, i.e. S. dykovae. K-stages measured 15–47 μm and occupied large areas of the heavily diseased swim bladders with parasites concentrating in and around the blood vessels , which had been reported in the past and which characterised heavy SBI cases in the present study, represented exclusively s Figure A-B. HeavS. dykovae but surprisingly high numbers of S. molnari. Thereby, presporogonic stages of S. molnari were considerably smaller (2–17 μm) than those of S. dykovae (see above) and were not found to accumulate but were dispersed. While most dominant in the blood and strongly vascularised areas of different organs , which was macroscopically noticeable only at this site. At non-SBI sites, S. molnari and Buddenbrockia sp. occurred almost exclusively intravascularly, while S. dykovae was undetectable by ISH.s Figure F and disM. encephalicus were rarely detected using ISH but were restricted to the vascular system, while T. hovorkai was found isolated in small spore-forming plasmodia within different connective tissue layers (not shown).Independent from the site, multicellular proliferative stages of S. dykovae blood stages occurred only during late spring/early summer and in a small percentage of fish . Importantly, while invading predominantly the kidney interstitium [S. dykovae in [S. molnari), S. molnari stages were also detected in the tissues of the swim bladder of all SBI carp and at one site (site 4), this was the only parasite detectable by ISH in pathologically modified swim bladders. It cannot be excluded that the pathology detected at site 4 involves a previous S. dykovae infection that has been cleared or if the sole cause of the inflammation is S. molnari. However, S. molnari was present in clearly elevated numbers at all sites with moderate to heavy SBI conditions when compared with non-SBI sites , although it is unclear whether its multiplication is based solely on temperature increase or on parasite-induced changes in host physiology [S. molnari infection levels with old records is difficult as PCR assays were unavailable at the time, however, in 1983, 70% (31/44) of stocks were infected with an overall prevalence of 46% [We assume that, in the past, the failure to develop tor fish , 12, 13 mirabile , is actuysiology . Howeverysiology . At highysiology . CompariS. molnari blood stages decreases only during winter and during the spore-forming season in spring. In contrast, S. dykovae has a very limited incubation time in the blood, which presumably coincides with the limited presence of fish-infective spore stages in the environment, providing only a small window for the infection of carp, in late spring/early summer. Our data furthermore show that S. molnari proliferates predominantly in the blood and subsequently invades, amongst other organs, the swim bladder tissues. The size of S. molnari stages in the swim bladder was the same as that of their blood stages, a size far inferior to that of S. dykovae K-stages. This suggests that S. molnari does not proliferate once histozoic. In contrast, S. dykovae multiplies further in the swim bladder and forms larger parasite stages with tertiary cells [S. dykovae represents the major contributor to a more severe pathological condition of SBI. S. molnari stages with a histozoic life style were most common in the kidney, the back of the eye and the swim bladder (in this order). These are likely good sites for parasite accumulations due to the occurrence of fine capillary networks (rete mirabile), which slow down the blood flow and trap multicellular parasite stages due to the diameter of the capillaries, allowing them to attach to and pass through the blood vessel walls into the surrounding tissues. S. molnari’s site preference for the kidney may furthermore be explained by a recent split from kidney-infecting sphaerosporids [We found that the prevalence of ry cells . This maosporids .Buddenbrockia sp. Small parasite stages, similar to initial stages of infection of Tetracapsuloides bryosalmonae Canning et al., 2002 in the gills and the blood of salmonids [S. dykovae[. However, the related electronmicrographs show that they represent cell doublets that contain sporoplasmosomes inside their primary cells, which are often membrane-associated. Both features are characteristics that clearly link these cells to intrapiscine proliferative stages of the class Malacosporea, including the genus Buddenbrockia[Buddenbrockia sp. However, since the biodiversity of malacosporeans in cyprinids in Central Europe is unexpectedly high [Another myxozoan that is potentially participating in some cases of SBI as it is capable of invading the swim bladder and other organs via the vasculatory system, is the malacosporean almonids –29 were almonids , 30, 31.nbrockia. As the dly high  and chanT. bryosalmonae is known to cause substantial proliferation of the host’s white blood cells resulting in a granulomatous inflammatory reaction, predominantly the kidney and in the spleen, thus causing a severe condition, proliferative kidney disease, PKD, in salmonids [Buddenbrockia sp. This indicates that Buddenbrockia sp. may be responsible for a general immunological response rather than swim bladder-specific changes. This could potentially worsen an immune-compromised condition caused by high numbers of S. dykovae in the swim bladder or vice versa. While the order of appearance of the two parasites has yet to be resolved, considerable swelling of all haematopoietic organs has been reported in a number of SBI cases [Haemogregarina cyprini in [S. dykovae infection is usually characterised by swelling of only the kidney [almonids , 35. SimBI cases , 37 withprini in , while Se kidney .Buddenbrockia and especially S. molnari likely represent an important co-factor or pre-condition for developing SBI in carp, at the sites investigated in this study, we could clearly exclude other myxozoans as causative or co-causative agents of SBI as they were restricted to well-isolated plasmodia in the connective tissue of the swim bladder (T. hovorkai) or occurred in very small numbers .While Plumatella repens serves as host for Buddenbrockia sp. [Buddenbrockia-related SBI when compared with ponds that offer exclusively muddy sediments with a different invertebrate microfauna for myxozoan life cycle completion.There is considerable evidence that the epizootics of SBI vary according to the fish rearing systems used, e.g. , 38. We ckia sp.  and carpS. dykovae and S. molnari, as previous life cycle data on these species are scarce [S. dykovae and S. molnari belong to the Sphaerospora sensu stricto phylogenetic clade [S. dykovae, S. molnari and Buddenbrockia sp. to SBI would be to determine when and where these parasites are present in the environment and to mirror the results from environmental screening with the course of the infections and the disease condition in fish, throughout the year, similar as performed for the marine myxozoan Ceratomyxa puntazzi Alama-Bermejo et al. 2011 [S. molnari blood stages, we urgently need to improve our scarce knowledge regarding the parasite’s preferred aquatic habitat, the periods of host infection, proliferation mechanisms in the host, the interplay with S. dykovae in the course of SBI and other pathological effects high numbers of S. molnari may have on common carp in Central European Aquaculture facilities.It would be of considerable importance for SBI management strategies to determine and confirm the invertebrate hosts of e scarce  and havee scarce , 18. S. ic clade  which isic clade , 43. Howal. 2011 . With emS. molnari proliferative stages and mild to severe SBI pathology, while only severe cases were characterised by large, proliferative swim bladder stages of S. dykovae, the species previously ascribed to SBI in common carp. We have noted emerging numbers of S. molnari blood stages at various carp culture sites, possibly in relation to rising water temperatures. While this leads us to predict increased re-occurrence of SBI cases in Central Europe in the near future, it cannot be excluded that further problems will emerge as S. molnari has also been related to eye, gill and skin pathology in common carp. It is thus of special importance to advance our scarce knowledge on the life cycle, transmission and development of S. molnari in common carp.The present study provides the first evidence that more than one myxozoan species may be involved in the etiology of SBI of common carp, depending on site, culture system setup and disease severity. Using, for the first time, molecular diagnostics to identify the pathological agents of SBI that belong to the Myxozoa, we were able to clarify ambiguities and misidentifications of proliferative myxozoan stages that occurred in the past, when SBI agents were analysed exclusively on the basis of their morphology and by relating life cycle stages based on experimental transmissions. We determined a strong correlation between high numbers of


Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA for treating cardiovascular disorders. The roles of cytochrome P450 enzymes (CYPs) in the metabolism of STS have remained unclear. This study aims to screen the main CYPs for metabolism of STS and study their interactions in vitro.Seven major CYPs were screened for metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. Phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as probe substrates to determine the potential of STS in affecting CYP-mediated phase I metabolism in humans. Enzyme kinetic studies were performed to investigate the modes of inhibition of the enzyme–substrate interactions by GraphPad Prism Enzyme Kinetic 5 Demo software.Km and Vmax values of STS were 54.8 ± 14.6 µM and 0.9 ± 0.1 nmol/mg protein/min, respectively, for the HLMs and 7.5 ± 1.4 µM and 6.8 ± 0.3 nmol/nmol P450/min, respectively, for CYP3A4. CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19 showed minimal or no effects on the metabolism of STS.Sodium tanshinone IIA sulfonate inhibited the activity of CYP3A4 in a dose–dependent manner by the HLMs and CYP3A4 isoform. The This in vitro study showed that STS mainly inhibited the activities of CYP3A4.The online version of this article (doi:10.1186/s13020-016-0083-z) contains supplementary material, which is available to authorized users. Salvia miltiorrhiza (Danshen) for treating cardiovascular disorders /(Km + [S]),where Km is the substrate concentration at which the reaction velocity is 50 % of Vmax. The activity of each CYP isoform was calculated by the MCR of its corresponding probe substrate to determine the inhibition of the CYP isoforms. The MCR of the probe substrate was considered to be 100 % when no specific inhibitor and STS were added to the incubation assay. The IC50 values were determined by analyzing the plot of the logarithm of the inhibitor concentration versus the percentage of activity remaining after inhibition, using the SPSS for Windows Version 11.5 . The inhibition data were fitted to different models of enzyme inhibition  by nonlinear least-squares regression analysis using GraphPad Prism 5 software  to calculate the Ki values.The MCR of the incubation solution without any specific inhibitor for STS was considered to be 100 % to determine the major enzymes responsible for STS metabolism in HLMs. The effects of the specific inhibitors on the MCR of STS were evaluated by one-way analysis of variance (ANOVA) , LSD test was used for the homogeneity of variance, otherwise Tamhane’s test. Values of Km and Vmax values for HLMs and CYP3A4 were 54.8 ± 14.6 µM and 0.9 ± 0.1 nmol/mg protein/min, respectively, and 7.5 ± 1.4 µM and 6.8 ± 0.3 nmol/nmol P450/min, respectively. The in vitro CLint values for STS with HLMs and CYP3A4 were 0.016 mL/mg protein/min and 0.902 mL/nmol P450/min, respectively.The metabolism of STS after incubation with HLMs and CYP3A4 was shown in Fig. IC50 or Ki values for the CYP isoforms to ensure adequate inhibitory selectivity, and maximal inhibitory potency [The inhibitory effects of the CYP specific inhibitors on the MCR of STS in HLMs were shown in Fig.  potency –29. In tIC50 of 6.4 µM. The inhibitory effects of STS on the activities of CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19 were negligible  on the activity of each CYP isoform were determined by metabolism of a single concentration of isoform-specific probe with HLMs . STS showed potent inhibition of CYP3A4 (midazolam 1-hydroxylation), with an IC50 values are qualitatively informative and helpful for addressing whether inhibition has occurred, but are of limited quantitative use because they can be influenced by the substrate concentration selected. Hence, it might not be accurate to use these parameters for quantitative prediction of drug interactions in vivo. Therefore, we performed additional experiments designed to estimate the Ki values. The preliminary inhibition data generated from a single probe substrate reaction were used to simulate the appropriate range of substrate and inhibitor concentrations for use in the construction of Dixon plots for inhibition of the CYP isoforms by STS in HLMs, from which precise Ki values were estimated.According to Jeong et al. , IC50 vaKi values were determined by midazolam as the probe substrate. Among all of the CYPs tested, CYP3A4 was the most sensitive to inhibition by STS  have been identified in the CYP3A4 gene [CYP3A4 gene such as CYP3A4*1B and CYP3A4*22 were reported to affect the pharmacokinetics of tacrolimus [Genetic variations in the 3A4 gene . In prev3A4 gene , while scrolimus . HoweverThis in vitro study showed that STS mainly inhibited the activities of CYP3A4.


Dasatinib, a potent and specific Src tyrosine kinase inhibitor, was found to decrease the levels of β-dystroglycan phosphorylation on tyrosine and to increase the relative levels of non-phosphorylated β-dystroglycan in sapje zebrafish. Furthermore, dasatinib treatment resulted in the improved physical appearance of the sapje zebrafish musculature and increased swimming ability as measured by both duration and distance of swimming of dasatinib-treated fish compared with control animals. These data suggest great promise for pharmacological agents that prevent the phosphorylation of β-dystroglycan on tyrosine and subsequent steps in the degradation pathway as therapeutic targets for the treatment of Duchenne muscular dystrophy.Identification of a systemically acting and universal small molecule therapy for Duchenne muscular dystrophy would be an enormous advance for this condition. Based on evidence gained from studies on mouse genetic models, we have identified tyrosine phosphorylation and degradation of β-dystroglycan as a key event in the aetiology of Duchenne muscular dystrophy. Thus, preventing tyrosine phosphorylation and degradation of β-dystroglycan presents itself as a potential therapeutic strategy. Using the dystrophic  Danio rerio has rapidly been adopted as an organism of choice for all aspects of the drug discovery pipeline  onwards . The loss of other DGC components in the absence of dystrophin is common with other models of Duchenne muscular dystrophy (DMD) such as the mdx mouse -MultiDsk , where inhibition of the intermediate stages of the proposed pathway . All recorded measures of swimming activity were significantly lower in sapje fish including total time spent swimming, time spent swimming at fast or slow speeds and time spent swimming for short or long distances (data not shown), and total distance swum in 10 min . This strengthens the notion that the mechanism by which dasatinib treatment is able to improve muscle integrity is dependent on dystroglycan expression and its phosphorylation in the absence of dystrophin, as no improvement was seen in a dystrophic fish that lacks dystroglycan .Having established that dasatinib treatment of normal zebrafish could alter the extent of dystroglycan phosphorylation, we next examined the effect of dasatinib in can Fig.  on the lor Pyr41   (sapje fish (data not shown). Combinations of multiple kinase inhibitors, or kinase inhibitors with proteasome inhibitors, may lead to greater efficacy and the possibility of reduced side-effects. However, dasatinib was clearly the most effective leading to a 40% rescue of the dystrophic phenotype. While this is a significant improvement in dystrophic pathology, one has to question, given the hypothesis, why the extent of recovery is not greater. However, considering the 100% efficacy of the mouse genetic model in preventing phosphorylation of tyrosine 890, with only a partial amelioration of the dystrophic phenotype, it is perhaps not so surprising that the pharmacological treatment in zebrafish did not result in a full recovery. Moreover, these analyses are based only on our ability to monitor tyrosine phosphorylation at Y890, as this is the only tyrosine phosphorylation site that has so far been identified unequivocally  –43. Thesivocally ,32, and ivocally . More reivocally . Currentt222asapje . For sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) anaylsis, sapje–/– fish were selected post-treatment by birefringence. For embryos treated between 3 and 5 dpf, homozygote sapje embryos were sorted using birefringence prior to drug treatment. Embryos were treated as described above, but using 10 embryos per well. Phenotypically normal siblings were used as controls. The degree of rescue of the dystrophic phenotype was determined as described previously . There were no survival or toxicity issues with any of the treatments used in the study. High-speed video-tracking motion analysis of zebrafish embryos was performed using a ViewPoint ZebraLab system  and as described in  containing 0.1% Triton X-100. For antibody staining, fixed embryos were permeabilized with pre-cooled acetone at −20°C, blocked in PBS containing 1% bovine serum albumin, 1% DMSO and 1% Triton X-100, and incubated overnight in blocking solution containing the appropriate dilution of antibody. Imaging was carried out on an Olympus fluoview-1000 confocal microscope. For western blot analysis of zebrafish larvae, whole larvae were homogenized in RIPA buffer and boiled in SDS sample buffer before separation by SDS–PAGE and transfer to PVDF. on Figs.  and 6.FH2kb-tsA58 myoblast cells were maintained and subjected to surface biotinylation pulse-chase assay as described previously  and Pyr-41 and MG132 from Sigma-Aldrich . The following antibodies were used in WB and/or immunofluorescence (IF) applications. Non-phosphorylated β-dystroglycan , β-dystHMG online.Supplementary Material is available at Muscular Dystrophy Campaign grant number RA/806 (L.L.) a PhD studentship from the MRC grant numbers G0900203-1/1 G1000405-1/1 (R.W.P.) and a project grant from the Duchenne Parent Project NL (S.J.W.). Funding to pay the Open Access publication charges for this article was provided by MRC (UK).The work presented in this paper was funded by a PhD studentship from the


Extracellular signal-regulated kinases (ERKs) play critical roles in numerous cellular processes, including proliferation and differentiation. ERK5 contains a kinase domain at the N-terminal, and the unique extended C-terminal includes multiple autophosphorylation sites that enhance ERK5-dependent transcription. However, the impact of phosphorylation at the various sites remain unclear. In this study, we examined the role of phosphorylation at the ERK5 C-terminal. We found that a constitutively active MEK5 mutant phosphorylated ERK5 at the TEY motif, resulting in the sequential autophosphorylation of multiple C-terminal residues, including Thr732 and Ser769/773/775. However, when ERK1/2 was selectively activated by an oncogenic RAS mutant, ERK5 phosphorylation at Thr732 was induced without affecting the phosphorylation status at TEY or Ser769/773/775. The Thr732 phosphorylation was U0126-sensitive and was observed in a kinase-dead mutant of ERK5 as well, suggesting that ERK1/2 can phosphorylate ERK5 at Thr732. This phosphorylation was also promoted by epidermal growth factor and nerve growth factor in HEK293 and PC12 cells, respectively. The ERK5–T732A mutant was localized in the cytosol under basal conditions. In contrast, ERK5 phosphorylated at Thr732 via the RAS-ERK1/2 pathway and ERK5–T732E, which mimics the phosphorylated form, were localized in both the nucleus and cytosol. Finally, ER–32A and U0126 blocked ERK5-dependent MEF2C transcriptional activity. Based on these findings, we propose a novel cross-talk mechanism in which ERK1/2, following activation by growth factor stimulation, phosphorylates ERK5 at Thr732. This phosphorylation event is responsible for ERK5 nuclear localization and ERK5-dependent transcription. Extracellular signal-regulated kinases (ERKs), also called mitogen-activated protein kinases (MAPKs), participate in various cellular processes, including cell proliferation, differentiation, migration and gene expression. The MAPK family includes the classical MAPKs, such as ERK1/2, c-Jun N-terminal kinase 1/2/3, p38MAPK α/β/γ/δ and ERK5, as well as the atypical MAPKs ERK3, ERK4, ERK7 and nemo-like kinase (NLK) ,7,8, neuERK5 gene knockout is lethal at E9.5–10.5 because of cardiovascular defects [ERK5 in adult neurogenic regions involved in hippocampus-dependent memory formation impairs fear extinction, the expression of remote memory and olfactory behavior [ERK5 is physiologically essential, as demonstrated by a report showing that  defects . These d defects ,17. Condbehavior ,19,20. Fbehavior ,22.We previously showed that ERK5 plays essential roles in neurite outgrowth, in the expression of the neurotransmitter synthesizing enzyme tyrosine hydroxylase in rat pheochromocytoma cells (PC12 cells) , and in Nerve growth factor (NGF), epidermal growth factor (EGF) and luciferin were purchased from Sigma Aldrich . Antibodies against the phospho-ERK5 TEY motif (which cross-reacts with the phospho-ERK1/2 TEY motif), ERK5 and myc, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody, and U0126 were purchased from Cell Signaling Technology . Anti-ERK2 antibody was purchased from Santa Cruz . Fetal bovine serum (FBS) was purchased from Cell Culture Laboratory . Lipofectamine 2000, horse serum and Alexa488-conjugated anti-rabbit IgG secondary antibody were purchased from Life Technologies . Polyvinylidene difluoride membrane, enhanced chemiluminescence (ECL) assay kits, hyper-film ECL and protein G sepharose beads were purchased from GE Healthcare . Another ECL kit was purchased from PerkinElmer . GST-tagged ERK5 and ERK2 recombinant proteins were purchased from SignalChem . A mycERK5’K83M kinase-dead mutant (KD), mycER–32A and mycER–32E were engineered from mycERK5 using the QuikChange site-directed mutagenesis kit . DNA plasmids encoding constitutively active MEK5 mutants ) were kindly provided by Dr. Eisuke Nishida . DNA plasmids encoding mycERK2, constitutively active RAS-G12V (RASV12) and β-actin promoter-driven β-galactosidase were a kind gift from Dr. Philip J.S. Stork . DNA plasmids encoding four × MEF2 response elements upstream of the minimal c-FOS promoter driving firefly luciferase (pMEF/Luc) were kindly provided by Dr. Ron Prywes . Anti-phospho-Erk5 (pThr732 and pSer769/773/775) rabbit polyclonal antibodies were developed at Cell Signaling Technology in Danvers, MA (USA).2 incubator at 37°C. PC12 cells were obtained from the Japanese Cancer Research Bank  and grown in DMEM supplemented with 10% FBS, 5% horse serum, penicillin (50 units/ml) and streptomycin (50 μg/ml) in a 5% CO2 incubator at 37°C.HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, penicillin (50 units/ml) and streptomycin (50 μg/ml) in a 5% COCells or immunoprecipitates were dissolved in Laemmli sample buffer  and heated at 95°C for 5 min. Electrophoresis was performed on 8–11% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes using the semi-dry blotting method. The blots were blocked for 30 min with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20, and incubated with primary antibodies overnight at 4°C , ERK5 and ERK2, and 1:500 dilution for antibodies against phospho-ERK5 (pThr732 or pSer769/S773/S775)). The blots were washed several times and then incubated at room temperature for 2 h with an HRP-conjugated secondary antibody . After washing, blots were developed using an ECL assay kit, and visualized by chemiluminescence on hyper-film ECL or with ChemiDocXRS . The densities of the bands corresponding to phospho-ERK5 at Thr732 were analyzed by densitometry (Image J64). The similar experiments were repeated at least three times, except for those in Figs. 3VO4, pH 7.4). After sonication, the lysates were precleared with protein G sepharose beads for 1.5 h at 4°C. The precleared lysates were mixed with or without anti-ERK2 antibody (1:50) and 20 μl of protein G sepharose, and incubated for 2 h at 4°C in 500 μl RIPA buffer. The beads were washed twice and the immunoprecipitates were eluted by the addition of 25 μl Laemmli buffer. For examining in vitro interactions, ERK5 (0.5 μg) and ERK2 (0.5 μg) recombinant proteins were incubated in 500 μl of RIPA buffer for 30 min at 37°C. Then, after preclearing with protein G sepharose, the supernatants were immunoprecipitated with ERK2 antibody as described above.Briefly, cells were lysed in RIPA buffer containing protease inhibitors , mycER–32A or mycER–32E were fixed with 4% paraformaldehyde. In addition, cells co-transfected with mycERK5-WT or mycERK5-KD and oncogenic RASV12 mutant were also fixed with 4% paraformaldehyde. Cells were then treated with Triton X-100 (0.5%) and blocked with 5% bovine serum albumin for 2 h, and subsequently incubated with anti-myc primary antibody (1:400 dilution) at 4°C overnight followed by Alexa488-conjugated anti-rabbit IgG secondary antibody (1:300 dilution) for 3 h at 37°C. The nuclei were also stained with Hoechst 33258 (1 μg/ml). Then, cells were observed with a fluorescence microscope . The cell whose fluorescence intensity of the mycERK5-WT or the mutants in nucleus was similar or stronger than that in cytosol was defined as ERK5 nuclear localization, this ratio was counted in randomly selected areas.5 cells/well. The next day, DNA plasmids, including ERK5 (WT or mutant) and reporter genes , were combined with Lipofectamine 2000 (1 μl/well) and mixed gently in serum-free DMEM (50 μl) and added to the culture plates for 4–6 h. Cells were then serum starved overnight and incubated with EGF in the presence or absence of U0126 at 37°C for 6 h. Cells were lysed in lysis buffer  (100 μl/well) and centrifuged to remove cell debris. The resulting supernatant (50 μl/tube) was mixed with 300 μl assay buffer . The luciferase reaction was started by the addition of 100 μl luciferin solution (150 μM), and luciferase activity was measured using a luminometer . As an internal control, β-actin promoter-driven β-galactosidase activity was measured to normalize for transfection efficiency. The cell lysate (20 μl) was incubated with 100 μl reaction buffer  supplemented with chlorophenol red-β-D-galactopyranoside (2 mg/ml) , and the absorbance at 595 nm was measured.For reporter gene assays, HEK293 cells were seeded into 24-well plates at a density of 0.5–1 × 10Data were expressed as the mean ± SEM, and significant differences were assessed using the Tukey-Kramer method.Upon phosphorylation of the ERK5 TEY activation motif by MEK5, multiple serine and threonine residues in the ERK5 C-terminal are autophosphorylated. It is thought that the C-terminal can, by itself, promote gene transcription when phosphorylated at Ser769/773/775 and Thr732 . Thus, wTo evaluate the specificity of the antibody against phospho-ERK5 at Thr732, ERK5-WT, ER–32A and ERK5-KD were expressed in HEK293 cells, and the phosphorylation status of these ERK5 mutants was examined. As expected, phosphorylation of ERK5 at Thr732 was diminished in ER–32A- and ERK5-KD-expressing cells , confirmin vitro ERK5-ERK2 protein binding experiments using recombinant proteins. Proteins were co-incubated for 30 min at 37°C, and ERK2 was immunoprecipitated. ERK5 was absent in the immunoprecipitates under our experimental conditions, suggesting that ERK5 binds to ERK2 indirectly (data not shown).It has been shown that ERK1/2 forms homodimers upon phosphorylation by the upstream kinase MEK1/2 . In addiWe previously demonstrated that a constitutively active mutant of RAS (RASV12) promotes phosphorylation of the TEY site in ERK1/2, but not the TEY site of ERK5 in PC12 cells . FurtherBecause growth factors such as EGF or NGF promote phosphorylation of both ERK1/2 and ERK5 at their respective TEY sites, we examined the phosphorylation status of ERK5 at Thr732 when both ERK isoforms were physiologically activated by these growth factors. HEK293 cells were co-transfected with mycERK2 and mycERK5-WT or mycERK5-KD, then stimulated with EGF (100 ng/ml) for 5 min in the presence or absence of U0126 (20 μM). EGF induced phosphorylation of ERK5 at Thr732 1.60-fold in addition to phosphorylation at the TEY sites of both ERK1/2 and ERK5 in HEK293 cells. U0126 blocked phosphorylation of ERK1/2 at the TEY site and ERK5 at Thr732 . ERK5-KD was also phosphorylated at both the TEY site and Thr732 . ExperimDiaz-Rodriguez et al. examined the subcellular localization of ERK5-WT and its mutants, including those in which four Ser and Thr residues including Thr732 were substituted with Ala or Glu . While HA-tagged ERK5-WT and ERK5–4xPA maintained an essentially cytoplasmic distribution, the phospho-mimetic ERK5–4xPE had a nuclear localization . In the Because phosphorylation of the ERK5 C-terminal is thought to enhance gene transcription independently of the phosphorylation of transcription factors , we nextIn this study, we demonstrate that ERK1/2 phosphorylates ERK5 at Thr732 upon growth factor stimulation, and that this phosphorylation is necessary for ERK5 nuclear localization and ERK5-dependent transcription. A putative signaling pathway is shown in Endogenous ERK5 co-immunoprecipitated with ERK2, although, surprisingly, the interaction between ERK5 and ERK2 (possibly ERK1 as well) did not change in response to NGF under our experimental conditions . It has It is thought that the interaction between ERK5 and ERK1/2 is essential for ERK1/2-mediated phosphorylation of ERK5 at Thr732, nuclear retention and the enhancement of MEF2 transcriptional activity. Drastic ERK5 conformational changes elicited by phosphorylation by MEK5 allow exposure of its constitutively active nuclear localization signal (509–539) and inhibition of its nuclear export signal by dissociation of intramolecular binding between the ERK5 N and C termini. This results in a predominantly nuclear localization ,36,37. IBecause MEF2 activation by EGF was significantly inhibited by U0126 and overexpression of ER–32A , we infein vitro kinase assay. However, because the phosphorylation site has the minimal consensus sequence for phosphorylation by ERK1/2 (i.e. Ser/Thr-Pro) [We found that the binding between ERK5 and ERK1/2 was indirect, and we did not determine whether ERK5 Thr732 phosphorylation by ERK1/2 was direct or indirect using Thr-Pro) , we surmin vitro, and it is necessary to carefully adjust immunoprecipitation conditions, including salt and detergent concentrations, to allow the immunoprecipitation of ERK5 but disturbing ERK2 binding.Reduced ERK5 electrophoretic mobility (phosphorylation shift) is often regarded as an index of ERK5 activation. However, the phosphorylation status of the ERK5 C-terminal, including Thr732, influences ERK5 electrophoretic mobility regardless of ERK5 phosphorylation at its TEY motif, as shown in In conclusion, we demonstrate a new cross-talk mechanism between ERK5 and ERK1/2 in which phosphorylation of ERK5 at Thr732 is induced by ERK1/2, and showed that this phosphorylation event is required for ERK5 nuclear localization and MEF2C activation. Further studies are necessary to clarify how phosphorylation status influences ERK5 structure and to elucidate the mechanisms underlying MEF2C activation.


Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The Km and Vmax values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC50 values were 16.00 μM and 9.83 μM, and Ki values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.Ginsenoside compound K (CK), a rare ginsenoside originating from  Panax Ginseng is a complementary and alternative medicine (CAM) that has many pharmacological activities, such as maintaining physical vitality and boosting resistance to stress and aging  axis in both plots. The asymptotic cases of competitive and uncompetitive inhibition can be generated by inserting the conditions Ki’ to ∞ for competitive inhibition, or Ki to ∞ for uncompetitive inhibition /(Km+[S]), where Km is the substrate concentration at which the reaction velocity is 50% of Vmax. To determine the inhibition of the CYP isoforms, the activity of each CYP isoform was calculated using the metabolic clearance rate of its corresponding probe substrate. The metabolic clearance rate of the probe substrate was considered 100% when no specific inhibitor and CK were added to the incubation assay. The IC50s were determined by analyzing the plot of the logarithm of the inhibitor concentration versus the percentage of activity remaining after inhibition using the SPSS software for Windows . To calculate the Ki values, the inhibition data were fit to different models of enzyme inhibition  by nonlinear least-squares regression analysis using the GraphPad Prism 5 software (GraphPad Co. Ltd), Ki values were calculated with the use of nonlinear regression according to the equation v = (VmaxS)/(Km(1+I/Ki)+S) for competitive inhibition and equation v = (VmaxS)/(Km+S)(1+I/Ki) for noncompetitive inhibition, where I is compound concentration, Ki is the inhibition constant, S is the substrate concentration, and Km is the substrate concentration at half of the maximum velocity (Vmax) of the reaction [Ki’ values were calculated using the secondary plot of slopes of the S/V versus [I]  .Km and Vmax values were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min for the HLMs, 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min for CYP2C9, 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min for CYP3A4, respectively. The in vitroCLint values of CK in the HLMs, CYP2C9, and CYP3A4 were 0.0033 mL/mg protein-1·min-1, 0.0130 mL/nmol P450-1·min-1, and 0.0252 mL/nmol P450-1·min-1, respectively.IC50 or Ki values for the CYP isoforms to ensure adequate inhibitory selectivity and maximal inhibitory potency. In the presence of sulfaphenazole and ketoconazole, the metabolic clearance rate (MCR) of CK decreased to 73.5±20.9% and 74.6±28.1% of that of the control, respectively  on the activity of each CYP isoform determined by the metabolism of a single concentration of isoform-specific probe were tested with the HLMs (or expressed CYPs when needed). CK showed the potent inhibition of CYP2C9 (tolbutamide 4-hydroxylation) and CYP3A4 (midazolam 1-hydroxylation), with ICKi values were determined using tolbutamide as the probe substrate. The Vmax and Km values estimated using a nonlinear regression model for the competitive enzyme inhibition of CYP2C9-catalyzed tolbutamide 4-hydroxylation in the HLMs, the representative Lineweaver-Burk plots . These findings are partly supported by the study of Hao et al.(2008) that ginsenosides, Rh2, CK, and all sapogenins exhibited moderate CYP3A4 inhibition in baculosomes. According to Kong et al[IC50 values, as potent, if IC50≤ 20 μg/mL or ≤10 μM; moderate, if IC50 20–100 μg/mL or 10–50 μM; or weak, if IC50≥100 μg/mL or ≥50 μM. CK is classified as a potent inhibitor of CYP3A4, a moderate inhibitor of CYP2C9, and a weak inhibitor of the other five CYPs tested in this study.In the present study, our data suggested that CYP2C9 and CYP3A4 were the major enzymes involved in the metabolism of CK ong et al, the potCYP2C9 is one of the most abundant CYP enzymes  . It metaIC50and Ki values of CK were 11-53-fold and 49-fold higher than those of sulfaphenazole in HLMs, respectively [IC50and Ki values of CK were 41-123-fold and 761-fold higher than those of ketoconazole in HLMs, respectively [max) and time to peak plasma concentration (Tmax) were directly obtained from the observed concentration-time data. The area under the plasma concentration-time curve (AUC) from time zero to last measured concentration (AUC(0−t)) was calculated according to the linear trapezoidal rule, the estimate of oral clearance(CL/F) was calculated as CL/F = Dose /AUC(0–t) [max of CK were 4.07 μM . The Cmax of CK of some volunteers were found to be near 5 μM through one-day supplementation of 800 mg, which is close to the IC50 and Ki values of CK for CYP2C9 and CYP3A4  . The resd CYP3A4 . Thus, w


The two estrogen receptors (ERs), ERα and ERβ, mediate the diverse biological functions of estradiol. Opposite effects of ERα and ERβ have been found in estrogen-induced cancer cell proliferation and differentiation as well as in memory-related tasks. However, whether these opposite effects are implicated in the pathogenesis of Alzheimer’s disease (AD) remains unclear. Here, we find that ERα and ERβ play contrasting roles in regulating tau phosphorylation, which is a pathological hallmark of AD. ERα increases the expression of miR-218 to suppress the protein levels of its specific target, protein tyrosine phosphatase α (PTPα). The downregulation of PTPα results in the abnormal tyrosine hyperphosphorylation of glycogen synthase kinase-3β (resulting in activation) and protein phosphatase 2A (resulting in inactivation), the major tau kinase and phosphatase. Suppressing the increased expression of miR-218 inhibits the ERα-induced tau hyperphosphorylation as well as the PTPα decline. In contrast, ERβ inhibits tau phosphorylation by limiting miR-218 levels and restoring the miR-218 levels antagonized the attenuation of tau phosphorylation by ERβ. These data reveal for the first time opposing roles for ERα and ERβ in AD pathogenesis and suggest potential therapeutic targets for AD. Extensive research has established the two most prominent pathological hallmarks in AD brains: senile plaques and neurofibrillary tangles (NFTs). The degree of cognitive impairment has been shown to significantly correlate with the presence of NFTs  α. The abnormal expression of PTPα resulted in the aberrant tyrosine phosphorylation and thus function of glycogen synthase kinase-3 (GSK-3) and protein phosphatase 2A (PP2A), causing a disruption in the phosphorylation balance of tau protein. Our data provide a novel mechanism for the epigenetic regulation of tau phosphorylation in AD, which may suggest new therapeutic targets.To explore the potential roles of ERα and ERβ in tau phosphorylation, we first examined the relevance of ERα and ERβ in tau phosphorylation in the prefrontal cortex of 18-month-old Tg2576 mice (pathogenic stage), which is a widely used AD mouse model. By analyzing fluorescence intensity, we found that ERα was negatively correlated with Tau1, which is a nonphosphorylated tau located at Ser198/199/202 sites, while the intensity of ERβ was negatively correlated with AT8, which is a phosphorylated tau at Ser202/Thr205 Fig.,B. In adTo validate the exact role of ERα and ERβ in tau phosphorylation, we overexpressed human ERα and ERβ in HEK293/tau cells to examine the phosphorylation levels of tau. The phosphorylation of tau at Thr205, Ser214, Thr231, Ser396, and Ser404 sites dramatically increased in response to ERα overexpression, while significantly decreased to ERβ overexpression Fig.–H. Theseet al., et al., We then asked whether blocking ERα and ERβ could reverse the abnormal tau phosphorylation induced by ERα and ERβ overexpression. ICI 182,780 (ICI), which is a nonspecific ER antagonist, reduced ERα-caused tau hyperphosphorylation and precluded ERβ-dependent attenuation in tau phosphorylation –c. MeanwWe then applied an effective small hairpin (sh) RNA plasmid that specifically targeted mouse ERα or ERβ. We transfected the effective shRNA with its scrambled control into Neuro2A cells and examined tau phosphorylation by Western blot. shRNAs selectively decreased the protein levels of ERα or ERβ but not altered another receptor level –d. SilenAs abnormally phosphorylated tau usually aggregates to form paired helical filaments, which are the dominant component of neurofibrillary tangles in AD, we then examined the tau aggregation in the HEK293/tau cells upon ERα or ERβ treatment. We found that ERα overexpression significantly increased the phosphorylation of tau in the insoluble fraction but not the soluble fraction, as well as the amount of aggregated tau in the insoluble fraction –c. In ceTo further understand the mechanisms underlying ER-regulated tau phosphorylation, we screened the main kinases and phosphatases that are involved in tau phosphorylation. The phosphorylation of Ser9 in GSK-3β was significantly decreased, while the phosphorylation of Tyr216 in GSK-3 and Tyr307 in PP2A was dramatically increased with ERα overexpression. In the cells overexpressing ERβ, an obvious increment in the phosphorylation of Ser9 in GSK-3β and a decrement in the phosphorylation of Tyr216 in GSK-3 and Tyr307 in PP2A were observed. In addition, no alterations were found in the total levels of GSK-3β, PP2A, protein kinase (PK) Aα, PKAβ, cdk5, and p35/25 Fig.. Treatmevia PTPα inhibition.As reportedly previously, tyrosine phosphorylation is mostly mediated by tyrosine kinases and phosphatases. Among those kinases and phosphatases, Src, fyn, PTPα, and PTP1B have been implicated in the pathogenesis of AD or tauopathy. We then examined the levels of those enzymes. In response to ERα overexpression, the levels of total Src, fyn, and PTP1B were not changed and the Tyr416 of Src was increased, but the Tyr527 of Src and the level of PTPα were decreased, suggesting the activation of Src and the inhibition of PTPα Fig.,B. We thIn response to ERβ overexpression, the levels of total Src, fyn, and PTP1B and the phosphorylation of Src were not changed, but the level of PTPα was increased, suggesting the activation of PTPα ,e. UsingTo further verify that ERα and ERβ differentially regulate PTPα pathway, we examined the tyrosine phosphorylation of GSK-3β, PP2A, and Src and the protein level of GSK-3β, PP2A, Src, fyn, and PTPα upon the ERα or ERβ silencing in N2a cells. We found that silencing ERα caused a decrease in tyrosine phosphorylation of GSK-3β and PP2A, which was accompanied with an elevation in PTPα protein level ,b, whilePTPRA, the gene for PTPα  and dissolved in DMSO to 5 mm for stock. Lipofectamine 2000 was purchased from Invitrogen . Cell culture media were from Gibco . Plasmids containing the human ERα and ERβ cDNAs were constructed according to the following sequences: NM_001122741 and NM_001437 to pEGFP-N1. shRNA-ERβ plasmid toward mouse ERβ (NM_207707.1) was constructed to vector GV102 by Neuron Biotech Inc. . si-ERα oligonucleotide toward mouse was synthesized using the sequence published before  and dissolved in DMSO to 100 μad libitum for at least 2 weeks before the day of experimentation. All animal experiments were performed according to the ‘Policies on the Use of Animals and Humans in Neuroscience Research’ revised and approved by the Society for Neuroscience in 1995.Eighteen-month-old male Tg2576 mice were purchased from the Jackson Laboratory  and housed in a room on a 12:12 hr light–dark cycle and 22 ± 2 °C with water and food −1 G418 with 10% fetal bovine serum , and mouse neuroblastoma 2a (N2a) cells (kindly gift by Dr. Huaxi Xu at Xiamen University) were seeded in six-well plates in DMEM with 10% fetal bovine serum . Both cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C. The cells were cultured for at least 24 h after plating, and when grown to 80–90% confluence, the culture medium was replaced with serum- and antibiotic-free DMEM prior to treatment.HEK293/tau cells (HEK293 cells stably transfected with the longest human tau (tau441) cDNA) were cultured in DMEM in the presence of 200 μg mLm PP2 for 1 h. Then, the media were removed and the cells were harvested and stored at −20 °C for further experiments.Plasmids used for transfection were amplified and purified by Qiagen kit  according to the manufacturer’s instruction. Briefly, HEK293/tau or N2a cells were seeded in six-well plates, grown to 60–70% confluence, and then cultured in serum- and antibiotic-free OPTI-MEM for 4 h. Plasmids were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Cells transfected with GFP constructs were visualized at 48 h after transfection by an Olympus IX70 microscope with a 209LCPlanF1 lens . For double transfection, the plasmid and oligonucleotides were added to OPTI-MEM, respectively, at the last step of transfection. 48–60 h after transfection, cells were treated with 100 nM ICI 182,780 for 1 h or 5 μ−1) and perfused through aorta with 100 mL 0.9% NaCl followed by 400 mL phosphate buffer containing 4% paraformaldehyde. About 2 h later, brains were removed and postfixed in perfusate overnight and then cut into sections (15–20 μm) with vibratome . The sections of mice brain were collected consecutively in PBS for immunofluorescence staining. Free-floating sections were incubated with bovine serum albumin (BSA) to block nonspecific sites for 30 min at 25 °C. Sections were then incubated overnight at 4 °C with primary antibodies rabbit polyclonal ERα or ERβ antibody for 48 h, and after washing with PBS, sections were subsequently incubated with mouse monoclonal Tau1, Tau5, or AT8 for 48 h. After washed with PBS for 30 min, sections were subsequently incubated secondary antibodies Alexa Fluor 488 (donkey anti-mouse) or Alexa Fluor 546 (goat anti-rabbit) for 1 h at 37 °C. The prefrontal cortex region was chosen for imaging using a laser confocal microscope  , 1 μL of each 2 μm primer (300 mm each), 5 μL of SYBR Green, and 2.5 μL RNase/DNase-free sterile water. Blank controls were run in triplicate for each master mix. The cycle conditions were set as follows: initial template denaturation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 5 s, and combined primer annealing/at 60 °C for 30 s, and elongation at 72 °C for 30 s. This cycle was followed by a melting curve analysis, ranging from 60 to 95 °C, with temperature increasing by steps of 0.5 °C every 10 s.The total RNA from the cells was extracted by TRIzol reagent (Invitrogen), and 1 μg RNA was reversely transcripted. qRT–PCR was performed on ABI StepOne Plus using SYBR Green ® Premix Ex Taq . MicroRNA was extracted using miRNA isolation kit . Reactions were prepared in a total volume of 10 μL containing 0.5 μL cDNA  followed by boiling for 5–6 min, and then sonicated for 5 s on ice. The cell lysates were then centrifuged at 12 000 g for 5 min at 4 °C; aliquots of supernatants were added to one-third volume of 4× sample buffer, 10% beta-mercaptoethanol (ME), and 0.05% bromophenol blue and then stored at −20 °C or used immediately. Protein concentration was quantitated using the BCA Protein Assay Reagent Kit   for 30 min–1 h and probed with primary antibodies overnight at 4 °C. Then, the blots were incubated with goat anti-mouse or anti-rabbit conjugated to IRDye 800  (1:15000) for 1 h at 25 °C. The protein bands were visualized and quantified by the Odyssey infrared imaging system . The levels of the phosphorylated protein tau, PP2Ac, GSK-3β, and Src were normalized against the total protein tau, PP2Ac, GSK-3β, Src, and PTPα. The amount of protein was expressed as relative level of the sum optical density against controls.Equal amounts of protein were separated by SDS–polyacrylamide gel electrophoresis (10% gel) and transferred to nitrocellulose membrane. The membranes were blocked with 5% nonfat milk dissolved in PBS  containing protease inhibitors (100 μg mL−1 PMSF) and centrifuged at 50 000 g for 40 min in 4 °C, and the supernatants were saved as the RAB-soluble fraction. The RAB-insoluble pellets were sonicated in sample buffer containing 0.2 g mL−1 sucrose, 18.5 mm Tris (pH 6.8), 2 mm EDTA, 80 mm DTT, and 2% SDS and centrifuged at 50 000 g for 20 min in 4 °C. The supernatant was discarded, and the pellet was homogenized in cold RIPA buffer  with protease inhibitor and centrifuged at 50 000 g for 20 min in 4 °C. The supernatant was saved as RIPA-soluble fraction, and the pellet was extracted in 70% formic acid as FA fraction. Fractions were analyzed by SDS-PAGE.For tau aggregation analysis . The one-way anova procedure followed by LSD’s post hoc tests was used to determine the different means among groups.Data were expressed as mean ± SD and analyzed using This work was supported in part by the National Natural Science Foundation of China , MOST International Collaboration Grant (2011DFG33250), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13016).L.Q.Z. initiated, designed, and directed this study. Y.S.X., F.F.L., D.L., H.Z.H., N.W., and L.T. performed the experiments. J.G.C. and J.Z.W. provided partial financial support. C.X.G., Y.L., and J.Z.W. provided some comments. Y.S.X., F.F.L., H.Y.M, and L.Q.Z. wrote the manuscript. All authors read and approved the final manuscript.All the authors declare that they have no conflict of interest in relation to this study.


The optimal iliac screw path was determined to provide references for lumbosacral-pelvic reconstruction.Radiographic data of 100 patients with normal pelvis were selected for this study. Four paths were designed. Paths A, B, and C were from the starting point of the crossing point of the chiotic line and posterior iliac crest  to the upper edge of the acetabulum, anterior inferior iliac spine, and acetabulum center, respectively. Path D was from the starting point of the posterior superior iliac spine to the anterior inferior iliac spine. The lengths of the different paths of screw passage and bone plate thicknesses of two narrow places were measured and analyzed.Paths A, B, and D were approximately equal in length, but the thickness of the iliac plate in path A was significantly thicker than those in paths B and D. No significant difference was found between the iliac thickness of paths A and C, but the passage length of path A was significantly longer than that of path C.Path A had the longest passage length and thickest iliac plate and could accommodate the relatively longest and thickest iliac screw. Thus, path A was the optimal iliac screw passage. Iliac screw fixation is used in the reconstruction of spinal-pelvic stability -6. IliacOne hundred outpatients or inpatients underwent pelvic CT  scan and three-dimensional (3D) reconstruction without positive performance for various reasons from February 2009 to June 2012. A total of 58 males and 42 females, aged 19 to 65 years (average age 42.2 years), were involved in this study. Their radiographic data were evaluated. The inclusion criteria were adults with pelvis under normal development and absence of abnormalities, such as iliac bone deformities, tumors, and trauma fractures. This study was conducted in accordance with the declaration of Helsinki. This study was conducted with approval from the Ethics Committee of the Sixth Affiliated Hospital of Wenzhou Medical College. Written informed consent was obtained from all participants.A line was drawn from the upper edge of the eminentia iliopubica to the closest point of the front edge of the auricular surface to the posterior iliac crest. This line, which was called the chiotic line, divided the posterior iliac crest into the front and back sections. The crossing point of the chiotic line and posterior iliac crest was called the CLIC point, which was located 24.0 mm above the posterior superior iliac spine (PSIS).Four iliac screw paths were designed. Paths A, B, and C were from the starting point of the CLIC to the upper edge of the acetabulum, anterior inferior iliac spine (AIIS), and acetabulum center, respectively. Path D was from the starting point of the PSIS to the AIIS. Specific methods were as follows:1. Path A represented the connection and screw passage direction from the CLIC point to the upper edge of the acetabulum  with spacing of 1.25 mm and 3D computer reconstruction. The distance between the two points was directly measured, with an accuracy of 0.1 mm. The lengths of screw passage and thicknesses of bone plates in two narrow places in different paths were measured and analyzed. Each radiographic datum was repeatedly measured twice within 1 week by the same surveyor, and the mean value was used as the final result.t test, with an inspection level of bilateral alpha = 0.05. P < 0.05 was considered statistically significant.SPSS13.0 software was used in this study. The measured data in accordance with normal distribution were presented as Four paths with different entrance points and screw passages were measured and analyzed. Paths A (from the CLIC point to the upper edge of the acetabulum), B (from the CLIC point to the AIIS), and D (from the PSIS to the AIIS) had similar lengths of screw passage, but the thickness of iliac plate in path A was significantly thicker than those in paths B and D. No significant difference was found between the iliac thickness of paths A and C (from the CLIC point to the acetabular center), but the passage length of path A was significantly longer than that of path C. Among the paths from the CLIC, path A had the longest passage length and thickest iliac plate, and could accommodate the relatively longest and thickest iliac screw. Thus, path A was significantly superior to the other three paths. The lengths of passages and thicknesses of bone plates in two narrow places in the different paths are shown in Table Lumbosacral-pelvic fixation without pelvic intensive long segment fusion had a clinically higher non-fusion rate, regardless of the number of sacral fixation screws. Application of iliac screw in combination with lumbar pedicle screw and rod could produce a better effect in maintaining lumbosacral-pelvic stability. McCord et al.  detectedIn the clinical application of iliac screws, orthopedic surgeons have difficulties in avoiding intraoperative nerve injury, blood vessels, and other important structures. Orthopedic surgeons also experience difficulties in the safe and effective implantation of iliac screws. As of this writing, the entrance point and path of iliac screws have no uniform standard. Two sclerotin-rich columnar zones are found in the ilium -13, whicThe entrance point of the iliac screw is controversial. Berry et al.  believedIliac screw path has an important value for lumbosacral-pelvic reconstruction. Of all paths in our design, path A (from CLIC point to the upper edge of the acetabulum) had the longest passage length and thickest iliac plate and could accommodate the relatively longest and thickest iliac screw. Thus, path A was the optimal iliac screw passage. In the future, we also need to be confirmed in the clinical work.The authors declare that they have no competing interests.BL reviewed the database and prepared the manuscript. JW and WG carried out the statistical analysis and assisted with preparation of the manuscript. LZ assembled the database and supervised the writing of the whole paper. All authors read and approved the final manuscript.


Information transmission in cells occurs quite accurately even when concentration changes are “read” by individual binding sites. In this paper we study ligand number and site occupancy fluctuations when ligands diffuse and react going beyond the analyses that focus on their asymptotic decay. In this way we show that, for immobile binding sites, fluctuations in the number of bound molecules decay on a relatively fast scale before the asymptotic behavior kicks in. This result can explain the observed co-existence of highly fluctuating instantaneous transcriptional activities with accumulated mRNA concentrations that have relatively small noise levels. We also show that the initial stages of the decay in the bound molecule number fluctuations have one or two characteristic timescales depending on the concentration of free molecules. This transition can explain the changes in enzyme activity observed at the single molecule level. Drosophila melanogaster embryos. The analysis of the variability of the concentrations of the protein Hunchback (Hb) and of the transcription factor Bicoid (Bcd) involved in its production shows that the resulting pattern is compatible with detecting [Bcd] with a 10% error [h) compared to the embryo developmental time [Bcd] could be inferred with this precision. In  can be read by those binding sites? This is one motivating question of our paper.The seminal work of Berg and Purcell  showed teraging)  was derieraging) . This dieraging) . These teraging) . In factfb(Tobs), that the binding site spends bound during an “interaction” interval, , and how it differs from the equilibrium probability, pb, that it be bound given the actual concentration of ligand. Thus, introducing the stochastic variable, Nb)((t), such that Nb) is equal to the average:pb is equal to the mean, fb(Tobs) and pb are defined similarly but divided by the total number of sites, NST. The squared difference between them is estimated asτi, of the ACF, Gb). In the case at hand var(Nb) depends on the mean number of free ligand molecules that are within an interaction distance of the binding site. The number of free ligands in this interaction volume is a (highly fluctuating) random variable. The problem is nonlinear and complicated and the derivation of Drosophila melanogaster embryos with some type of spatial averaging: not that of the “product” (the mRNA), as suggested in [S] = KD[Pb)(]), and an observation volume, Vobs, where the number of molecules, Nf)( and NS)(〉 = [Pf)(〉 = [Pb)(]Vobs and 〈NS) total number of binding sites in Vobs, NST ≡ Nb)(+NS) after an observation time, Tobs. These differences reflect by how much the concentrations that can be estimated after a time, Tobs, by counting how many particles of each species are, on average, in the observation volume, Vobs, differ from the bulk concentrations in the total volume, VT, which are proportional to the mean of the number of particles of each species. We estimate these differences using Nb)( by the corresponding Ns) in Vobs to compute Ns) in VT but with a Gaussian weight: a is half the waist of the Gaussian and s) and t. In this way, it is Gs)((τ) we calculate the differences, δcs) and the index, (m), labels the eigenvalues, X is the matrix of eigenvectors, λm) = 〈Nf) = (1 − pb)〈Nb) or proportional to ξ2 (something that corresponds to a diffusive decay) the integrals can be performed exactly. Thus there are simple algebraic expressions for the terms (or components) of the ACF that correspond to these types of eigenvalues. As discussed in ∼(aD[Bcd]Tobs)−1/2 with D ∼ 1μm2/s, the Bcd diffusion coefficient obtained with FRAP ∼5/μm3. The authors argued that this expression gave a lower bound of Tobs  = 〈Nf)(〉 = pb = 〈Nf)(〉/(〈Nf)(〉+KDVobs). This is the approach followed in  = 1/Vobs so that Nb) = (1 − pb)〈Nb)/〈Nf)(〉/(〈Nf)(〉 + KDVobs) we estimate that the sharp transition in [Hb] occurs where 〈Nf) and it is these other interactions that introduce the main limitations to the transport of Bcd, we expect the resulting collective diffusion coefficient to be the one that determines the arrival times of free molecules and the precision with which their concentration can be “read” by the regulatory sites. This needs, however, a separate study that will be done in the future.In  the timeith FRAP , a ≈ 3nmevice of . For Tobμm3 volume. The experiments showed a ∼45% fluctuation level at each transcription locus which was compared with that of the accumulated mRNA. The experiments were done for Hb and for the gene, Krüppel (kr), because the accumulated mRNA of the latter is proportional to the elapsed time. To analyze the observations the authors argued that if transcription has standard deviation, σnuc, per N0 mRNA molecules produced, the maximum noise reduction in the accumulated mRNA is achieved by independently running the process m times. Taking into account the measurement noise, η, the fractional noise, μ is the mean of the accumulated mRNA and where the subscript L is used to distinguish this expression from the one we derive later that takes into account a “nonlinear” correction to estimate the error. From the observations the authors obtain N0 = 100±20 and η ≈ 0.03. According to this theory, by the time the mean expression level reaches 800 molecules per nucleus, The work of  addresseNb) are given, respectively, by Tobs for long enough time, which is the type of reduction produced by time averaging. The early decay of Eqs Tobs  expression for Nb) = (1 − pb)〈Nb)(〉 regardless of whether we use Nb) = (1 − pb)〈Nb) variance of Nb). After this initial reduction the fractional noise is correctly described by fd or fr limits, i.e., it decays as prescribed by time-averaging only, but with m, with Tobs/(2τb). Inserting Eqs m = μ/N0 as in . The dynamics of this type of process was observed at the single molecule level in [Pf) an additional step with respect to Pb)( to the product) increasing [Pf) time, the latter is characterized by a diffusive correlation time that is responsible for the long tail of the dwell-time distribution. Therefore, the transition can be associated to whether the reaction or the diffusive steps determine the dwell-time distribution without the need of having many different enzyme conformers.The scheme level in . The obstally in . AlthougS1 TextIn this text we give a more detailed description of the model and of the calculations that lead to the various formulas presented in the paper.(PDF)Click here for additional data file.


Aspergillus hypersensitivity (AH) were shown to be associated with severe symptoms or worse lung function in COPD patients. The prevalence of elevated total IgE (T-IgE) and its association with clinical symptoms and lung function in COPD have not been studied. The prevalence of AH and its correlation with clinical characteristics in a COPD cohort of larger sample size is also lacking.Allergy and Aspergillus specific IgE. Patients with T-IgE ≥ 1000 KU/L were further investigated for allergic bronchopulmonary aspergillosis (ABPA).273 patients with COPD were evaluated by respiratory symptoms, blood test, chest HRCT, lung function, serum detection of T-IgE and The prevalence of elevated T-IgE and AH in patients with COPD was 47.3% and 15.0%, respectively. Eight patients (2.9%) met the diagnostic criteria for ABPA. Compared with the normal T-IgE group, patients with elevated T-IgE had a longer history of dyspnea (p < 0.01), an earlier onset of dyspnea after chronic cough/expectoration (p < 0.01), and were more likely to wheeze (p < 0.01). They also showed worse lung functions and more severe GOLD staging (p < 0.01). Analysis of the clinical data in male patients with smoking as the risk factor showed the same results. To evaluate the clinical characteristics of COPD with AH, patients with elevated T-IgE were further divided into subgroups with and without AH. When compared with the normal T-IgE group, both the two subgroups showed longer history of dyspnea (p < 0.01), an earlier onset of dyspnea (p < 0.01) and a worse status of lung function (p < 0.05). Correlation analysis demonstrated that T-IgE was correlated positively with the time length of dyspnea , and the ratio of duration of dyspnea to that of chronic cough/expectoration , but negatively with FEV1/FVC% , and FEV1%predicted .Aspergillus may have similar effects on disease expression or progression of COPD.There was a high prevalence of elevated serum T-IgE and AH in patients with COPD. Serum T-IgE level was correlated with symptoms such as dyspnea and impairment of lung function. Allergens other than  Aspergillus fumigatus (A. fumigatus) sensitization is of clinical importance in both asthma and COPD. The prevalence of Aspergillus hypersensitivity (AH) and allergic bronchopulmonary aspergillosis (ABPA) in asthma was 28% and 12.9% respectively in one study . No statistical difference was shown in gender (p = 0.96), age (p = 0.08), and smoking history (p = 0.31) of the control population when compared with those of COPD patients. Elevated T-IgE level was found in 18.0% of the population (27/150), and the average level of T-IgE was 48 kU/L.A. Fumigatus hypersensitivity and ABPA was shown in Figure A. Fumigatus hypersensitivity was confirmed in 15.0% (41/273) patients, all of whom had an increased level of serum T-IgE, i.e. 31.8% of the COPD patients with elevated serum T-IgE (41/129) were hypersensitive to A. Fumigatus , 12.2% (5/41) and 7.3% (3/41) respectively. The clinical data of the 8 patients with combined COPD and ABPA were shown in Table The prevalence of increased serum T-IgE, 15.0% 41/73 patienThere was no significant difference in gender, age, the percentage of smokers, smoking index of the smokers and history of chronic cough/expectoration between the group with elevated T-IgE n = 129) and that with normal T-IgE (n = 144). Compared with the normal IgE group, patients with elevated T-IgE had a longer history of exertional dyspnea, an earlier onset of dyspnea after chronic cough/expectoration, and were more likely to wheeze. COPD patients with elevated serum T-IgE also showed worse pulmonary functions, as demonstrated by lower values of FEV1/FVC% and FEV1%predicted, and by more severe GOLD staging  and that with normal T-IgE (n = 67), but patients with increased serum T-lgE were more likely to wheeze, had a longer history of dyspnea and earlier onset of dyspnea on the background of chronic cough/expectoration history. They also had a worse pulmonary function   and another without AH , and compared the clinical data of the Af(+)/IgE(+) subgroup, the Af(−)/IgE(+) subgroup, and the group with normal serum T-IgE . There was no significant difference in age, smoking index and history of chronic cough/expectoration. When compared with the Af(−)/IgE(−) group, both the Af(+)/IgE(+) subgroup and the Af(−)/IgE(+) subgroup showed longer history of dyspnea, an earlier onset of dyspnea and a worse status of lung function (FEV1%predicted). The severity by GOLD stages and the prevalence of wheezing were significantly different among the three groups, but no statistical difference was found between any two groups. There was also no difference in the clinical data between the Af(+)/IgE(+) subgroup and the Af(−)/IgE(+) subgroup (see Table P< 0.001), and the ratio of duration of dyspnea to that of chronic cough/expectoration  , and FEV1%predicted   between patients with elevated serum T-IgE and those with normal T-IgE, we performed correlation analysis of serum T-IgE with these parameters. Serum T-IgE was shown to be correlated positively with the time length of dyspnea history r = 0.401, ) Figure , but neg01,  FiguAspergillus sensitization was common in COPD patients with increased levels of IgE, and some patients were confirmed to have combined COPD and ABPA.Previous studies have demonstrated that allergy was associated with decreased lung function in general population, smokers and mild COPD patients -18. HoweAspergillus-specific IgE showed elevated serum T-IgE, and no positive result of Aspergillus-specific IgE was found in patients with normal serum T-IgE, suggesting a high consistency between serum T-IgE and airborne allergen specific IgE in our cohort. The results demonstrated that the prevalence of elevated serum T-IgE was 47.3% (129/273) in patients with COPD, implying that even among COPD patients without obvious atopy, hypersensitive inflammation of the lower airways may exist, probably representing the real proportion of the allergic phenotype in patients with COPD, a much higher prevalence as compared to those reported by Jamieson et al. [The increased serum level of T-IgE may result from allergic inflammation at other parts of the host, and may be associated with genetic factors and even smoking history ,20. In on et al. . The reaAspergillus sensitization and occurrence of ABPA in COPD patients are receiving considerable interests. Agarwal et al. reported that the prevalence of Aspergillus sensitization and ABPA in a cohort of COPD patients without atopy was 8.5% and 1% respectively [Aspergillus sensitization in COPD was 13% [Aspergillus sensitization in patients with COPD was 15%, similar to that reported by Bafadhel et al., but higher than that by Agarwal et al. Besides the different study subjects, different methods to determine Aspergillus sensitization may be a main reason for the discrepancy, because detection of specific IgE (used in our study) has been shown to be more sensitive than SPT  to identify Aspergillus sensitization [Aspergillus sensitization was 2.9%, 6.2%, and 19.5% respectively, indicating that the occurrence of ABPA in COPD was lower than that in asthma [ectively , while itization ,21,22. WWe found no significant difference in the history length of chronic cough/expectoration between patients with elevated serum IgE and those with normal serum IgE, but the former group was more likely to wheeze, which was similar to the result of Jamieson et al. . BecauseIgE is not only a sensitive marker for the presence of allergic inflammation, but also correlated positively with airway inflammation and remodeling in asthma -8. ZedanAspergillus sensitization [Aspergillus sensitization and those with normal serum IgE. However, no significant difference in dyspnea history and lung function was found between patients with Aspergillus sensitization and those with elevated serum IgE but without Aspergillus sensitization. This suggests that allergens other than Aspergillus may have similar effects on COPD, but further study is needed for a final conclusion.Previous studies have shown that lung function is more severely impaired in COPD patients with tization . Our stuCOPD combined with ABPA is believed to be rare, although more cases were reported recently -30, possAspergillus specific IgE were not followed in all the patients, although allergic sensitization in adults is generally believed to be stable. Because of the retrospective nature of the study, more measures of patient reported outcome (PRO) such as COPD Assessment Test (CAT), and history of exacerbations which might have an association with allergy in a proportion of patients, had not been collected and analyzed. We did not analyze the difference in long-term drug therapy, particularly inhaled corticosteroids in these patients. Medication use and compliance of the patients may affect symptoms and lung function, although it is possible to have a similar effect on level of serum IgE [There are several limitations to our study. As a cross-sectional study, serum T-IgE and erum IgE . Finallyerum IgE -33. One erum IgE . In our Aspergillus sensitization in COPD patients. There was a high prevalence of elevated serum T-IgE in patients with COPD but without obvious atopy. Serum T-IgE level was found to be associated with symptoms such as dyspnea and impairment of lung function. Recurrent exacerbation of COPD is believed to accelerate disease progression and impairment of pulmonary function, and related to increased disability and mortality [In conclusion, to our knowledge, this is the first study to investigate serum T-IgE level in patients with COPD and its association with the symptoms and lung function. It is also a study of the largest sample size for the investigation of the prevalence of ortality ,36. Acutortality . Howeverortality . Becauseortality , whether