It is enriched in the basal ganglia, hippocampus, olfactory bulb, and cortical areas, and plays a role in motor and cognitive functions. Muscarinic M1 agonists are potential therapeutic agents for cognitive disorders. The aim of this study was to evaluate [11C]AF150(S) in rat brain were measured using a high-resolution research tomograph, both under baseline conditions and following pre-treatment with various compounds or co-administration of non-radioactive AF150(S). Data were analysed by calculating standard uptake values and by applying the simplified reference tissue model (SRTM).Regional kinetics of [11C]AF150(S) was rapidly taken up in the brain, followed by a rapid clearance from all brain regions. Analysis of PET data using SRTM revealed a binding potential (BPND) of 0.25 for the striatum, 0.20 for the hippocampus, 0.16 for the frontal cortical area and 0.15 for the posterior cortical area, all regions rich in M1ACh-R. BPND values were significantly reduced following pre-treatment with M1ACh-R antagonists. BPND values were not affected by pre-treatment with a M3ACh-R antagonist. Moreover, BPND was significantly reduced after pre-treatment with haloperidol, a dopamine D2 receptor blocker that causes an increase in extracellular acetylcholine (ACh). The latter may compete with [11C]AF150(S) for binding to the M1ACh-R; further pharmacological agents were applied to investigate this possibility. Upon injection of the highest dose (49.1 nmol kg−1) of [11C]AF150(S) diluted with non-radioactive AF150(S), brain concentration of AF150(S) reached 100 nmol L−1 at peak level. At this concentration, no sign of saturation in binding to M1ACh-R was observed.[11C]AF150(S) was rapidly taken up in the brain and showed an apparent specific M1ACh-R-related signal in brain areas that are rich in M1ACh-R. Moreover, binding of the agonist PET ligand [11C]AF150(S) appears to be sensitive to changes in extracellular ACh levels. Further studies are needed to evaluate the full potential of [11C]AF150(S) for imaging the active pool of M1ACh-R in vivo.The agonist PET ligand [ Acetylcholine (ACh) is a core neurotransmitter that controls vital functions via the peripheral nervous system  and the central nervous system (CNS) (e.g. cognitive and motor functions). Cholineq/11 proteinscopolamine, [11C]NMPB, [N-11C-methyl]-benztropine and [11C]QNB(+)-PHNO, revealed that the agonist PET ligand labelled in vivo a smaller receptor pool than an antagonist PET ligand, and moreover, agonist PET ligands appeared more sensitive to displacement by released endogenous dopamine[A non-invasive molecular imaging technique such as positron emission tomography (PET) could be useful to elucidate the function of M1ACh-R [11C]QNB-12. Thesxanomeline and [11C]butylThio-TZTP, appeared not selective for the M1ACh-R subtype and show high binding affinity for σ sites, leading to substantial non-selective binding. In addition, they suffer from poor pharmacokineticsAF150(S) as a potential M1ACh-R agonist PET ligand.In the search for a suitable selective M1ACh-R agonist PET ligand, AF150(S) was selected based on its pharmacological properties. AF150(S) has moderate affinity with an apparent equilibrium dissociation constant for high-affinity binding sites, ed cells. AF150(Sed cells. Cevimeled cells-29. AF15ed cells. Such im11C]AF150(S) has been synthesised successfully. Its binding properties and specificity for M1ACh-R were evaluated by in vitro autoradiography; high uptake in rat brain, possibly by facilitated transport mechanisms, was demonstrated in studies ex vivoAF150(S) were studied in rats using PET in order to explore its suitability as an agonist PET ligand for M1ACh-R. In vivo specificity of binding and sensitivity to changes in extracellular levels of ACh were examined by treatment with various pharmacological agents.In the present study, brain uptake and binding of AF150(S) was synthesised as previously described by methylation of the desmethyl precursor (AF400) using [11C]methyl iodide[−1 at the end of synthesis. [18F]NaF was prepared as described previouslyAF150(S) was injected. At the first injection for baseline scanning, the SA was 71 ± 16 GBq μmol−1; at the second injection, following pre-treatment, SA was 9 ± 2 GBq μmol−1. A three-dimensional (3D) emission scan was acquired, starting immediately prior to the IV injection and lasting for 45 min. At the end of the [11C]AF150(S) scans, [18F]NaF (15.2 ± 4.5 MBq) was injected, and 30 min later, an emission scan was acquired for 30 min.An IV bolus of 15.5 ± 4.2 MBq of [11C]AF150(S), were subsequently histogrammed into 21 frames with progressive duration . The [18F]NaF data were histogrammed into a single frame of 30 min. Data were reconstructed using 3D ordered subsets weighted least squaresAF150(S) in the brain. Rats were randomised into five groups of four rats, and for each rat, two consecutive [11C]AF150(S) scans were acquired with a 30-min interval. The first scan was performed under baseline conditions, providing information on regional distribution and kinetics of [11C]AF150(S) in the brain. The second scan was performed following pre-treatment with either the M4/M1ACh-R agonist xanomeline (5 or 30 mg kg−1 subcutaneous (SC))[−1 SC)[−1 SC)[−1 IV)[11C]AF150(S) injection.A total of 20 rats were used to examine the binding specificity of AF150(S) binding. Rats were randomised into three groups of four rats. In each animal, two consecutive scans were performed with a 45-min interval. The first scan was performed under baseline conditions, and the second after pre-treatment with haloperidol (1 mg kg−1 SC), the combination of AF-DX 384 ) and rivastigmine (2.5 mg kg−1 SC), or AF-DX 384 (5 mg kg−1 IP). Haloperidol and AF-DX 384 were administered 30 min and rivastigmine 45 min prior to the [11C]AF150(S) injection.Twelve rats were used to examine indirect effects of haloperidol (a dopamine D ligand) and AF-D ligand), the lat ligand), on [11C11C]AF150(S) in the syringe, right before intravenous injection. Rats were randomised into three groups of four animals; each animal underwent two consecutive scans with a 30-min interval. First, each rat received an injection of undiluted [11C]AF150(S) to register a baseline scan; before the second scan, rats received an injection of [11C]AF150(S) diluted with cold AF150(S).Twelve rats were used to investigate the effect of co-injection of cold AF150(S) on the total amount of AF150(S) taken up into the brain. Cold AF150(S)  was added to the dose of [18F]NaF scans were co-registered with a standard magnetic resonance (MR) rat brain template to delineate regions of interest (ROIs); this method was previously described and validated with [11C]AF150(S) in ratsAF150(S) uptake in brain regions under baseline conditions was examined in a total of 44 rats. Average time-activity curves for five brain regions are shown in Figure11C]AF150(S), radioactivity in all brain regions rapidly increased and reached its maximum within the first minute after injection. Uptake in the striatum, hippocampus and the two cortical areas was higher than in the cerebellum. Clearance of radioactivity from the brain was relatively fast, especially in the cerebellum. The maximum brain concentration measured was, on average, 155 ± 26 kBq mL−1 corresponding to 2.9 ± 0.5 nM [11C]AF150(S).1CAF150(SSUV ratios (SUVr) of the brain region over the cerebellum as a function of time are shown in Figure11C]AF150(S) specific binding in M1ACh-R-rich brain areas was determined using SRTM with the cerebellum as reference tissue. BPND values are presented in Table ND under baseline conditions ranged from 0.25 to 0.15, with a regional rank order of striatum > hippocampus > frontal cortical area ≈ posterior cortical area. Findings were quite consistent across the five groups of four rats. Representative images of baseline [11C]AF150(S) uptake and corresponding parametric BPND are shown in Figure1CAF150 BPND significantly increased by 20% and 19% in the left striatum and hippocampus, respectively. Xanomeline at a higher dose (30 mg kg−1) did not result in significant changes in BPND in any of the brain areas investigated. This high dose caused severe side effects, such as strongly increased heart rate, salivation and irregular breathing. Pre-treatment with pirenzepine and trihexyphenidyl caused significant reductions in BPND. With pirenzepine, reductions of 37%, 32% and 16% were seen in the frontal cortical area, posterior cortical area and left striatum, respectively. Trihexyphenidyl caused reductions in the frontal cortical area, posterior cortical area and hippocampus of 53%, 56% and 13%, respectively. Pre-treatment with darifenacin had no measurable effect on [11C]AF150(S) binding in any brain region.In Table ND values in five brain areas following pre-treatment with haloperidol, AF-DX 384 and AF-DX 384 plus rivastigmine. Haloperidol pre-treatment resulted in significant reductions in BPND in the right striatum and hippocampus of 27% and 15%, respectively. The animals showed severe rigidity following the haloperidol treatment. Pre-treatment with AF-DX 384, however, did not show any significant changes in BPND. The addition of rivastigmine to AF-DX 384 pre-treatment resulted in significant increases in BPND of [11C]AF150(S) in the right striatum, hippocampus and frontal cortical area of 22%, 12% and 24%, respectively. These treatments did not have an apparent effect on the animal's behaviour or body posture.Table 11C]AF150(S) are shown. The aim of the experiment was to explore whether an AF150(S) concentration occupancy curve could be achieved. Effects of co-injection of cold AF150(S) on the regional brain uptake of total AF150(S) , measured at 5.8 min after injection, are shown in FigureND values under baseline conditions and after co-administration of cold AF150(S) were not significantly different, see Table In Table 11C]AF150(S) were very rapid with peak values being reached within 1 min after injection, followed by a rapid decline. The calculated maximal brain concentration of [11C]AF150(S) of 2.9 nM remained approximately 70 times below the Kd,H (200 nM), of AF150(S) for M1ACh-R, under baseline conditions. In consecutive scans (baseline and following pre-treatment), the same synthesis batch of [11C]AF150(S) was used. As a consequence, the SA was about eight times lower at the second injection, and to keep the injected amount of radioactivity constant, a larger dose of AF150(S) was injected.Under baseline conditions, brain kinetics of [in vitro in rat brain[11C]AF150(S) uptake measured ex vivo[11C]AF150(S) uptake in the hippocampus  PET data. Furthermore, a reference tissue model was chosen for data analysis as no arterial input function could readily be obtained from rats.SUVr data showed increased retention in M1ACh-R-rich areas. The rank order of uptake, striatum ≥ hippocampus > frontal cortical area > posterior cortical area >> cerebellum, corresponds with the rank order of M1ACh-R density measured at brain. These fd ex vivo. Note ths Figure. BPND va M1ACh-R. This paND of [11C]AF150(S) in the frontal and posterior cortical areas. In addition, pirenzepine caused a significant reduction in BPND in the left striatum, and trihexyphenidyl in the hippocampus, indicative of a reduction in specific binding to M1ACh-R in these regions.Pre-treatment conditions, i.e. time of injection and dose of the applied compounds, were chosen according to their maximal pharmacological activity in rats. Pre-treatment with the M1ACh-R selective antagonists pirenzepine and trihexyphenidyl caused a decrease in BP−1 caused an increase in BPND in the striatum and hippocampus, whereas a decrease would have been expected if [11C]AF150(S) and xanomeline are competing for the same binding site. Since GPCRs are dynamic structures that undergo configurational changes upon agonist binding, several explanations are possible for this apparent anomalous finding. Xanomeline is reported to induce M1ACh-R activation via both orthosteric and ectopic binding sites[11C]AF150(S) binding, leading to higher BPND of [11C]AF150(S). An alternative explanation could be the effect of xanomeline on M4ACh-R. M4ACh-R is an autoreceptor, predominantly present in the striatum and hippocampus[11C]AF150(S), which in turn may result in increased binding of [11C]AF150(S). However, a higher dose of xanomeline (30 mg kg−1) that caused severe peripheral and cardiac effects, and glandular secretion, did not show any significant effect on BPND of [11C]AF150(S) in M1ACh-R-rich areas. Therefore, the observed effects of xanomeline seem to be dose dependent, and pharmacokinetic effects may play a role.The M4/M1ACh-R agonist xanomeline, selected to investigate agonist-agonist competition, at a dose of 5 mg kgng sites,46. Xanopocampus. Agonistpocampus, potentiIn vitro experiments have demonstrated partial agonist/antagonist effects of AF150(S) on M3ACh-R . M3ACh-R is present in glands and can also be found on smooth muscles of blood vessels and at low density in the brain[11C]AF150(S). Pre-treatment with darifenacin, however, did not significantly reduce BPND of [11C]AF150(S) in any brain region. Hence, [11C]AF150(S) appears not to show measurable binding to M3ACh-R in the brain in vivo.he brain. Darifen11C]AF150(S) in order to have maximal competition. Haloperidol pre-treatment causes blockade of D2 receptors, which in turn leads to reduced inhibition of the cholinergic system, in particular in the striatum and hippocampus. This results in increased extracellular levels of acetylcholine[in vivo microdialysis studies[ND of [11C]AF150(S) in the right striatum (−27%) and hippocampus (−15%) following haloperidol treatment can likely be ascribed to increased extracellular ACh levels, which may have competed with [11C]AF150(S) for M1ACh-R binding in these particular brain areas.Pre-treatment conditions, i.e. time of injection and dosing, were chosen to give maximal ACh release at 5.8 min post intravenous injection of [ studies. Further studies. Also in11C]AF150(S) for the M1ACh-R, located postsynaptically. In microdialysis studies in rats, AF-DX 384 given IP at a dose of 5 mg kg−1 resulted in a long-lasting significant increase in acetylcholine release, 30 min after administration, in both the cortex and hippocampusAF150(S) in M1ACh-R-rich brain areas, e.g. the right striatum, hippocampus and frontal cortical area. A tentative explanation for these observed increases in BPND could be agonist-mediated changes in M1ACh-R affinity/availability. This has been proposed for the dopamine D2 receptor radioligand [11C]raclopride which demonstrated increased BP after pre-treatment with l-dopa[11C]AF150 binding appears to be sensitive to changes in extracellular ACh levels, in particular when behavioural effects are apparent. Ideally, and in analogy with the dopamine D2 agonist PET ligand studies, the sensitivity of [11C]AF150(S) to changes in extracellular ACh should be assessed in direct comparison with an M1ACh-R antagonist PET ligand to investigate difference in sensitivity between them. Indeed for the dopamine D2 agonist PET ligands [11C]MNPA, [11C]NPA and [11C](+)-PHNO, a larger reduction in binding, by increased endogenous dopamine levels following amphetamine pre-treatment, was found as compared to the antagonist PET ligand [11C]raclopride[11C]AF150(S) binding in brain regions to alterations in extracellular ACh levels.Pre-treatment with AF-DX 384 combined with rivastigmine, surprisingly, resulted in significant increases in BPh l-dopa. Unfortuclopride,55-58. TND values were slightly increased compared to baseline conditions, yet due to variability, these values were statistically not significantly different (Table 11C]AF150(S) in M1ACh-R-rich brain areas is probably not due to diffusion but is a result of facilitated transport of AF150(S) in the brain that was hypothesised from analyses of results in our previous study of [11C]AF150(S) uptake into the brain ex vivo[11C]AF150(S), logDpH 7.4 = 0.05, which hampers diffusion through lipid cell membranes, (2) the eight-times-higher brain level as compared to plasma levels of [11C]AF150(S) and (3) the structural similarity between AF150(S) and nicotine, for which facilitated transport into the brain has been demonstrated.Increasing amounts of co-injected cold AF150(S) resulted in a more-than linear increase in corresponding brain concentrations of total AF150(S) in M1ACh-R-rich brain areas Figure, whereast Table . The morn ex vivo. The hyp−1) resulted in a brain concentration of approximately 100 nM AF150(S) in vivo, a value that still lies 50% under the in vitro Kd,H of AF150(S) (200 nM). At the highest applied dose, no sign of saturation in binding to M1ACh-R was observed; indeed, the latter is only expected to occur at a concentration of 4× Kd,H. Therefore, saturation should be investigated using a substantial higher dose of non-radiolabelled AF150(S).The highest administered molar dose of non-radiolabelled AF150(S) (approximately 50 nmol kg11C]AF150(S) in rat are indicative of the potential of [11C]AF150(S) for specific labelling of M1ACh-R. However, PET studies in rat suffer from several limitations such as the difficulty of getting an arterial input function, the small brain regions and the resolution of the PET camera. Therefore, [11C]AF150(S) should be further explored in PET studies in primates and humans. The chances of success and merit of such studies are supported by the following. M1ACh-R density in primate and human brain is about 50% higher than in rat brain, according to in vitro autoradiography studies[The findings in this preliminary PET study with [ studies. No spec studies.11C]AF150(S) was rapidly taken up in the brain and showed fast kinetics with a significant M1ACh-R-related signal in brain areas that are rich in M1ACh-R. Moreover, binding of the agonist PET ligand [11C]AF150(S) appears to be sensitive to changes in extracellular ACh levels. Further preclinical and clinical studies are needed to evaluate the full potential of [11C]AF150(S) for imaging the active pool of M1ACh-R in vivo.The agonist radioligand [The authors declare that they have no competing interests.HJCB performed the carbon-11 tracer synthesis and all animal studies. Data analysis and modelling were performed by HJCB, MCH and MY. Statistical analysis of the outcome measures was performed by HJCB and DLK. HJCB, ADW, MCH and JEL participated in the study design and discussion and interpretation of findings. HJCB drafted the manuscript. ADW, MCH, AF, JEL, and AAL revised and gave advice for improvement of the manuscript. All authors read and approved the final manuscript.


Hirschsprung's disease (HSCR) is a classic oligogenic disorder. Except inactivating mutations of RET, some single nucleotide polymorphisms (SNPs) are identified to be associated with the risk of HSCR. This study was conducted to examine the impact of the haplotypes profile of the reported associated SNPs of RET on the risk of HSCR in a Southeastern Chinese population.Genotypes of -5G > A (rs10900296), -1A > C (rs10900297), c135G > A (rs1800858), c1296A > G (rs1800860), and c2307T > G (rs1800861) were analyzed in 123 HSCR patients and 168 controls by polymerase chain reaction amplification and direct sequencing. Associations with risk of HSCR were estimated by odds ratio (OR) and their 95% confidence intervals (95% CI) using logistic regression.We observed a significantly increased risk of HSCR associated with the RET -5AA , -1CC , 135AA , 1296GG  or 2307GG  respectively. The five SNPs were in strong linkage disequilibrium. The haplotype A-C-A-G-G  and diplotype A-C-A-G-G/A-C-A-G-G  was also associated with the increased risk of HSCR, indicating a cumulative effect of these SNPs on the susceptibility of HSCR.These results support the hypothesis that common variations in RET pathway might play an important role in development of HSCR. Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of enteric ganglion cells in the myenteric (Auerbach's) and submucosal (Meissner's) plexuses of the variable lengths of gastrointestinal tract. The lack of enteric neurons is attributed to the defective proliferation, differentiation, survival, and migration of enteric nervous system (ENS) towards the end of the gut, whereby neural crest cells (NCC) fail to innervate the lower gastrointestinal tract during embryonic development, presenting in clinic with failure to pass meconium, bilious vomiting, enterocolitis-associated diarrhoea, chronic severe constipation and progressive abdominal distension in the neonatal period ,2. Basedet al [HSCR is a heterogenic disorder with a number of genes reported to be associated, most of which involved in the signaling pathway implicated in the proper ENS development. RET, a receptor tyrosine kinase, is the major susceptibility gene for HSCR, which locates in 10q11.2 and encodes a transmembrane tyrosine kinase receptor, responsible for triggering a number of downstream signaling transductions -5. ThereIn this study, we conducted a large case-control study to evaluate the contribution of the genotypes and haplotypes of these aforementioned five SNPs in the pathogenesis of sporadic HSCR in a Southeastern Chinese population.This case-control study consisted of 123 sporadic HSCR  and 194 controls . Cases were histological diagnosed with either biopsy or surgical resection material for absence of enteric plexuses in Children's Hospital Affiliated to Zhejiang University Medical School, among which twelve (10%) of patients were affected with long-segment aganglionosis (LSA) and the rest with short-segment aganglionosis (SSA). Controls were normal children taking physical examination in the same hospital during the same time period as the patients were enrolled and frequency matched to cases by sex and age. Both cases and controls were Chinese Han in Zhejiang and informed consent was obtained from their parents. The study was approved by the Institutional Review Board of the Children's Hospital Affiliated to Zhejiang University Medical School.Genomic DNA was extracted from blood sample of all study subjects by using QIAamp-Blood Kit . Genotypes were determined by PCR and direct sequencing without knowledge of the subjects' case/control status. Fifteen percent of the masked sample was randomly selected and genotyped twice by different people independently. The reproducibility was 100%.2 and 1.0 units of Tag DNA polymerase with 1 × reaction buffer . The PCR profile consisted of an initial melting step of 5 minutes at 95°C, followed by 35 cycles of 40 seconds at 95°C, 30 seconds at 63°C (57°C for c135G > A and c2307T > G), and 45 second at 72°C, and a final elongation step of 7 minutes at 72°C. PCR products were then sequenced using an ABI PRISM® Big Dye™ Terminator v3.1 Cycle sequencing kit  and an ABI 3130 automated sequencer according to the manufacturer's instructions.RET SNPs  were genotyped as described previously ,12. PCR Chi-square test was used to compare the allele and genotype frequencies between cases and controls, as well as LSA and SSA. The associations between the genotype and the risk of HSCR were estimated by odds ratios (ORs) and their 95% confidence interval (CI), which was calculated by unconditional logistic regression adjusted for gender. PHASE v2.1  was usedP > 0.05, data not shown). The frequency of A allele in -5G > A, C allele in -1A > C, A allele in 135G > A, G allele in 1296A > G and G allele in 2307T > G in cases were 82.5%, 88.6%, 78.9%, 90.7% and 82.1%, which was higher than that of 45.5%, 63.1%, 43.8%, 80.4% and 49.7% in controls, with the P for Cochran-Armitage trend test of 1.12 × 10-16, 3.43 × 10-11, 4.20 × 10-15, 5.70 × 10-4, and 2.32 × 10-14, respectively. All those markers were shown to be strongly associated with the HSCR risk  according to the smallest AIC (Akaike information criteria) value. Subjects carrying the RET -5AA, -1CC, 135AA or 2307GG genotype had a 17.75-fold (95% CI = 7.34-42.92), 10.89-fold (95% CI = 3.13-37.85), 13.61-fold (95% CI = 6.14-30.14) or 9.79-fold (95% CI = 4.28-22.43) elevated risk for the development of HSCR, compared with the counterpart wild genotype, respectively. The much higher OR value in the present study and previously published association studies indicated that HSCR is a classic oligogenic disorder, which is different from the common complex diseases, such as sporadic cancers, cardiovascular diseases and type 2 diabetes with OR less than 2,. Because the frequencies of wild genotype for 1296A > G were low, we combined it with heterozygous-type genotype together for analysis. Similarly, the homozygous RET 1296GG genotype was also linked to risk of HSCR, with the OR of 2.40 (95% CI = 1.38 - 4.18) compared with the AA or AG genotype.P = 0.001). Intriguingly, the haplotype composited with more HSCR-risk allele associated with more elevated risk of HSCR, indicating there is a cumulative effect of these 5 SNPs on the risk of HSCR . We also investigated the associations between RET diplotypes and risk of HSCR. Since the low frequencies of haplotype G-A-G-A-T and G-A-G-G-T, we combined them as reference in the following analysis. In consistent with the results from haplotype, the genetic risk of HSCR was increased in subjects with less copies of the G-A-G-A(G)-T haplotype and more copies of the A-C-A-G-G haplotype, with the P values for the Cochran-Armitage test of 3.09 × 10-15. Only diplotype with two copy of risk haplotype, A-C-A-G-G/A-C-A-G-G, was found to be associated with increased risk of HSCR , when compared with wild type of diplotype, suggesting a haplotype-dosage effect in the genetic susceptibility of HSCR.We further analyzed the association of the haplotypes (MAF > 0.05) comprising the five HSCR-associated SNPs with HSCR risk. The haplotype and diplotype frequencies are presented in Table This genetic epidemiological study investigated whether genetic polymorphisms in RET, alone or in combination, could have effects on the risk of HSCR in a Southeastern Chinese population. In this case-control study including 123 HSCR cases and 168 age- and sex- frequency-matched controls, we demonstrated that five polymorphisms in RET gene, including two SNPs in the regulatory region and three SNPs in the coding region, had substantial impact on risk of HSCR, separately or collectively. Subjects carrying the homozygous variant genotypes of -5G > A, -1A > C, c135G > A, c1296A > G or c2307T > G SNPs were at an increased risk for developing HSCR. Moreover, we also observed cumulative effects of these five polymorphisms on HSCR risk, demonstrating the haplotype composited with more HSCR-risk alleles rendered the hosts more susceptible to HSCR. To our best knowledge, this is the first study to investigate the contribution of the haplotypes of these five SNPs to the pathogenesis of sporadic HSCR in Chinese population. It lends supports to the previous findings that these SNPs were associated with the risk of HSCR ,14-16.Liu CP et al. reported C135 G/A and C1296 A/G at exon 2 and the combination of AG of these two SNPs were associated with susceptibility of HSCR in Southeast Chinese population ,18. AlsoIntriguingly, we found that the frequencies of risk alleles of-5G > A, -1A > C, c135G > A, c1296A > G or c2307T > G in Chinese Han were much higher than those in European , partialThese results support the hypothesis that common variations in RET pathway might play an important role in development of HSCR.The authors declare that they have no competing interests.JT, LW, LL, YW and RZ designed the study; WL, QX, QG, SD, HY and HL collected blood samples and epidemiological data and performed the experiments; JT and LW performed the statistical analysis and wrote the paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/32/prepub


Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced.Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions.We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts.This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of  Escherichia coli is one of the most intensively studied organisms and has been widely employed in scientific studies and industrial applications. The most widely used E. coli have been those derived from strains B and K-12, the result of pioneering work using K-12 for genetic and biochemical studies and B for studying virulent bacteriophages, restriction systems, mutagenic assays, and bacterial evolution [volution  and compvolution . We havevolution ,6 - REL6Previously, we showed that comparison of the genome sequences of strains B and K-12 could provide plausible explanations for some long-known differences between them . HoweverE. coli B REL606 and K-12 MG1655 to decipher the whole-organism characteristics that differentiate these two strains. An in silico genome-scale metabolic model of E. coli B REL606 was reconstructed and used to determine the genetic basis of the phenotypic differences.Here, we systematically combined the results of comparative analyses of the genomes, transcriptomes, proteomes, and phenomes of hpa cluster for degradation of 3- and 4-hydroxy phenyl acetic acid in the B strain and the paa cluster for catabolism of phenyl acetic acid in the K-12 strain. We found numerous strain-specific gene disruptions caused by deletions, frameshifts, or insertions of insertion sequence (IS) elements that might affect bacterial phenotypes  and branched-chain amino acids (ilvGMEDA), a subunit of the L-arginine transporter (artJ), cytochrome b562 (cybC), subunits of the histidine ABC transporter (hisPJ), cytotoxins (hokED), outer membrane porin (ompF), L-arginine decarboxylase (speA), and a cell division inhibitor (sulA). In MG1655, highly expressed genes included those involved in chemotaxis , Lon protease (lon), C4-dicarboxylate-sensing histidine kinase (dcuS), chaperones , the major subunit of type 1 fimbriae (fimA), a regulator of flagellar biosynthesis (flhC), glycerol-3-phosphate-dehydrogenase (glpABCD), glycerophosphoryl diester phosphodiesterase (glpQ), glycerol-3-phosphate transporter (glpT), hydrogenase 2 (hybCBO), outer membrane porins , and galactitol transport and metabolism (gatYZC).Many genes showed highly distinct expression levels in REL606 and MG1655 irrespective of growth conditions  revealed that the B strains expressed large amounts of OmpF but not OmpC, whereas K-12 strains expressed both OmpF and OmpC  tests on REL606 and MG1655 revealed dramatic phenotypic differences between the two strains ; however, in the exponential growth stage, up-regulation of genes involved in the glyoxylate shunt, tricarboxylic acid (TCA) cycle cycle, and gluconeogenesis was observed in K-12 strains  were virtually the same at the nucleotide level in B and K-12 strains. This was also the case for the transcriptional regulator gene nditions . Interes pathway , is disrgatYZC) and the protein level (GatYZAB) in MG1655, possibly because gatR, a repressor of the gat genes, is truncated in MG1655 but intact in REL606.Many of the phenotypic differences between B and K-12 strains in utilization of carbon sources, as shown in PM tests , II (ilvGM) and III (ilvIH) exhibited complicated expression patterns  LPS α1,2-galactosyltransferase. The B strain showed high expression of glmS, encoding a protein involved in both cell wall and LPS biosynthesis [The cell envelope is the principal stress-bearing and shape maintenance element in  strains . Structu strains . Sequenc strains . In bothynthesis , at bothfliY-R) were absent from the B genome, rendering B strains non-motile [cheZYRWA, tap, trg, and tsr) and of flhC, encoding the master regulator of flagellar biosynthesis, are much lower in B than in K-12 , the major mechanism regulating these processes [relBE-class loci  and two mazEF-homologous loci (chpRA and chpSB), whereas B strains possess only two TA loci . TA modules play an important role in bacterial stress physiology and might contribute to multidrug resistance [dnaK, groES, htpG, ibpA, and clpB) and ATP-dependent proteases  were expressed at lower levels in the B strain , carbon starvation protein (cstA), osmotically inducible peroxidase (osmC), phage shock protein (pspE), and universal stress protein (uspA)  Figure , which mrpoE gene, encoding heat-inducible sigma factor σE, and some genes in the operon (rseABC) regulated by this sigma factor, were highly expressed in REL606  is silenced by the H-NS protein [gspB-O genes, except for gspE, were relatively low in both E. coli B and K-12 strains throughout the culture period. Expression of the additional T2S genes present in the B strain was detected by transcriptome analysis. Regarding secretion capacity, both REL606 and BL21(DE3) secreted larger amounts of protein than the K-12 strains did  was provided by Richard E Lenski, Michigan State University, and E. coli BL21(DE3) was obtained from F William Studier, Brookhaven National Laboratory. E. coli K-12 MG1655 was purchased from American Type Culture Collection (ATCC) and E. coli W3110 from the Korean Collection for Type Cultures (KCTC). Cells were grown in 250-ml flasks containing 100 ml LB medium or minimal R/2 medium (pH 6.8) supplemented with 10 g/L glucose in a shaking incubator at 37°C and 200 rpm. R/2 medium contains 2 g/L (NH4)2HPO4, 6.75 g/L KH2PO4, 0.85 g/L citric acid, 0.7 g/L MgSO4·7H2O, and 5 ml/L of a trace metal solution [4·7H2O, 2.25 g/L ZnSO4·7H2O, 1 g/L CuSO4·5H2O, 0.5 g/L MnSO4·5H2O, 0.23 g/L Na2B4O7·10H2O, 2 g/L CaCl2·2H2O, and 0.1 g/L (NH4)6MO7O24.solution . Trace m2SO4 at 60°C at a flow rate of 0.6 ml/minute.Cell growth was monitored by measuring absorbance at 600 nm using an Ultrospec 3000 spectrophotometer . The concentration of glucose and organic acids was determined by high-performance liquid chromatography  with UV/visible-light (ProStar 320) and refractive index detectors . A MetaCarb 87H column  was eluted isocratically with 0.01 M HP < 0.05) were both aberrant. A genome scan was then performed using a ten-gene window, and regions containing four or more foreign genes were identified as genomic islands, with neighboring islands merged into larger ones.Detailed procedures for genome sequencing and annotation of REL606 have been described previously . BrieflyE. coli strains, 382 genes found uniquely in REL606, and 534 genes unique to MG1655. The remaining probes were positive and negative controls. The probes were spotted in duplicate onto a GAPSII glass slide  by Digital Genomics in Seoul, Korea.Oligonucleotide microarrays were constructed in which both REL606 and MG1655 could be tested for the synthesis of both common and unique mRNAs. Probes (70-mers) were designed by Array Designer  and cross-hybridization was checked by BLASTN. A total of 4,819 probes were synthesized by Illumina. The probes represented 3,762 genes that shared over 80 nucleotides of sequence between the two 2 of the intensity ratio with the two dyes for each spot was calculated from up to four values from the duplicated spot on the microarray and the dye-swap experiment. The microarray experiments were highly reproducible: the correlation coefficients between the same spots in each microarray experiment were about 0.98 and correlations between dye-swap paired microarrays were over 0.91. Genes showing growth stage-specific differences in expression levels of ≥2- or ≤0.5-fold were considered to be differentially expressed genes. The microarray data have been deposited at the Gene Expression Omnibus (GEO) [The transcriptome of REL606 cells at the exponential growth stage was compared with that of MG1655 cells at the same stage, and with the transcriptome of both REL606 and MG1655 cells in stationary phase. Total RNA was isolated from cells grown in LB medium using the Qiagen RNeasy column . Fluorescently labeled cDNA was synthesized by reverse transcription of total RNA with aminoallyl-dUTP and random primers followed by the coupling of Cy3 or Cy5 dyes . Hybridization and washes were performed according to the manufacturer's instructions . The hybridized slides were scanned with GenePix 4000B  and the scanned images were analyzed by GenePix Pro 6.0. Following background subtraction, spot signal intensities were calculated as percentages of total signal intensities of Cy3 or Cy5 signals on each microarray as a means of normalization. Spots with low intensities were excluded. All microarray experiments were performed in duplicate with dye-swapping. The logus (GEO)  under acE. coli B database, UniProt Knowledgebase [Whole cellular, outer membrane, and extracellular proteins were extracted and analyzed by two-dimensional electrophoresis using the IPGphor isoelectric focusing system  and a Protean II xi electrophoresis cell  as described previously  see det. For proedgebase , and NCBedgebase .PM tests on REL606 and MG1655 strains were carried out essentially as described previously . The PM et al. [Based on the metabolic network model of MG1655 (iAF1260) and genome annotation of REL606 [et al. . ReactioFBA  was usedFBA: flux balance analysis; COG: Clusters of Orthologous Group of proteins; IS: insertion sequence; LB: Luria-Bertani; LPS: lipopolysaccharide; PM: phenotype microarray;T2S: type II secretion; TA: toxin-antitoxin; TCA: tricarboxylic acid.The authors declare that they have no competing interests.in silico complementation tests; JHS performed the phylogenetic analysis of type II secretion systems; SYL and TKO participated in conceiving the project and analyzing the biological implications of omics data; SHY and M-JH wrote the manuscript; and JFK, SYL, and D-HL revised the draft. All authors have read and approved the manuscript for publication.JFK conceived, organized and supervised the project, and analyzed the omics information; SHY designed DNA microarray and phenotype microarray experiments, and analyzed and accessed the biological significance of genome, transcriptome, proteome, and phenome data; M-JH and X-XX designed and performed proteome experiments, and analyzed the data; HJ contributed to analyzing the genomic differences between B and K-12; CHL reconstructed the metabolic model of B and performed Supplementary Methods, Figures S1 to S4, Tables S1 to S5, and Supplementary References. Supplementary Methods: preparation of whole cellular, membrane and extracellular proteins, two-dimensional gel electrophoresis and image analysis, and metabolite abbreviations in Figure E. coli B strains (REL606 and BL21(DE3) and K-12 strains (MG1655 and W3110) in complex LB medium and minimal R/2 medium supplemented with 10 g/l glucose. Figure S2: total cellular, outer membrane, and extracellular proteomes of the E. coli strains. Figure S3: phenotype microarray comparison of E. coli B REL606 and K-12 MG1655. Figure S4: phylogenetic position of two T2S systems in E. coli B REL606. Table S1: pseudogene comparison between E. coli B REL606 and K-12 MG1655. Table S2: genes that were highly expressed at both the exponential and stationary growth phases during growth of E. coli B REL606 and K-12 MG1655 in LB medium. Table S3: proteins exhibiting significant quantitative differences between E. coli B and K-12 strains. Table S4: metabolic reactions modified in the metabolic network model for E. coli B REL606 compared to the model for E. coli K-12 MG1655. Table S5: phenotypic differences of E. coli B REL606 and K-12 MG1655 in PM1 and PM2 and in silico predictions of cell growth on each carbon source. Supplementary References.Click here for file


The introduction of HAART has significantly prolonged the life span of HIV-infected patients. However, the error-prone nature of HIV-1 reverse transcriptase (HIV-1 RT) results in the emergence of drug-resistant viruses and threatens the effectiveness of HAART. HIV-1 RT is a primary target of two classes of drugs: nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Recent phase III clinical trials have shown that two HIV-1 RT mutations, E138K and M184I, were the most frequent mutations found in patients that experienced virological failure during therapy that included rilpivirine (RPV), emtricitabine (FTC), and tenofovir (TDF).To investigate the mechanistic basis for resistance caused by the E138K and M184I mutations we used transient kinetics to characterize the enzymatic properties and drug susceptibility of RTs with these mutations and determined the biochemical mechanism of resistance to RPV. Specifically, we compared wild-type (WT) RT to RTs mutated in one or both of the enzymatic subunits: p66M184I/p51M184I, p66E138K/p51E138K, p66E138K/p51M184I, p66E138K/p51WT, p66WT/p51138K.Our results show that M184I reduces the catalytic efficiency of RT by more than two-fold (p66M184I/p51M184Ihas more than 2-fold reduced kpol/Kd.dNTP with respect to WT). This defect is compensated by mutation E138K either in both subunits or only in p51 subunit (p66WT/p51138K).None of the mutations affected the template-primer binding affinity of RT. As expected, M184I does not reduce the susceptibility to RPV. Instead, RPV resistance is achieved by reduction in the binding affinity of the drug to RT because of the E138K mutation in the p51 subunit.


Continuous P input increased soil total organic C and N by 1.3–9.2% and 3%–13%, respectively. P input induced C and N limitations as indicated by the decreased ratio of C:P and N:P in the soil and microbial biomass. A synergistic mechanism among the ecoenzymatic stoichiometry, which regulated the ecological function of microbial C and N acquisition and were stoichiometrically related to P input, stimulated soil C and N sequestration in the paddy field. The lower emissions of N2O and CH4 under the higher P application (P-60 and P-90) in July and the insignificant difference in N2O emission in August compared to P-30; however, continuous P input enhanced CO2 fluxes for both samplings. There is a technical conflict for simultaneously regulating three types of GHGs in terms of the eco-stoichiometry mechanism under P fertilization. Thus, it is recommended that the P input in paddy fields not exceed 60 kg ha−1 may maximize soil C sequestration, minimize P export, and guarantee grain yields.Agricultural fertilization may change processes of elemental biogeochemical cycles and alter the ecological function. Ecoenzymatic stoichiometric feature plays a critical role in global soil carbon (C) metabolism, driving element cycles, and mediating atmospheric composition in response to agricultural nutrient management. Despite the importance on crop growth, the role of phosphorous (P) in compliance with eco-stoichiometry on soil C and nitrogen (N) sequestration in the paddy field remains poorly understood in the context of climate change. Here, we collected soil samples from a field experiment after 6 years of chemical P application at a gradient of 0 (P-0), 30 (P-30), 60 (P-60), and 90 (P-90) kg ha The balance of elements has been a main focus of global change ecology and biogeochemical cycling research. Phosphorus (P) application remains an indispensable practice for agricultural crop production. However, P export from soil to surface waters may stimulate outbreaks of water eutrophication Ecological stoichiometry (Eco-stoichiometry) is based on stoichiometric theory and the metabolic theory of ecology, which involves the balance of energy and multiple chemical elements in ecological interactions at the subcellular to ecosystem scale Microorganisms drive Earth’s biogeochemical cycles Eco-enzyme activity represents an intersection of the ecological stoichiometry, wherein eco-enzyme activity (EEA) links environmental nutrient availability with microbial production 2O, ∼39% of CH4, and ∼1% of CO22, N fertilization may generate intermediate nitrogenous gases (N2O) The interactions among C, N, and P cycling also determine the effect of GHG emissions on the Earth’s climate through their influences on C and N sequestration in soil −1.y−1 was conducted in the Yangtze River delta in southeastern China to understand P driving soil eco-stoichiometry in a paddy field ecosystem. In 2011, we collected soil samples from these experimental paddy plots to investigate the stoichiometry of soil chemical, microbial biomass, and eco-enzyme activities in response to P fertilization. Data on GHG fluxes in July and August were also probed to understand the role of P on typical GHG emissions. We hypothesized that P application could change both soil C-N-P balance and eco-stoichiometric features in paddy soil and thus stimulate soil C sequestration.To date, few investigations have probed the role of P fertilization on soil eco-stoichiometry in paddy fields, which hinders the optimization of C-N-P biogeochemical cycles. In 2005, a paddy field experiment with annual applications of chemical P fertilizer at rates of 0.0, 30, 60, and 90 kg P hap<0.05) increased by 1.3% to 9.2% for P input compared to P-0, while soil N increased from 3–13% (p<0.05). Similarly, soil MBC was increased by 20% to 27% under P-60 and P-90; MBN was also increased under P-60 and P-90 by 28–50%, while an increase in MBP of 52–195% was observed. Due to P application, the ratios of soil MBC:MBP, MBC:MBN, and MBN:MBP were significantly increased with increased ratios of soil C:P (p  = 0.001), soil C:N (p  = 0.039), and soil N:P (p  = 0.002), respectively (p<0.01) and NAG+LAP showing no significant changes  with increasing P application  lower by 14%–57% under P input compared to P-0. However, the highest CH4 flux occurred with P-30 in August. The N2O-N flux in our study ranged from −0.02 to 0.05 mg·m−2 h−1. N2O emission in July was significantly (p<0.05) reduced by 34–75% in the tested paddy field with increasing P fertilization compared with P-0; no significant difference was found in August  with increasing P applications, and the ratio of soil N:P was reduced from 11.4 to 7.3 (p<0.05) decreased with increases in P application, while the MBC:MBN ratio remained unchanged, except for P-90 (p<0.01) increased compared to P-0. However, ln(NAG+LAP):ln(AP), which refers to the acquisition of N relative to organic P, showed no significant differences with P application (The stoichiometric ratios of soil C:P declined (from 156 to 97) significantly (4 to 7.3 . Howeverfor P-90 . The acqfor P-90 . The ln(lication . Scatterlication . This relication .p  = 0.001), MBC:MBP and ln(BG):ln(AP) (p  = 0.000), and MBN:MBP and ln(NAG+LAP):ln(AP) (p  = 0.050) were significant and showed a negative trend (The linear relationships between soil C:P and ln(BG):ln(AP) (ve trend . However−1) could increase ∼10% higher soil organic C.Phosphorus amendment increased the decomposition of soil organic C through increased soil respiration The fact that the soil C:P declined  while sop<0.05) negative relationship between the N:P eco-enzymatic and microbial stoichiometries  was found to stimulate root exudation and root aerenchyma development by more than two-fold compared to a high P supply (5–10 mg P L−1) 4 emissions were found for P-0 and P-30 sampling in July, while the highest emission occurred for P-30 sampling in August (4 flux and an increase in N2O flux in paddy soil. CH4 emissions (except P-0) were commonly found to be higher for sampling in July (water-logging) than in August (oxygen secretion through developed roots); however, an overwhelmingly opposite trend occurred for N2O for the two samplings. The reason for the latter phenomenon is unclear and will require additional evaluation. The decrease (p<0.05) and mostly unchanged N2O flux was found upon P application in July and August, respectively  or application of organic fertilizers are well-known means for enhancing soil C and N fixation −1) was significantly higher than that of P-0 (6200 kg ha−1), while no significant difference was found between P-60 (9100 kg ha−1) and P-90 (9070 kg ha−1). Grain yield under P-60 is close to the highest level among a survey overview on rice yield −1 is recommended for maximizing soil C sequestration, minimizing P export, and guaranteeing grain yields.Enhanced soil C sequestration induced by P input  is worth2O and CH4 emissions under P-60 and P-90 compared to P-30 or showed no difference for N2O emissions in August, while enhanced CO2 fluxes were observed in both July and August samplings. Therefore, these results suggest that technical conflicts would occur for the simultaneous regulation of three major GHGs through an eco-stoichiometry mechanism under the conditions of P fertilization. The integration of soil C sequestration, minimization of P export, and guarantee of the grain yield translate into a recommended P paddy field ecosystem input rate of no greater than 60 kg ha−1.In summary, we found that continuous P application increased the soil C and N pools. P input directly modified soil C:N:P ratios, which shifted the microbial stoichiometry of the biomass to be both C-limited and N-limited. Moreover, a synergistic mechanism among the eco-enzymatic stoichiometry regulates the ecological function of microbial C, N, and P acquisition, thus stimulating C and N sequestration in soil receiving P application in paddy fields. The P application significantly mitigated N.Mollic Endoaquoll). The soil before experimental plots construction was composed of 3% sand, 47% silt, and 50% clay in the top 150 mm, while contents of soil total P, total C and total N were found 13.7, 2087, and 148 mmol kg−1, respectively. Local farmers in this region routinely apply approximately 25–50 kg P ha−1 of inorganic P fertilizer or compound fertilizer in late July or early August in order to support one crop of rice and an over-wintering crop, such as wheat (Triticum aestivum) or rape (Brassica Napus).A plot experiment on P management for paddy field was established in April 2005 at the demonstration park of YuHang County Agricultural Research Station  in ZheJiang, China. The experimental region has a subtropical monsoon climate with an average temperature of 17.8°C and an average annual rainfall of 1450 mm. The soil type is typical clay, blue-purple paddy soil. The dominant soil type in this region is a blue-purple paddy soil  and 50 kg K ha−1 (KCl) each year. In 2011, 25-day-old rice seedlings (Oryza sativa L.) were transplanted at 150 mm×150 mm spacing, and rice was harvested on November 10, 2011. Details on rice grain yield and yield components are presented in The construction of the field experimental plots, including specific designs on plot ridges, trenches, berms and inlets/outlets, was previously described by Zhang After application of P for 6 years from 2005 to 2011, the experimental paddy field enters a relatively stable state for soil biosphere. Therefore, soil sampling in 2011 was chosen for a comparison investigation on soil eco-stoichiometric characteristics under different P treatments. On May 21, 2011, one month before rice transplantation, each plot was divided into six subplots of grab-sampling to minimize edge effects. The arable soil samples for each plot consisted of six composited 2 cm diameter×10 cm deep cores (cultivated horizon) 2, N2O, and CH4 within 24 h using an Agilent 6890D GC  equipped with an automated gas chromatographic system with an 63Ni electron capture detector for N2O analyses and flame ionization detector for CO2 and CH4.Sampling of GHGs was set in July and August 2011 following fertilization, representing the early-phase of rice tilling and middle-phase of rice jointing during the high temperature season. A series of lab-made gas collection apparatuses  was cons2, N2O, and CH4 fluxes were calculated using linear regression of the change in gas concentration, based on the following equation:F is the flux rate of CO2/CH4/N2O (mg⋅m−2⋅h−1), 2, CH4 and N2O gas density in standard state , P is the atmospheric pressure (kPa), T the air temperature (K), 2 and N2O flux rates, and H is the height of the chamber (m).The CO4-H2SO4 and measured spectrophotometrically using a continuous flow analyzer  set to 880 nm. The fresh soil samples used for determining microbial biomass C and N as well as the P measurements used the fumigation-extraction method as described by Wu et al. −1 K2SO4 for microbial C and N or 0.5 mol L−1 NaHCO3 for microbial P, while the other sample was fumigated with chloroform and then extracted. Following centrifugation, C, N, and P concentrations as well as microbial biomass element content were calculated from the difference between the fumigated and non-fumigated soil samples.Air-dried samples were analyzed for total organic C (TOC), total N, and P concentrations according to standard methods described by Bao −1 sodium acetate buffer using a vortex shaker for 1 min. The pH of sodium acetate buffer for the soil slurries is 6.0, which is the mean soil pH of the environmental samples −1 NaOH were added to each well to stop the reaction and increase the fluorescence of the substrates. Different buffers respond to NaOH addition in different ways. Thus, a time frame of 1 min between NaOH addition and the reading of plates in a fluorometer was used to reduce analytical variation −1 g −1.Four eco-enzymes  were selected as indicators of microbial nutrient demand in the cycles of C, N, and P, respectively . Fluoresp<0.05 levels. All assays were conducted in triplicate. A principal component analysis (PCA) was used to transform these variables to two factors. The acquisition ratios of ln(BG):ln(AP), ln(BG):ln(NAG+LAP), and ln(NAG+LAP):ln(AP) activities were also calculated and referred to the acquisition of organic C relative to organic P and N as well as organic N relative to organic P, respectively A completely randomized design was performed to examine significant differences in soil chemical composition, microbiological properties, eco-enzymes, and their stoichiometry among the various P fertilization treatments  using two-way analysis of variance (ANOVA) at Table S1Rice grain yield and yield components of experimental paddy field under P fertilization.(DOC)Click here for additional data file.Table S2Extracellular enzymes with corresponding substrate and the corresponding function.(DOC)Click here for additional data file.Table S3The order of pipetting buffer, slurries, references, and substrates in fluorometric enzyme assays.(DOC)Click here for additional data file.Text S1Rice grain yield and yield components.(DOC)Click here for additional data file.Text S2Gas collection apparatus fabrication.(DOC)Click here for additional data file.


HIV-1 infects non-dividing cells. This implies that the virus traverses the nuclear pore before it integrates into chromosomal DNA. Recent studies demonstrated that TNPO3 is required for full infectivity of HIV-1. The fact that TNPO3 is a karyopherin suggests that it acts by directly promoting nuclear entry of HIV-1. Some studies support this hypothesis, while others have failed to do so. Additionally, some studies suggest that TNPO3 acts via HIV-1 Integrase (IN), and others indicate that it acts via capsid (CA).To shed light on the mechanism by which TNPO3 contributes to HIV-1 infection we engineered a panel of twenty-seven single-cycle HIV-1 vectors each bearing a different CA mutation and characterized them for the ability to transduce cells in which TNPO3 had been knocked down (KD). Fourteen CA mutants were relatively TNPO3-independent, as compared to wild-type (WT) HIV-1. Two mutants were more TNPO3-dependent than the WT, and eleven mutants were actually inhibited by TNPO3. The efficiency of the synthesis of viral cDNA, 2-LTR circles, and proviral DNA was then assessed for WT HIV-1 and three select CA mutants. Controls included rescue of TNPO3 KD with non-targetable coding sequence, RT- and IN- mutant viruses, and pharmacologic inhibitors of RT and IN. TNPO3 KD blocked transduction and establishment of proviral DNA by wild-type HIV-1 with no significant effect on the level of 2-LTR circles. PCR results were confirmed by achieving TNPO3 KD using two different methodologies ; by challenging three different cell types; by using two different challenge viruses, each necessitating different sets of PCR primers; and by pseudotyping virus with VSV G or using HIV-1 Env.TNPO3 promotes HIV-1 infectivity at a step in the virus life cycle that is detectable after the preintegration complex arrives in the nucleus and CA is the viral determinant for TNPO3 dependence. Upon fusing with a target cell membrane, retroviruses release the virion core into the target cell cytoplasm. The core consists of a capsid (CA) protein lattice within which are located the viral genomic RNA, reverse transcriptase (RT), and integrase (IN), among other viral components. RT generates double-stranded DNA using the viral genomic RNA as template, though the exact intracellular location of these reactions, or the structural transformations undergone by the core, are not clear. Recent studies indicate that the CA disassembles (uncoats) in response to reverse transcription . The resThough the provirus is an essential intermediate in the retroviral replication cycle, not every PIC reaching the nucleus integrates into host cell chromosomal DNA . The terThe mechanism by which retroviruses gain access to the nucleus is not the same for all retrovirus genera. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) infect non-dividing cells -16, indiThe HIV-1 PIC is at least ~56 nm  and therTNPO3 is an importin-β-like karyopherin that promotes the nuclear import of serine/arginine-rich splicing factors (SR proteins) . Two genBy examining the effect of TNPO3 on the formation of 2-LTR circles, some studies have suggested that TNPO3 promotes nuclear transport of the HIV-1 PIC, while others have not. The first report on the effect of TNPO3 KD on HIV-1 replication  showed aTo examine the role of TNPO3 in HIV-1 infection, lentiviral vectors were used to generate a panel of HeLa cells with TNPO3 knockdown (KD) and optimal controls Figure . A firstThe KD and rescue of TNPO3 in the four pools of cells was verified by western blot Figure . If anytSince previous studies suggest that CA is the viral determinant for HIV-1 replication-dependence on TNPO3 ,33,34, t+ cells by flow cytometry. As compared to the control KD cells, transduction of the TNPO3 KD cells by WT HIV-1 was 11-fold lower  KD HeLa cells were challenged with three-part lentiviral vectors encoding a GFP reporter gene and pseudotyped with VSV Glycoprotein (VSV G). Individual vectors bearing each of the CA mutants were compared side-by-side with the WT CA. Three days after challenge with the vectors the extent of transduction was determined by assessing the percent GFPr Figure . CA mutar Figure , indicatWhen absolute infectivity relative to WT virus was considered, the TNPO3-independent CA mutants clustered into two groups. N74D, A105T, and most double-mutants involving these residues had absolute infectivity as high as the WT , the location of most of the CA residues that, when mutated, render HIV-1 TNPO3-independent, is near the point of contact between two CA monomers in the CA hexamer. Figure +-HeLa cells were challenged with the same CA mutants in the context of HIV-1 reporter virus bearing the HIV-1 Env glycoprotein, the results were identical to those obtained with VSV G-pseudotyped vector (data not shown).Having screened the panel of 27 CA mutants, three mutants with TNPO3 phenotypes, but good absolute infectivity, were selected for more detailed characterization. Each of the four pools of TNPO3 KD and rescue cells described above Figure  were chaPrevious studies concur that TNPO3 depletion results in a block to HIV-1 replication that occurs after reverse transcription has been completed, but before establishment of the provirus ,28,29,34loxP site in the U3 region of the 3'LTR. PCR reactions using a primer complementary to the loxP site were able to distinguish pWPTs-GFP-derived signals from those of the two proviruses that were already present in these cells . As described above, TNPO3 levels in these cells had been modified by sequential transduction with two different HIV-1 vectors, one for TNPO3 knockdown and one for rescue with ntTNPO3 cDNA . Since CD4s Figure . P90A/A9s Figure .The early steps of HIV-1 infection in TNPO3 KD Jurkat cells were then characterized using the qPCR method described in Figure TNPO3 KD in Jurkat cells had no effect on the amount of full-length viral cDNA produced by WT or CA mutant viruses Figure . SimilarA previous study demonstrated that CA, and not IN, is the viral determinant of TNPO3-dependence, at least when these components of HIV-1 were exchanged with those of the TNPO3-independent virus MLV . Though A significant fraction of the TNPO3-independent CA mutants have been reported previously to have effects on CA stability ,44-46. AAmong the TNPO3-independent mutants, N74 and A105 localize to a pocket on the CA surface that is near the dimer interface. This pocket is the target of a drug that destabilizes the CA core . The N74Mutants N74D and A105T might perturb binding to the CA pocket of a cellular factor that alters CA stability. TNPO3 itself has been reported to bind to CA  and it pCypA, a peptidylprolyl isomerase that directly binds HIV-1 CA , also seSince TNPO3 is a karyopherin that binds HIV-1 IN, most studies have attempted to demonstrate a role for TNPO3 in nuclear import of the HIV-1 PIC. Some of them have been able to show it, some others not ,29,34,35Using a range of approaches we demonstrated that TNPO3 has no detectable effect on the amount of 2-LTR circles produced during HIV-1 infection. Given the limitations of this indirect assay for nuclear import of the PIC, our results suggest that TNPO3 acts on HIV-1 after the PIC arrives in the nucleus. It remains a possibility, though, that TNPO3 acts in the cytoplasm to promote maturation of the PIC via some unknown mechanism. One group reported that TNPO3 is required for nuclear import of PICs that were visualized directly by GFP-labeled IN  but the According to generally accepted models of the HIV-1 replication cycle, HIV-1 CA uncoats in the cytoplasm to permit reverse transcription, or perhaps uncoats in response to reverse transcription ; this wo+ T cells were grown in RPMI-1640 (Invitrogen) supplemented with 10% FBS. Azidothymidine  and Raltegravir  were used at concentrations of 25 μM and 10 μM, respectively.293T, TE671 and HeLa cells were grown in Dulbecco's modified Eagle medium (D-MEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (PAA), 20 mM L-glutamine, 1000 U/ml penicillin, and 1000 mg/ml Streptomycin (GIBCO). Jurkat CD4gag and pol expression [- [env-inactivating mutation and EGFP in place of nef. pAPM is an HIV-1 based knockdown vector in which a single transcript driven by the spleen focus-forming virus (SFFV) LTR contains a miR30 framework modified to target a gene of interest and the puromycin N-acetyltransferase gene [pWPTs-GFP is an HIV-1-based transfer vector with EGFP expression under the control of the EF1α promoter . p8.9NdSpression . pMD2-G pression . pNL4-3.ssion [-  bears HIase gene . pAIB isase gene . pETSUMOhttp://cancan.cshl.edu/RNAi_central/RNAi.cgi?type=shRNA - lab of Dr. Gregory Hannon, Cold Spring Harbor Laboratory), we designed three miRNA-based shRNA targeting sequences against the TNPO3 transcript. Three 97-mer oligonucleotides were synthesized and PAGE purified: ts1, 5'-TGCTGTTGACAGTGAGCGACGCCAGAATCTGTGGACTCAATAGTGAAGCCACAGATGTATTGAGTCCACAGATTCTGGCGCTGCCTACTGCCTCGGA-3'; ts69, 5'-TGCTGTTGACAGTGAGCGCCCTCAATATGAGGTAGTAGAATAGTGAAGCCACAGATGTATTCTACTACCTCATATTGAGGATGCCTACTGCCTCGGA-3'; ts72, 5'-TGCTGTTGACAGTGAGCGCGGACAGTAACTTCATGGCTAATAGTGAAGCCACAGATGTATTAGCCATGAAGTTACTGTCCATGCCTACTGCCTCGGA-3'. The 97-mer oligonucleotides were then amplified by PCR using the following primers: miR-30 XhoI 5': 5'-AAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAG-3' and miR-30 EcoRI 3': 5'-AGCCCCTTGAATTCCGAGGCAGTAGGCA-3'. The PCR reaction was carried out with AccuPrime Pfx SuperMix, 1 mol⁄L Betaine(Sigma-Aldrich), 0.4 μmol⁄L each primer, and 100 ng 97-mer oligonucleotide template. The PCR product was column purified, digested with XhoI and EcoRI, and ligated in pAPM to create the pAPM-TNPO3 constructs . The function of TNPO3 KD vectors  was checked by generating stable, puromycin-selected HeLa cells. Efficiency of the TNPO3 KDs was analyzed by looking at the expression levels of TNPO3. Both ts69 and ts72 strongly inhibited the expression of TNPO3, but ts72 was selected for most experiments since of the three constructs it was the most potent.Using the RNAi Central web-based shRNA design program  in 1 ml of DMEM without serum for 20 min at room temperature. 6 hrs after transfection the transfection medium was replaced with fresh target-cell medium. 48 hrs after transfection the supernatant was collected, centrifuged at 1200 × rpm for 5 min, filtered through a sterile 0.45 μm syringe filter (Millipore), and stored in 1 mL aliquots at -80°C. When comparing viruses or vectors, samples were normalized by RT activity present in the viral supernatant using the PCR-based assay described below. For three-part vector systems, the following DNA ratio was used: 3 parts transfer vector: 2 parts packaging plasmid: 1 part envelope. For two-part virus systems a 4:1 ratio was used (7 parts env- virus: 1 part envelope)Viruses and minimal vectors were produced by transfection of 293T cells using Polyethylenimine (PEI) . 4 × 10Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay (SGPER) that possesses both high sensitivity and an extraordinary dynamic range. The assay is a modified version of one described previously . BrieflyTo generate stable microRNA-based shRNA KDs, HeLa cells were transduced with pAPM microRNA-based shRNA vectors  targetinFor transient siRNA, 100 nM of Gene Solution siRNA (Qiagen) targeting TNPO3 was complexed with 5 μl of Lipofectamine RNAiMAX (Invitrogen), following the manufacturer's instruction, and added to the cells. KD was assessed by SDS-PAGE/western blot 72 h after transfection.6 cells using the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer's instructions.Total DNA was extracted from 4 × 10Quantitative PCR for pWPTs-GFP full-length linear cDNA and 2-LTR circles were detected with Sybr green (Invitrogen), while Alu-PCR used a TaqMan probe. The primers used for the detection of late RT products were: pWPT J1B fwd and pWPT J2 rev Table . The pri4SO4, 5 mM MgCl2, 0.1 mg/ml BSA, 0.2 mM dNTPs, 1× Sybr green), 300 nM each primer, 100 to 200 ng of template DNA, and 1 μl of Hot Start Taq Polymerase (Promega) in a volume of 20 μl. Alu PCR reaction mix contained 1× TaqMan Universal Master Mix (Applied Biosystems), 50 nM pWPT J1B fwd primer, 900 nM SB704 rev primer, 100 nM MH603 probe and 100 to 200 ng of template DNA in a volume of 20 μl.Late RT and 2-LTR circle PCR reaction mixes contained 1× Sybr green mix .qPCR for NL4.3 GFP E- full-length linear cDNA, 2-LTR circles and Alu PCR was performed as described previously   and mouse anti-actin antibody (Sigma). The secondary antibodies were HRP-linked donkey anti-rabbit IgG and HRP-linked sheep anti-mouse IgG .CA: Capsid;IN: Integrase; RT: reverse transcriptase; KD: knockdown; WT: wild-type; PIC: preintegration complex; LTR: long terminal repeat; HIV-1: human immunodeficiency virus type 1; MLV: Moloney murine leukemia virus; NPC: nuclear pore complex; CypA: cyclophilin A; CsA: cyclosporine; CPSF6: cleavage and polyadenylation factor 6; SFFV: spleen focus forming virus; IRES: internal ribosome entry site; CMV: cytomegalovirus.The authors declare that they have no competing interests.AD and JL conceived and designed the experiments, analyzed the data, and wrote the paper. AD performed the experiments. Both authors read and approved the final manuscript.We would like to thank Massimo Pizzato and Federico Santoni for technical assistance, and Peter Cherepanov for reagents. This work was supported by NIH grant RO1AI59159 and Swiss National Science Foundation grant 3100A0-128655 to J.L. The funders had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication.


Persistent infection of human papillomavirus (HPV) types 16 and 18 causes cervical cancer. To better understand immune responses to the prophylactic vaccine, HPV 16/18 L1 virus-like particles (HPV-VLPs), we investigated B cell epitopes of HPV16 L1-derived peptides.Sera from mice immunized with HPV-16/18 L1 VLPs were analyzed for their IgG titers against 10 different HPV16 L1-derived peptides (20-mer) that contain human leukocyte antigen (HLA)-class I A-2, A-24 and class II DR.d) and C57BL/6 (H-2b) mice. Mapping analysis showed that the 10-amino-acid sequence at positions 304to 313 was an immunogenic portion. It is of note that the binding capability of this 10-mer peptide to the HLA-A2 and HLA-A24 molecules was confirmed by the HLA class I stabilization assay. In addition, one unique 20-mer was determined as a B cell epitope in each strain.One 20-mer peptide at positions 300 to 319 was identified as a common B cell epitope in both Balb/c (H-2These results might provide new information for better understanding of immune responses to HPV 16 L1. Cervical cancer is the second most prevalent cancer in women worldwide. HPV 16 is the most common type associated with cervical cancer ,3. AlthoSerum of the Balb/c mice was obtained before immunization, and 3, 5, 8, 11, 14 weeks after the first immunization. Each group consisted of 6 mice, and serum from each mouse was independently measured for IgG level. A 100-fold dilution of samples was used to determine the levels of IgG reactive to each of 10 different HPV16 L1-derived peptides (20-mer), and the results were given in fluorescent intensity units (FIU)  mice before and 5 weeks after the immunization to address any difference between Balb/c and C57BL/6N mice. As a result, IgG against both peptide 6 and peptide 8, but not any others, were detected at 5 weeks (P < 0.05)  that had binding motifs to both HLA-class I (A2 or A24) and HLA-class II (DR) in animal model. We used BALB/c and C57BL/6N mice that have been regarded as Th2- and Th1-skewed strains, respectively, and widely known to express different immune responses in normal and pathological states [Mapping analysis of peptide 6 showed 10-mer (aqifnkpywl) at positions of 304 to 313 of HPV16 L1 as an immunogenic portion. In addition, the 9-mer peptide (qifnkpywl) at position 305 to 313 with binding motifs to HLA-A2 and -A24 molecules was also recognized by immunized sera. It is of note that both these 9-mer and 10-mer peptides showed substantial binding capability to HLA-A2 and HLA-A24 molecules, the two dominant HLA-class I A types among Japanese and other ethnics . The pepWhether the vaccine-induced IgG to the peptide 6 or 8 possesses biological activity to either neutralize HPV infection or facilitate the prophylactic effect of the HPV 16/18L1 vaccine needs to be studied in near future. In addition, since we have known that the same epitopes are often recognized by both B cells and T cells , T cell d) and C57BL/6N (H-2b) mice. These results might provide new information for better understanding of immune responses to HPV 16L1.In summary, one 20-mer peptide at positions 300 to 319 was identified as a common B cell epitope in both Balb/c (H-2d) and C57BL/6N (H-2b) mice used in this study were 6 weeks of age and were maintained in a pathogen-free environment. Balb/c mice (H-2d) were mainly provided for the study because they have been regarded as a Th2- skewed strain [d class I-A molecules were similar to those of human leukocyte antigen (HLA) class I-A2402, which are expressed in 60% of Japanese [Female Balb/c .in vitro biases.The blood of each mouse was collected from the orbital sinus. Blood collection was performed before vaccination, and 3, 5, 8, 11, and 14 weeks after the first immunization in Balb/C (n = 6 in each group). In C57BL/6 mice, blood was collected before (n = 9) and 5 weeks after the first immunization (n = 9). Collected blood was allowed to coagulate by keeping it at room temperature for 1 h followed by centrifugation and collection. Serum from each mouse was sealed and stored at −80°C until use. Measurement of IgG titers was conducted simultaneously to avoid possible Ten different HPV16 L1-derived peptides 20-mer) with binding motifs to both HLA-class I (A2 or A24) and HLA-class II (DR) were selected by the web software (MULTIPRED)  as reported previously . The 96-Blood samples were obtained from each of the mice at each scheduled point. Peptide-specific IgG levels in serum were measured by flowmetry assay using the Luminex system as reported previously . In brieb gene (RMA-S/A24), according to a previously reported method with several modifications [5 cells per well in a 24-well plate) were cultured for 18 hours at 26°C in 1 ml of RPMI 1640 medium  containing 10% FBS  in the presence of synthetic peptides (25 μg/ml) and β2-microglobulin . After washing, the cells were cultured for 3 hours at 37°C, and then stained with anti-HLA-A2 mAb  or anti-HLA-A24 mAb , followed by incubation with PE-conjugated rabbit anti-mouse IgG Ab . After washing, the cells were suspended with 1 ml of PBS containing 1% formaldehyde, and analyzed with FACSCanto (BD Bioscience). The binding capability of each peptide to HLA-A2 or HLA-A24 molecules was evaluated by the increase in mean fluorescence intensity (MFI) assessed by flow cytometry, as follows: MFI increase (%) = (MFI with a given peptide – MFI without peptide)/(MFI without peptide) X 100. As positive controls, an HLA-A2-binding peptide derived from influenza virus M1  or an HLA-A24-binding peptide derived from Epstein-Barr virus LMP2  was used. As a negative control, a peptide derived from oncogene K-ras (KLVVVG AGGV) was used.The actual binding of the peptides to HLA-A2 or HLA-A24 molecules was evaluated by MHC class I stabilization assay with the TAP2-deficient RMA-S cells stably transfected with the HLA-A*0201 gene (RMA-S/A2) or with the HLA-A*2402/Kications . BrieflyThe statistical significance of the data was determined using Friedman`s test and the Wilcoxon rank sum test. P-values less than 0.05 were considered statistically significant.HPV: Human papillomavirus; VLPs: Virus-like particles; HLA: Human leukocyte antigen; FIU: Fluorescent intensity units.The authors declare that they have no competing interests.A.F. performed most of the experiments and involved in manuscript preparation. T.S. and N.K. coordinated laboratory manipulation. KK proposed the hypothesis of this study edited the manuscript. K.K., N.T., K.U., K.I. and T.K. designed this study. S.S. and A.F. designed the peptides analyzed. A.F., S.M. gave vaccination to mice and obtained blood sample, A.F., S.M., N.K. and T.S. measured the antibody. A.F., T.S., S. H., and K.I. analyzed the data. KI and TK are the project leaders and were involved in project design, manipulation, data analysis and finalization of the manuscript. All authors read and approved the final manuscript.


EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS . Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis.Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between  The EWS protein is mainly located in the nucleus, accumulated in Cajal bodies and central regions of nucleoli, but it is also present in cytoplasm and associated with cell membrane , 2. We hBrk (breast tumour kinases) phosphorylate tyrosine residues present in the NLS of Sam68 , which iExpression of the EWS-YFP fusion protein resulted exclusively in nuclear accumulation, with high concentration in nucleoplasmic speckles and a fraction in the subnuclear central region , therebyTo demonstrate a possible phosphorylation of Y656, GFP-Zf protein was constructed by fusion of His-GFP with a part of EWS protein (aa 525–656). This part of the C-terminal RNA-binding domain of the EWS protein consists of the Zinc finger (Zf) motif followed by the arginine-glycine rich box 3 (RGG3) and C-NLS . This frGFP-Zf and GFP-Zf(Y656A) fusion proteins containing 6His-tag at the N-terminus of GFP were expressed in eukaryotic HEK 293(T) cells, extracted, subjected to SDS-PAGE, and analyzed on Western blots by using antiphosphotyrosine antibody. Phosphorylation was detected in GFP-Zf (band at ~40 kDa) but not in GFP-Zf(Y656A) and not in the untransfected sample . The WesAs nuclear import of the EWS protein is mediated by transportin-1 , we haveRecently, mutations in FUS/TLS have been shown to be responsible for familial amyotrophic lateral sclerosis (ALS), and mutants of the FUS/TLS protein accumulate in the cytoplasm of cortical neurons and lower motor neurons in the brain of ALS patients , 10. TheIt is possible that different kinases are able to phosphorylate the EWS protein individually in a cell cycle-dependent and a cell compartment-dependent manner, as it has been found with Sam68 containing PY-NLS. Sam68 is phosphorylated in the nucleus by Brk (breast tumour kinases) and at the cell membrane by Src kinases . Remarkahttp://us.expasy.org/, SUMOplot) and is a potential subject for further investigation.Although phosphorylation of Y656 in the EWS protein seems to be essential, it might not be the exclusive regulating factor for nuclear localization, since other known or predicted mechanisms can be recruited to cooperatively or sequentially control nucleocytoplasmic distribution of a particular protein. SUMO-ylation, another reversible posttranslational modification of Sam68  and of hEWSR1 cDNA as template and Zf-XhoI-NotI-forward and EcoRI-stop-reverse or Y656A-stop-reverse primers 5′-TCTCTCGAGCGGCCGCGCCACCATGAATCCGGGTTGTGGAAACCAGAA-3′ and 5′-CCGAATTCTCAGTAGGGCCGATCTCTGCGCTCCTG-3′, or 5′-CCGAATTCTCAGGCGGGCCGATCTCTGCGCTCCTG-3′, respectively. The PCR products were treated with restriction enzymes XhoI and EcoRI and subcloned into pcDNA3.1(−)B/myc-His (Invitrogen) to generate in-frame fusions. Finally, the His-GFP-Zf and His-GFP-Zf(Y656A) vectors were constructed by amplification of the His-GFP fragment from EWS-Myc-6xHis/pEGFP-N2, a derivative of pEGFP-N2 plasmid (Clontech) using the NheI-His-forward and NotI-GFP-reverse primers 5′-TCTGCTAGCGCCACCATGGCCGTCGACCATCATCATCATCATCAT-3′ and 5′-TGCGTCGCGGCCGCTCTTGTACAGCTCGTCCATGCCGAG-3′, respectively, and digestion with the appropriate restriction enzymes, and by cloning the resulting product into the pcDNA3.1(−)B-Zf and pcDNA3.1(−)B-Zf (Y656A) plasmids. To obtain His-GFP-Zf(Y656A)-SV40NLS, His-GFP-Zf(Y656A) vector was fused with an NLS from SV40 large T antigen (PKKKRKV). The oligonucleotides 5′-AATTCCCAAAAAAGAAGAGAAAGGTCAGG-3′ and 5′-AGCTTCTGACCTTTCTCTTCTTTTTTGGG-3′ were annealed, and the resulting fragment was digested with restriction enzymes EcoRI-HindIII and was inserted into His-GFP-Zf(Y656A).The eukaryotic vectors for expression of the EWS-YFP, EWS(Y656A)-YFP, and EWS(Y656F)-YFP fusions were constructed as described . To prod2 atmosphere at 37°C. For visualization analysis, HEK 293 T cells were cultured on glass cover slips to 40% confluency and transiently transfected with mammalian expression constructs using the method of calcium phosphate precipitation. For protein visualization, the cells were fixed 24 h after transfection with 4% paraformaldehyde (PFA) for 5 min and stained with 4′,6-diamidino-2-phenylindol (DAPI) (Roche). Cover slips were mounted using Vectashiled medium (Vector) onto glass slides, and the cells were analyzed by fluorescence and confocal microscopy.Human embryonic kidney (HEK) 293 (T) cells, kindly provided by Professor P. Sonderegger , were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Life technologies) and 1% (w/v) each of penicillin and streptomycin (Life technologies) in a humidified 10% COPrimary rabbit anti-C-terminal EWS antibody SE 680, kindly provided by Dr. O. Delattre , was used with 1 : 5000 dilution. Primary mouse anti-transportin-1 antibody  was kindly provided by Professor I. Stamenkovic . Primary rabbit antiphosphotyrosine antibody (Zymed) was used with 1 : 2000 dilution. The results were confirmed by using primary mouse antiphosphotyrosine antibody  with 1 : 2000 dilution (data not shown). The secondary goat antirabbit and goat antimouse antibodies, respectively, coupled to horseradish peroxidase (Sigma) were used with 1 : 2000 dilution. His-GFP-Zf and His-GFP-Zf(Y656A) protein purification by His-SELECT Nickel affinty gel (Sigma) was performed as described [Western blotting was performed as described  with somLaser-scanning confocal fluorescence microscopy was performed using a Leica SP2 AOBS UV CLSM microscope and HCX PL APO lbd.BL 63.0x NA 1.40 OIL UV objective. Images were acquired using excitation wavelengths of 405 nm and 514 nm and the emission wavelengths of 470 nm and 528 nm for DAPI and YFP, respectively. Images were captured digitally using Leica software and processed using Adobe Photoshop 8.0. The stacks of images were imported into Imaris (Bitplane) software for the 3D rendering of the images.


Klebsiella sp. 601 multicopper oxidase, which is highly homologous to E. coli CueO with a similarity of 90% and an identity of 78%.Structural comparison between bacterial CueO and fungal laccases has suggested that a charged residue Glu (E106) in CueO replaces the corresponding residue Phe in fungal laccases at the gate of the tunnel connecting type II copper to the protein surface and an extra α-helix (L351-G378) near the type I copper site covers the substrate binding pocket and might compromise the electron transfer from substrate to type I copper. To test this hypothesis, several mutants were made in Km (2.4-7fold) and kcat (1-4.4 fold) values for all three substrates DMP, ABTS and SGZ as compared with those for the wild-type enzyme. Its slightly larger kcat/Km values for three substrates mainly come from the decreased Km. Deleting α-helix (L351-G378) resulted in the formation of inactive inclusion body when the mutant Δα351-378 was expressed in E. coli. Another mutant α351-380M was then made via substitution of seven amino acid residues in the α-helix (L351-G378) region. The α351-380M mutant was active, and displayed a far-UV CD spectrum markedly different from that for wild-type enzyme. Kinetic studies showed the α351-380M mutant gave very low Km values for DMP, ABTS and SGZ, 4.5-, 1.9- and 7-fold less than those for the wild type. In addition, kcat/Km values were increased, 9.4-fold for DMP, similar for ABTS and 3-fold for SGZ.The E106F mutant gave smaller The Glu residue at position 106 appears not to be the only factor affecting the copper binding, and it may also play a role in maintaining enzyme conformation. The α-helix (L351-G378) may not only block access to the type I copper site but also play a role in substrate specificities of bacterial MCOs. The α351-380M mutant catalyzing oxidation of the phenolic substrate DMP effectively would be very useful in green chemistry. Multicopper oxidases (MCOs) contain a multiple copper center, and catalyze the oxidation of a variety of substrates along with the four-electron reduction of dioxygen to water . LaccaseB. subtilis CotA and E. coli CueO, were also determined at high resolution  version 1.11a (SHIMADZU Corporation), and kinetic parameters were determined by Lineweaver-Burk plot [2) value was equal to or large than 0.997. All data were also analyzed using the statistic software SPSS based on the non-linear regression method INVERSE, and analysis of variance gave P values of less than 0.005 in each case.Specific activity was measured with a Shimadzu UV/visible spectrophotometer (UV-2550). The extinction coefficients used in active assays were 14.8 mMurk plot  and alsoAll authors read and approved the final manuscript. X-GW conceived the study; YL, ZG and XL contributed to the design of the study; YL, ZG and XL performed the experiments and analyzed the results; X-GW and ZG wrote the paper.