Phosphatase of regenerating liver 3 (PRL-3) promotes cancer metastasis and progression via increasing cell motility and invasiveness, however the mechanism is still not fully understood. Previous reports showed that PRL-3 increases the phosphorylation of many important proteins and suspected that PRL-3-enhanced protein phosphorylation may be due to its regulation on cytokines. To investigate PRL-3’s impact on protein phosphorylation and cytokine secretion, we performed antibody arrays against protein phosphorylation and cytokines separately. The data showed that PRL-3 could enhance tyrosine phosphorylation and serine/threonine phosphorylation of diverse signaling proteins. Meanwhile, PRL-3 could affect the secretion of a subset of cytokines. Furthermore, we discovered the PRL-3-increased IL-1α secretion was regulated by NF-κB and Jak2-Stat3 pathways and inhibiting IL-1α could reduce PRL-3-enhanced cell migration. Therefore, our result indicated that PRL-3 promotes protein phosphorylation by acting as an ‘activator kinase’ and consequently regulates cytokine secretion. The human genome contains about 500 genes encoding protein kinases, and the majority of them are serine/threonine (S/T) kinases and about 90 are tyrosine (Y) kinases . ReversiThe Phosphatase of Regenerating Liver (PRL) phosphatases are a distinct sub-family of prenylated protein-tyrosine phosphatases consisting of three members  that share over 75% of amino acid sequence identity . SubsequTo date, only few phosphorylated proteins were reported as PRL-3’s substrates, i.e., Ezrin , ElongatAntibody microarray has been widely used for comprehensive proteomic analysis in various cancers and other diseases . Here inThe study using human tissue samples was approved by the Biomedical Ethical Committee of Peking University Cancer Hospital & Institute. Colorectal cancer tissue samples were obtained from the Tissue Bank of Peking University Cancer Hospital. These samples were surgically dissected, pathologically verified, and stored in liquid nitrogen frozen. Written informed consent was obtained from all patients.The human colon cancer cell lines HCT116 and LoVo were obtained from ATCC . HCT116 and LoVo cells stably expressing PRL-3 and control cells (NC) were previously established . NF-κB i2). For collecting culture supernatants, cells were harvested at about 80% density and washed by PBS twice gently, and then cultured in RPMI-1640 without FBS for 24 hours.Cells were normally grown in RPMI-1640 , supplemented with 10% bovine serum, 100 U/ml of streptomycin and penicillin in a Thermo cell incubator  and Western blotting were performed as previously described .Cells was collected and delivered to Wanyen Biotechnologies Inc  in liquid nitrogen. Protein was extracted and was examined for phosphorylation using an antibody array  containing well characterized site-specific antibodies against 582 phosphoproteins and their 452 unphosphorylated counterparts. Antibody array slides were scanned by GenePix 4000B (Axon Instruments) and data were calculated by GenePix Pro 6.0 (Axon Instruments). The phospho ratio of the specific phospho-site was calculated as below:PRL-3/unphospho PRL-3)/ (phospho NC/unphospho NC).Phospho ratio = (phospho Significantly changed phosphoproteins (p < 0.05) over 1.5-fold up- or 0.5-fold downregulated were included. The entire data of phosphoprotein array was shown in For the secreted cytokine analysis, cells were grown in RPMI-1640 without FBS for 24 hours and the supernatants were delivered to RayBiotech Inc  in liquid nitrogen. Culture supernatants were added to antibody arrays against 1000 unique cytokines  and processed according to the manufacturer’s protocol. The array slides were also scanned by GenePix 4000B (Axon Instruments) and data were calculated by GenePix Pro 6.0 (Axon Instruments). Significantly changed cytokines (p < 0.05) over 1.5-fold up- or 0.5-fold down-regulated when the signal value in HCT116-NC or HCT116-PRL-3 was greater than 200 were included. The data of cytokine array was shown in http://david.abcc.ncifcrf.gov/), as well as the GO (Gene Ontology) analysis of the differential phosphoproteins and cytokines based on the biological process, protein class, molecular function and cellular component. We further used DAVID to investigate expressively regulated pathways and p-values were corrected for multiple testing by using a B-H (Benjamini-Hochberg) procedure. The data generated by DAVID was shown in http://string-db.org/) and further visualized by Cytoscape 3.2.1.The translation of Gene ID of significantly changed phosphoproteins was performed using DAVID Bioinformatics Resources , and then 1 μg of RNA was used to synthesize cDNA with GoScriptTM Reverse Transcription system . qRT-PCR was performed using a StepOne Real-time PCR system (Applied Biosystems) and SYBR Green PCR master mix reagents (TOYOBO). Expression data were normalized to that of GAPDH. Primers used are listed in After culture supernatants were collected as described above, GDF-15, IL-1α and Neuropeptide Y (NPY) in supernatant was assayed by using the enzyme immunoassay kits according to the procedures described by the manufacturers. IL-1α (DLA50) and GDF-15 (DGD150) were purchased from R&D Systems , while NPY (E-EL-H1893c) was from Elabscience .5’-GUUCCUCCAUUGAUCAUCUTT-3’, antisense: 5’-AGAUGAUCAAUGGAGGAACTT-3’; si-IL-1α-2#, sense: 5’-CUGAAGAAGAGACGGUUGATT-3’, antisense: 5’-UCAACCGUCUCUUCUUCAGTT -3’; si-negative control (NC): sense: 5’-UUCUCCGAACGUGUCACGUTT-3’, antisense: 5’-ACGUGACACGUUCGGAGAATT-3’. Cells were plated at a density of 20 × 104 cells/well in six-well plates. 50 nM IL-1α or NC siRNA was added to the cells by using Oligofectamine from GenePharma  according to the manufacturer’s protocol. Fresh medium was replaced 48 hours later, and then the cells were collected and used for migration assays or for preparation of total RNA extraction.The IL-1α and negative control small interfering RNAs (siRNAs) were purchased from GenePharma . The sequences of each siRNA are as follows: si-IL-1α-1#, sense: 5/ml, with 800 μl medium containing 10% FBS added to the lower chamber. Cells were allowed to migrate at 37°C for 48 hours. The transwells were fixed in methanol and stained in 0.1% crystal violet solution for 30 min at room temperature. Cells in the upper chamber were removed with a cotton wool. The polycarbonate membrane was then removed and sealed on slide by resin, and the cells that penetrated to the lower side of the membrane were counted in six randomly selected fields under a ×100 or ×200 microscope.200 μl re-suspended cells containing 5 ng IL-1RN or after knockdown of IL-1α were seeded on the upper chamber of each transwell  at a density of 2×10Each experiment was done independently at least three times with similar results. The data were analyzed by using Prism 5 . Results expressed as the mean ± SD. Significant differences were assessed with the Student’s t test and p < 0.05 was considered significant.We constructed PRL-3 overexpressing cells in HCT116 and LoVo cells by stably transfecting pcDNA3.1-myc-NC/PRL-3 as described earlier , 40 and Given the importance of protein phosphorylation in cellular signal transduction , we perfTo gain insight into the biological processes, molecular function and the subcellular locations of these differentially regulated proteins, we identified associated Go terms using DAVID. As shown in We further used DAVID to examine the regulated phosphoproteins’ participation in signal transduction pathways and the top 13 signal pathways predicted to be expressively represented from our data set are shown in It is worth to note that the analysis of phosphoprotein array also revealed that mutation diminishing the phosphatase activity (PRL-3/C104S) could increase protein phosphorylation as the wild type PRL-3 did , suggestAs indicated in phosphoproteins-associated signaling pathway , PRL-3 iIL-1 family is crucial for innate inflammatory and immune responses and can be induced by a wide variety of factors . RecentlOur data showed that PRL-3 could affect multiple signaling pathways by enhancing tyrosine phosphorylation and serine/threonine phosphorylation in cultured cells and colorectal cancer tissues. Besides, PRL-3 could have impact on the secretion of several cytokines. Additionally, we found that PRL-3 promotes the secretion of IL-1α through activating NF-κB and Jak2-Stat3 pathways and the inhibition of IL-1α signaling could decrease PRL-3-enhanced cell migration.It has been accepted that PRL-3 is associated with cancer metastasis and progression, however, the detailed function of PRL-3 remains unclear. As a phosphatase, there have been limited substrates of PRL-3 discovered so far. Contrary to the molecular nature of PRL-3 itself, PRL-3 could activate Src, Rho-family GTPases, PI3K-AKT, MAPK, STAT3/5, NF-κB, EGFR, and mTOR, and their downstream molecules by increasing phosphorylation levels , 40, 53,However, there is still no clear clue to explain how PRL-3 functions as an upstream activator of kinase. We hypothesize that PRL-3 may increase protein phosphorylation through activating several important kinases, and among them, IKBKB, LYN, PRKCA, JAK2, MTOR and SRC may be the crucial ‘relay stations’ and ‘amplifiers’ for PRL-3’s wide impact on protein phosphorylation.Previous report conjectured that PRL-3 might regulate protein phosphorylation via extracellular factors . In our Due to the important role of IL-1 in innate inflammatory and immune responses, we studied the mechanism of PRL-3 regulating the secretion of IL-1α, and found that PRL-3-increased IL-1α was influenced by NF-κB and Jak2-Stat3. Both NF-κB and Stat3 could function as transcriptional factors when they regulate cytokines’ expression such as IL-1β, and they share the same binding site on the promoter of IL-1β , so we pTaken together, our study provided detailed and valuable data about PRL-3’s impact on protein phosphorylation and cytokine secretion, and revealed that PRL-3 is associated with many important signaling pathways, providing the clue of investigating the mechanism of PRL-3’s function in tumor progression and other physiological processes.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY-deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period , and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of the mutants  exhibited reduction in repression activity compared with wild-type (WT) CRY1, whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells. After translation, CRY and PER form heterodimers and translocate to the nucleus where they act as repressors and inhibit their own expression. When nuclear localized CRY and PER proteins are degraded, the inhibition is relieved; the next circadian cycle is subsequently initiated  to better understand the role of phosphorylated CRY1 in clock function. We identified phosphorylation sites that cause long periods, short periods, or even arrhythmicity.Although the mechanism by which CRY and PER repress the transcriptional activation of CLOCK:BMAL1 is not well understood, post-translational modifications, such as the phosphorylation of CRY and PER proteins, are closely linked to the inhibition of transcription . The phoCry1)-CRY1 was constructed by replacing the P(CMV) promoter of pcDNA3.1-Cry1-Flag with the mCRY1 native promoter (1.5 kb) and the first intron (P(t intron . All of 4 DKO cells were plated onto 35-mm culture dishes. Cells were cotransfected using the X-treme GENE HP DNA transfection reagent (Roche) with 1 μg of pGL3-P(Per2)-dLuc reporter plasmid and 50 ng of a CRY expression plasmid. Three days after transfection, the cells were treated with 0.1-mM dexamethasone (Sigma) for 2 h and then placed in XM medium as previously described -dLuc was combined with 5 ng of a CRY expression plasmid, 10 ng of BMAL1, and 15 ng of the CLOCK plasmid. Empty vector pcDNA3.1 was added as necessary to obtain total DNA concentration of 200 ng per well. Twenty-four hours after transfection, cells were prepared for the Dual-Luciferase Reporter Assay System (Promega).HEK293 cells were grown and transfected in 96-well plates. For transfection, 10 ng of the reporter plasmid pGL3-P system were performed as described, with minor modifications . The GPSHEK293 cells were transfected with a plasmid encoding GFP-mCRY1 (WT or mutant). Twenty-four hours after transfection, the cells were stained with Hoechst 33258 (Sigma) and fixed with 4% paraformaldehyde in PBS. Samples were observed using Zeiss confocal LSM800 with a 63× water-immersion objective, and the data were analyzed using Image J software.n ≥ 3 for each experiment). Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test when comparing each mean to a control mean. All statistical analyses were performed using GraphPad Prism 6 . *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.In all experiments, unless noted, error bars represent SEM  exhibited lack of effect on the circadian period ] or the native mCRY1 promoter [P(Cry1)] . However, the other four Asp mutants  exhibited no obvious change in repression activity , although the repression activity of S404D was slightly stronger than WT  promoter [P(t1/2 = 1 h) and d4EGFP (t1/2 = 4 h) represent the half-life markers. Our results show that the half-life of WT mCRY1 is similar to that of d1EGFP (Figure Previous reports have shown that phosphorylation of mCRY1 at S247 does not affect protein stability . TherefoP Figure B, top. TP Figure B, middleP Figure B, bottomThe crystal structure of mCRY1 reveals that binding sites for mPER2 and FBXL3, which partially overlap, are involved in transcriptional repression and protein stability , 23. To The stability of CRY1 protein is regulated by two competing SCF E3 ligase complexes: FBXL3 mediates degradation in the nucleus, while FBXL21 protects CRY1 in the nucleus and facilitates degradation in the cytoplasm , 14. TheAlthough the mutants S158D, S247D, and T249D showed similar periodical phenotypes with Y266D, Y273D, and Y432D, the subcellular location of the proteins had no obvious change, suggesting that the mechanisms were different. Indeed, the identified phosphorylated sites were located in different functional regions of mCRY1 (Figure In the basic TTFL model, both positive and negative regulatory elements are important for generating the autoregulatory feedback loop. Post-translational regulation of the activity, degradation, and localization of these regulators, most notably phosphorylation influences the circadian rhythms , 25. AltPreviously, we have demonstrated that the proper ratio of intercellular CRY proteins determines the normal clock period length . In thisIn summary, we identified critical CRY1 sites where mutations disrupted circadian rhythmicity. Although some enzymes correspond to specific modifications in mCRY1, such as MAPK at S247 and AMPK at S71 and S280 , 11, theNL and EZ conceived the study, designed the experiments, and analyzed the data. NL performed the experiments. NL and EZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aggressive behaviors are highly prevalent in children. Given their negative consequences, it is necessary to look for protective factors that prevent or reduce their progress in early development before they become highly unshakable. With a sample of 147 children, the present study aimed to assess the relation between parental education and inhibitory control in the aggressive behavior of children aged from 7 to 10 years. The participants completed a go/no-go task to assess inhibitory control, whilst their parents reported their education level, and their teachers rated the aggressive behavior of the children through the Teacher Rating Scale (TRS) of the Behavior Assessment System for Children 2 (BASC-2). The results showed that both parental education and inhibitory control determined aggressive behavior in children. In addition, inhibitory control partially mediated the associations between parental education and aggressive behavior after accounting for age. However, a moderated mediation model revealed that lower parental education was associated with higher levels of aggressive behavior, which, in girls occurred independently of inhibitory control. In contrast, inhibitory control mediated this relation in boys. These results suggest the importance of parental education and inhibitory control in the aggressive behavior of children, supporting the idea that both constructs are relevant for understanding these conduct problems in schools, particularly in boys. The clinical implications of these findings are discussed, along with possible future lines of investigation. The high prevalence of aggressive behavior in children and its negative consequences for individuals and society make it a relevant topic on which to focus . When weThe General Aggression Model GAM;  providesGiven the prevalence and undesirable consequences of aggression, looking for protective factors that prevent or reduce its progress during childhood is an essential goal. There is already some evidence to suggest that certain variables could be important in reducing these behaviors, such as parental education and the capacity for inhibitory control, both of which have been linked to aggressive behaviors, particularly in adolescents .Parental education refers to the level of academic studies achieved by the parents of the children in question. According to the structure of the GAM, parental education belongs to the first stage as a person variable. It has been related to a variety of outcomes such as academic performance, diet, and mental health . It has Inhibitory control is the ability to control or stop pre-potent and automatic actions or thoughts, allowing for more flexible and goal-directed behaviors . This abA link has also been found between both of these variables . In particular, higher levels of parental education appear to be related to a greater level of inhibitory control . This reFurther, inhibitory control also appears to be influenced by gender. Specifically, higher scores on inhibitory control are usually found in girls  from 2 yFigure 1). We chose to study these effects in childhood given that this is a critical period for modifying maladjusted behaviors before they become highly unshakable. Given the previous findings reported in the literature, we firstly hypothesized that parental education and inhibitory control would be negatively related to aggression. Secondly, we expect inhibitory control to mediate the relation between parental education and aggressive behavior. Finally, we expect to find a moderator effect of gender when analyzing this mediator role of inhibitory control.On the basis of these considerations, the aim of the present study was to assess the relation between parental education and inhibitory control in the aggressive behavior of children. In particular, we intend to analyze the role of inhibitory control as a mediator in the relation between parental education and aggression, as well as the moderator role of gender in this mediator effect . Inclusion criteria were the absence of any  disability or cognitive/language deficits. The parents were informed about this research and no compensation, reward, or incentive was offered in exchange for participation in the study. Of the 155 families who were asked for their consent to participate in the study, 147 (95%) answered positively and completed the questionnaires.The sample was composed of 147 children from various Spanish public schools (50% boys), with an age range between 7 and 10 years  provided written informed consent. The Research Ethics Committee of the University of Málaga approved the study protocol as part of the projects SEJ-07325 and PSI2012-37490. Trained undergraduate researchers collected information relevant to the study from parents, children, and qualified teachers throughout the course of 1 week. Specifically, the parents provided data on their own levels of education, which could be classified as primary, secondary, or university level. The children were assessed on inhibitory control through the go/no-go task. Finally, the teachers completed the aggressive behavior scale of the Behavior Assessment System for Children (BASC) and the time given to complete it was the same for each teacher.Parental education was classified according to the classification system of the Spanish National Institute of Educational Evaluation , which dThe Go/no-go task was employed in order to provide a measure of inhibitory control. Our experimental paradigm consisted of a serial presentation of screen-centered blue and red colored circles for the Go stimuli and green and yellow colored circles for the No-go stimuli. The No-go trials were preceded by sequences of 0, 1, 2, 3, 4, 5 Go trials . The stiThe Behavior Assessment System for Children 2 BASC-2; , TeacherFigure 1), we used Model 14 in PROCESS to develop and analyze the gender role in our previous mediational model.All statistical analyses were carried out using the SPSS package . Preliminary analyses were conducted to compute descriptive statistics. The analysis of the relation between age, gender, parental education, inhibitory control and aggressive behavior scores was conducted with Pearson’s coefficients and Kendall’s Tau-b. To investigate the validity of parental education and inhibitory control for determining aggressive behavior, we conducted three-step hierarchical regression analyses. We also conducted regression analyses to examine the role of inhibitory control as a mediator of the link between parental education and aggressive behavior, based on the recommendations of Table 1. For all variables except aggressive behavior, skewness and kurtosis values were within an acceptable range of ±2 , with higher parental education determining lower aggressive behavior. Inhibitory control, added at the last step of the model, proved to be a significant determinant of aggressive behavior (ΔR2 = 0.06) over and above the effects of parental education.The results of the regression model are shown in Previous analyses of regression showed that when simultaneously including parental education and inhibitory control in the model, these variables accounted for 18% of the variance in aggressive behavior when age and gender were controlled. This result — which suggests that both constructs are relevant in determining the aggressive behavior of children — led us to construct a mediation model and moderate mediation model to test the relation among these variables. To do so, we first investigated the hypothesis that inhibitory control can be an important mechanism in the relation between parental education and aggressive behavior in children.c in Figure 2, the mediation analysis showed that aggressive behavior was lower in children whose parents had a higher educational level. Moreover, path a in Figure 2 shows that parental education was positively associated with inhibitory control, suggesting that those children whose parents have a higher parental educational level reported higher inhibitory control. Finally, as can be seen in path b in Figure 2, inhibitory control was negatively related to aggressive behavior. Using an indirect procedure, the bootstrapped 95% CI confirmed that parental education exerted a significant negative indirect effect on aggressive behavior through inhibitory control . This result points to the possibility that inhibitory control partially mediates the association between parental education and aggressive behavior.In particular, we tested whether inhibitory control mediates the relation between parental education and aggressive behavior. As can be seen in path Table 3 presents coefficients, standard errors, and a summary of the moderated mediation model.Given that previous research indicates that inhibitory control could be influenced by gender, with higher scores of inhibitory control being reported for girls , a moderTable 3 for coefficients and confidence intervals). Parental education was in turn related to aggressive behavior. When controlling for inhibitory control, the relation between parental education and aggressive behavior was less significant. The 95% bias-corrected bootstrap-CI indicated conditional indirect effects of parental education on aggressive behavior through inhibitory control, although the magnitude of these effects was only significant for boys (Table 4). The moderated mediation model accounted for 23% of the variance in aggressive behavior scores.Inhibitory control was associated with aggressive behavior, and a gender by group interaction was found for this association  or the ability to perceive, use, understand and regulate emotions  may alsoWe also found that the mediation effect was moderated by gender. Thus, in our study gender emerged as a moderator of the link between inhibitory control and aggressive behavior, which is consistent with our third hypothesis. Specifically, for the girls in our study, lower parental education was a determinant of aggressive behavior independently of inhibitory control, whilst for the boys inhibitory control played a mediatory role in this relation. Our results are consistent with those previously reported in the literature , along wIn addition to all of the the results reported here, it is important to note that our sample was composed of children, on which there are considerably fewer studies and where prevention is easier than in older individuals. Indeed, childhood represents an ideal period for training and modifying undesirable behaviors. Moreover, in our study we avoided relying on the subjective perceptions of the children by using external reports for assessing aggressive behavior. Finally, inhibitory control was also measured through an objective laboratory task. Taken together, these aspects of our methodology strengthen the validity of our findings.These findings demonstrate that both parental education and inhibitory control are determinants of aggressive behavior in children aged between 7 and 10 years, supporting the idea that both factors are critical for understanding these behavior problems. Furthermore, inhibitory control played a role in mediating the relation between parental education and aggression, but only for the boys in our sample. Future studies are needed to replicate these findings and to further explore the effects of gender on the links between parental education, inhibitory control, and aggression.RC conceived of the study, participated in the data collection, analyzed the data, led preparation and wrote the first draft of the manuscript. MG-C wrote the first draft of the manuscript. PF-B conceived of the study, analyzed the data and contributed to writing the manuscript. All authors contributed to the interpretation of data, helped to draft and revise the manuscript and have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer EC and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


We report on the formation and stability of induced solitons in parity-time (PT) symmetric periodic systems with the logarithmically saturable nonlinearity. Both on-site and off-site lattice solitons exist for the self-focusing nonlinearity. The most intriguing result is that the above solitons can also be realized inside the several higher-order bands of the band structure, due to the change of nonlinear type with the soliton power. Stability analysis shows that on-site solitons are linearly stably, and off-site solitons are unstable in their existence domain. It is shown that the form of this logarithmic nonlinearity satisfies the condition of the separability of noninteracting subsystems, and a neutron interferometer experiment was proposed to test the physical reality of such a nonlinearity15I is scales with ln(I), has been employed as a relevant equation of nonlinear wave mechanics including Gausson, periodic and quasiperiodic evolution18Theory and experiment have demonstrated that a saturable nonlinear medium is promising for the propagation and control of spatial solitons. Among these optical solitons, photorefractive optical solitons forms a specific branch15181920212223I and is scales with ln(σ + I), where σ = 1 and 0 is an relevant saturable parameter, corresponding to the self-focusing nonlinearity and changing nonlinearity , respectively. Note that a logarithmic nonlinearity of this sort given by ln(I) was employed by Snyder and Mitchell in their study of mighty morphing spatial solitons and bulletsI ≫1, negligible power exists in the tails for intensities below I < 1. In this model σ = 0, the limit I → 0 might appear unphysical for the realistic materialsI) can support stable and localized Gaussian soliton solutions of the form A is the soliton amplitude, k and φ represent the soliton frequency and phase, respectivelyIn this paper, we address the existence and stability of solitons in PT-symmetric periodic systems with a saturable nonlinearity, the perturbed index nonlinear change of which varies logarithmically incident optical beam normalized intensity q :We consider the propagation of an optical beam in a PT-symmetric lattice with logarithmically saturable nonlinearity. The propagation dynamic of the beam is governed by the following one-dimensional nonlinear Schrödinger (NLS) equation for the dimensionless complex amplitude of the light field x and longitudinal z coordinates are scaled to the characteristic beam width and diffraction length, respectively. In V0 and ω0 are amplitudes of real and imaginary parts of periodic potential, respectively; φ = 0, π/2 correspond to two different complex refractive index distributions, namely on-site and off-site nonlinearity, depending on whether the real part of modes is symmetric and antisymmetric, in x. Note that there is no difference between the above two refractive index landscapes except a π phase shift, as shown in x = 0), in fact, both on-site (odd mode) and off-site (even mode) solutions exist for the two values of φ at appropriate initial conditions. V0 = 4 and ω0 = 0.8 with φ = 0 and π/2, respectively. σ is an relevant saturable parameter. According to the values of σ one can distinguish two different cases. (i) σ = 1: it represents a self-defocusing nonlinearity; (ii) σ = 0: the logarithmic function is positive at |q|2 > 1 and negative at |q|2 < 1; that is to say, the type of nonlinearity would be changed with light intensity.where the transverse q = f(x)exp(iμz) that can be characterized by the propagation constant μ, f(x) = h(x) + ie(x) is a complex-valued function. We search the stationary soliton solutions of The stationary solutions can be solved numerically by the Newton’s iteration, spectral renormalizationg(x) and t(x) are small perturbations that can grow with the complex rate δ = δr + iδi upon propagation and the asterisk means complex conjugation. Linearization of f(x) leads to the following eigenvalue problem,where δr>0, solitons are unstable, otherwise, they are stable.which we solve numerically to find the growth rate. If μ form the Bloch bands. Based on the Floquet-Bloch theory, we can obtain the Bloch spectrum by solving the above equation withk is the Bloch wave number bounded between −1 ≤k ≤ 1, w(x) = w(x + T) is a Bloch function with the same periodicity as the lattices. A typical lattice spectrum obtained by the plane wave expansion method is depicted in V0 = 4 and ω0 = 0.8. All possible propagation constant values are arranged into bands (gray regions), where μ ≥ 2.657), first finite band gap (2.584 ≥ μ ≥ 0.8407), second finite band gap (0.138 ≥ μ ≥ −0.5137), and so on in this paper. It should be pointed out that there exists a phase transition point (or threshold) ω0 exceeds this critical value, an abrupt phase transition occurs because of the spontaneous symmetry breaking, the first two bands start to merge together and form an oval-like structure and a complex band diagram forms, as shown in ω0, it is shown that the real parts of the first two bands begin to overlap with each other, with the imaginary parts taking nonzero opposite signs.It is instructive to analyze the linear propagation of light beams in the optical lattice. When the energy flows of soliton beams are small, the optical lattices induce a bandgap structure in the linear Schrödinger spectral problem, σ = 1). Numerically, we find two types of lattice solitons bifurcating from band edge in the semi-infinite band gap.To begin with, in this section, we investigate solitons and their stability supported by PT-symmetric periodic lattice with logarithmical self-focusing nonlinearity  plane is presented in q|2>1), q|2<1, and solitons can be found in finite band gaps. As a result, these solitons can exist in several bands and gaps, due to the change of nonlinear type with the soliton power. It should be note, for |q|2 → 0, P = 0.01 can also be readily found. μ = 0.5 and 4.5, respectively. Different from lattice solitons in the previous section, these inband solitons here do not localize at high power. The stability of these solitons is tested by direct simulation and linear stability analysis. It is shown that, when these inband on-site soliton do not have extremely low powers, they are found to be stable in whole existence domain. In the model with μ = 0.5 and 4.5, whose evolution propagations are shown in z = 500 are shown in the insets of φ = π/2 and σ = 1, shown in I) model is indistinguishable from that of the ln(σ + I) nonlinearity.In this subsection, we examine off-site inband solitons inside the higher-order band of the linear spectrum. The power diagram is shown in σ = 0, both the on-site and off-site inband solitons can also be realized in the PT-symmetric periodic systems, where the type of nonlinearity would be changed with light intensity. The most intriguing property is that the two families of solitons can exist inside the high-order bands of the band structure. This property is attributed to the change of nonlinear type with the soliton power. It is shown that existence curves of inband solitons penetrate inside the relatively high-order band gap, where solitons have very low power. Similar to lattice solitons under a self-focusing nonlinearity, on-site inband solitons are linearly stably, and off-site inband solitons are unstable in their existence domain. At high power domain, it also always exhibits chain-type evolution, the low power soliton can also shift its energy into one lattice site but not be found to evolve into an on-site soliton.To conclude, Solitons and their stability in PT-symmetric periodic systems with the logarithmically saturable nonlinearity have been analyzed. Two families of solitons, i.e., on-site and off-site solitons supported by the PT-symmetric periodic potential can be formed for both the self-focusing nonlinearity and varied nonlinearity. Both on-site and off-site lattice solitons for the case of self-focusing nonlinearity have been found in the semi-infinite band gap, and bifurcate from the edge of Bloch band into the corresponding band gap. On-site lattice solitons belonging to the semi-infinite band gap can propagate stably in whole existence domain, but off-site lattice solitons belonging to this band gap are unstable in the entire domain of their existence. These off-site lattice solitons with high power exhibit chain-type unstable evolution under random initial perturbation, whereas an off-site soliton in low power domain would evolve into an on-site lattice soliton after a distance. In the case A critic might question whether our model can be realized physically, indeed, the logarithmic Schrödinger equation has no direct application in nonlinear optics. This law arises in various fields of contemporary physics. Various meaningful physical interpretations have been given to the presence of the logarithmic potentialHow to cite this article: Zhan, K. et al. Solitons in PT-symmetric periodic systems with the logarithmically saturable nonlinearity. Sci. Rep.6, 32990; doi: 10.1038/srep32990 (2016).


Avian infectious bronchitis (IB), caused by a gammacoronavirus, is an OIE-listed (List B) disease and characterized by respiratory and renal involvements, causing high mortality, and economic loss in both layers and broilers. In comparison with other diagnostic methods, real-time polymerase chain reaction (RT-PCR) and conventional RT-PCR are potent, more sensitive and faster techniques for infectious bronchitis virus (IBV) detection. This research was conducted to detect IBV using specific primers of IB in three governorates  in the south of Iraq. Tracheal specimens were collected from 46 IB suspected commercial broiler flocks. XCE2+ and XCE2- Primers, which amplify all IBV serotypes, were used. Primers MCE1+, BCE1+ and DCE1+ were used to amplify the specific nucleotide sequences of Massachusetts, 793/B and D274 genotypes, respectively. The results of real-time RT-PCR of this study showed that 34 (74.00%) out of 46 infected flocks were positive to IBV. The results of nested PCR showed that 50.00% and 5.89% of positive samples were belonged to genotypes 793/B and Massachusetts, respectively, and the remaining positive (44.11%) were unknown. The results indicate presence of Massachusetts and 793/B IBV strains in commercial broilers in southern Iraq. It possesses a positive sense single–stranded RNA genome that ranges from 27 to 31 Kb.2 The number of IBV serotypes that exist throughout the world is unknown. More than 50 different serotypes have been listed and new IBV variants continue to emerge.3 It is now well documented that a considerable number of different serotypes with antigenic and pathogenic differences exist in poultry industry of different parts of the world.4Infectious bronchitis (IB) is a highly contagious viral disease of the upper respiratory and urogenital tract of chickens, which is caused by infectious bronchitis virus (IBV). The disease is prevalent in all countries with an intensive poultry industry, affecting the performance of both broilers and layers, thereby causing the considerable economic loss in poultry industry worldwide.5 IBV strains of the 4/91 type, which are also known as 793B, were first reported and characterized in Britain, 1991,6 and have been the dominant genotype in Europe.7 The Serological survey has revealed a high incidence of IBV infection of the 793/B type in layer and broiler chickens worldwide.8 Infectious bronchitis still causes serious problems in the Iraqi poultry industry due to the inability of the vaccines to protect the different genotypes. Due to the limited network of poultry diagnostic laboratories in Iraq, differential diagnosis is only made according to clinical signs and gross lesions.The D274 type was the most common type of IBV in several western European countries in the early and mid-1980s.9,10 In Iraq, the first report of identification and genotyping of IBV isolates has been from Kurdistan-Iraq, which indicated the circulation of 793/B  along with a new IBV variant (Sul/01/09) in vaccinated  broiler farms.10 So far, there has been no report on the prevalence rate of IBV genotypes in the south of Iraq.The characterization of IBV has raised additional problems in terms of both epidemiology and control. Although IBV in the poultry farms in Iraq (with H120 and 4/91 strains) is presently controlled by both inactivated and live attenuated vaccines, the outbreaks of IB have still been observed on broiler farms.The aim of the present study was to detect of three IBV genotypes including , 793/B and D274 in the south of Iraq.Sampling. Tracheal tissue samples were collected from 46 IB suspected broiler farms, located in Basra, Thi-Qar and Muthana governorates. All chicks were 20-35 day-old and vaccinated with H120 and 4/91. Five tracheal tissue samples per flock were selected for RNA extraction.RNA extraction and cDNA synthesis. Total RNA was extracted from the tracheas using CinnaPure RNA  based on the kit instructions. The extracted RNA was used in reverse transcription (RT) reaction to generate cDNA through cDNA synthesis kit . The cDNA was stored at –20 ˚C until use.Real-time PCR for IBV detection. Real-time PCR was carried out to detect IBV. TaqMan® probe and primers  were used in this study according to Callison et al. method.11 Forward primer 5’ GCTTTTGAGCCTAGCGTT3’, reverse primer 5’ GCCATGTTGTCACTGTCTATTG 3’, and TaqMan® dual-labeled probe FAM-CACCACCAGAACCTGTCACCTC-BHQ1, were used to amplify and detect a 143-bp fragment of the 5’-untranslated region (5’UTR). The mixture for each tube was consisted of 13 μL TaqMan Master Mix, 0.2 μL of 2 pM IBV probe, 1 μL of 10 pM forward primer, 1 μL of 10 pM reverse primer, 2.8 μL distilled water, to reach a total volume of 18 μL. Then, 2 μL cDNA was added. A negative control containing nuclease-free water instead of cDNA were included in each run. The thermal profile for the PCR was 95 ˚C for 10 min, followed by 40 cycles of 94 ˚C for 15 sec, 50 ˚C for 30 sec and 72 ˚C for 30 sec. PCR amplification was performed using Rotor Q  system.Nested PCR for IBV genotyping. For determination of genotypes, a type specific nested PCR was conducted.12 Oligonucleotide primers included MCE1+, DCE1+, and BCE1+ which specifically amplified a hyper-variable region of the S1 gene of Mass, D274 and 793/B serotypes, respectively. XCE3- primer was a common primer for all IBV genotypes detection. Primer sequences and their expected band size are shown inIn the current research, 46 broiler flocks were selected for tracheal tissue sample collection. The results of real-time RT-PCR of this study showed that 34 (74.00 %) out of 46 infected flocks were positive to IBV . 35.00% 13 Since it was first described in the early nineties in the United Kingdom, 4/91 type IBVs was identified in many other countries and became one of the most predominant genotype in Europe.14,15 Recombination with field isolates and reversion to virulence has been reported for the IBV vaccines.16,17 Therefore, it is important to verify the relationship between field isolates and the vaccine strain.18 Despite the availability of an attenuated vaccine against 793/B serotype viruses, recent studies suggest that viruses belonging to the 793/B sero-group are still prevalent in European flocks, and their continual presence is still an important concern for the poultry industries in many different countries.19 It is now recognized that IBV comprises more than one serotype. The original serotype Mass has now been spread worldwide and is included in most of the commercially available vaccines.20Infectious bronchitis (IB) has a significant economic impact on the poultry industry causing the loss of millions of dollars worldwide each year due to the condemnation of infected chickens.The results of the present study showed that 34 (74.00%) out of 46 sampled flocks were positive to IBV by real-time PCR and that 35.00%, 35.00% and 30.00% of the sampled flock in Basra, Thi-Qar and Muthana, respectively, governorates were positive to IBV. In addition, the results showed the distribution of 793/B and Mass genotypes with the prevalence rate of 50.00% and 5.89%, respectively, yet no D274-type virus was detected in the south of Iraq. The presence of other IBV types is probable, however, they were not detectable as applied primers were specifically amplified three types of IBV. Unknown positive IBV strains must be sequenced to be characterized.21 Detection of 793/B serotype has been reported from Saudi Arabia, Japan, Denmark, Poland, France, Italy, and Argentine.22 Also, the 793B type was reported in Iran; the co-existence of Mass and 793/B type’s viruses at the same time in 33 flocks in Iran revealed that Mass type vaccine viruses have not conferred a proper immunity against 793/B type viruses.23 There was a study in the middle of Iraq, showed that 92.10% of samples collected from suspected flocks were infected with IBV, 20.00% of samples were infected with IB alone and 45.71% of samples with IB combined with both Mycoplasma galisepticum and avian influenza virus subtype H9 and 25.71% of samples were positive to both IBV and AIV(H9).9 Mahmood et al. isolated infectious bronchitis virus from trachea and kidney tissues of eight broiler farms in Kurdistan region of North Iraq from 2008 to 2010. Their results showed that two farms were just infected with 793/B type, one with an unknown strain and five with Sul/ 01/09.10 Jahantigh et al. conducted a work to identify the infectious bronchitis virus with group-specific primers in Zabol, south-east of Iran. Their results showed that 36.36% of the sampled flocks were positive to IBV by RT-PCR.24 Also, in Jordan, there are studies that have detected the presence of IBV, like Ark, DE-072, and Mass-like serotypes.25 Another study of Jordan showed that sampled flocks have Mass, 4/91, and D274 isolates, respectively.26 However, D274 genotype was not detected in the present study.In Israel, Mass was the only type detected for many years until the 793B type of IBV (Israel/793B/variant 1/96) was identified in 1996.et al. results who found no Mass-type IBV in the North of Iraq.10 In our research, 50.00% of farms in each governorate were infected with type 4/91, which was two times higher than its prevalence in Kurdistan region of Iraq. According to 4/91 based vaccination strategies applied in Iraq, 4/91-type IBVs could be vaccine-derived, however, this high prevalence rate also may reflect the weak biosecurity systems and uncontrolled entering of broiler chicks from other Iraqi governorates or intense trading with neighboring countries .Our results showed the low prevalence (5.89%) of Mass type in Basra that was in consistent with Mahmood 27 like geese, ducks, and pigeons, might also play roles in the spread of IBV strains over the world. Specific IBV strains that were able to infect another bird species, especially if it is a migratory bird, would spread more easily over long distances than a strain that could not replicate in that bird species.The large number of IBV variants exist worldwide, some being unique to a particular area and others having a more general distribution. The reason why some strains spread readily over major parts of the world whereas others remain local is unknown. Some IBV variants, such as 793/B or QX that have been spread over Asia, Europe, and Africa in a short period, have not been reported in the USA or Australia. It seems likely that geographical isolation and control measures employed in countries may play a significant role in preventing entry of IBV variants. The recent discoveries of IBV and IBV-like strains in other species of birds other than the chicken,In conclusion, the current study clearly demonstrated that there was the presence of 793/B and Mass IBV genotypes (Vaccine or field strain) in commercial chicken flocks in south of Iraq. By utilizing diagnostic techniques such as those were used in this study, it is possible to conduct a detailed epidemiological investigation of this disease. Despite the use of H120 and 4/91 vaccines in Iraq, it is common to find IB problems in vaccinated chickens. In the present study, the sequencing of PCR products was not performed, therefore, the origin of these types is not clear at present. Future work should include the sequencing of IBV strains in the region in order to determine the type of unknown strains and to choose the suitable vaccination program.


Boehmeria nivea L.) phytocystatin gene, designated as BnCPI, was isolated from a ramie cDNA library and its full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence (691 bp) consisted of a 303 bp open reading frame (ORF) encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical isoelectric point (pI) of 6.0. The alignment of genome DNA (accession No. MF153097) and cDNA sequences of BnCPI showed that an intron (~104 bp) exists in the coding region. The BnCPI protein contains most of the highly conserved blocks including Gly5-Gly6 at the N-terminal, the reactive site motif QxVxG (Q49V50V51S52G53), the L79-W80 block and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N (L22G23R24 F25A26V27 D28D29H30 N31) block that is common among plant cystatins. BLAST analysis indicated that BnCPI is similar to cystatins from Glycine max (77%), Glycine soja (76%), Hevea brasiliensis (75%) and Ricinus communis (75%). The BnCPI was subcloned into expression vector pSmart-I and then overexpressed in Escherichia coli BL21 (DE3) as a His-tagged recombinant protein. The purified reBnCPI has a molecular mass of 11.4 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Purified reBnCPI can efficiently inhibit the protease activity of papain and ficin toward BANA (Nα-benzoyl-L-arginine-2-naphthyamide), as well as the mycelium growth of some important plant pathogenic fungi. The data further contribute to our understanding of the molecular functions of BnCPI.Phytocystatins play multiple roles in plant growth, development and resistance to pests and other environmental stresses. A ramie ( Helicoverpa armigera, Erwinia carotovora and Pythium aphanidermatum -[AGT]-[RKE]- [FY]-[AS]-[VI]-x-[EDQV]-[HYFQ]-N block (L22G23R24 F25A26V27 D28D29H30 N31) (The cDNA sequence of the sequence . The matH30 N31) . The secH30 N31) . The terH30 N31) .BnCPI were amplified from different ramie cultivars using the primers cpif and cpir  were identified in cultivar ZZ1 that differed from those in QDY and HPD  and Ricinus communis (accession No. XP_002523225), followed by sequences of cystatin from Glycine soja (accession no KHN35444), G. max (accession No. NP_001239817) and Hevea brasiliensis (accession No. ACZ02398), all of which shared 75% identity with BnCPI. These were followed by sequences of cystatin from Vigna unguiculata (accession No. CAA79954) and Cajanus cajan (accession No. XP_020236689), both of which shared 74% identity with BnCPI.Analysis of the BLAST results (11 May 2017) revealed that the deduced amino acid sequence of BnCPI shared the highest identity (76%) with sequences of cystatin from BnCPI with sequences of phytocystatins from other plants confirmed that BnCPI and R. communis cystatin were most similar. BnCPI contained most of the typical features of other phytocystatins, such as the GG doublet and LARFAV motif in the N-terminal region, and the central signature motif QxVxG , but it lacked the PW motif contained in some of the other cystatins from plants and animals. Here, it was revealed by multiple alignments that the PW motif was replaced by the LW motif in the BnCPI amino acid sequences, which is identical to the phytocystatin of papaya (accession No. CAA50437) and ragweed (accession No. AAA32672).As shown in E. coli BL21 (DE3). Overexpression of BnCPI was induced by adding IPTG  to the medium. Expressed recombinant BnCPI was not secreted to the liquid medium. Bacterial cells were collected by centrifugation, homogenized by ultrasonic crash and purified by affinity chromatography. After removing SUMO and further purification, the resultant protein was analyzed using SDS–PAGE electrophoresis. A single protein band with a molecular mass of about 11.4 kDa was obtained  were tested as above. For papain and ficin, only about 18.2% and 29.0% enzyme activities remained after treatment, respectively, whereas for bromelain, reBnCPI showed no obvious inhibition activity  was pre-incubated with papain, ficin and bromelain, and then the residual enzyme activities on activity . These dF. oxysporum, A. aternata, B. cinerea and P. vexans) cultures. The results indicated that reBnCPI inhibited the growth of all four phytopathogens, but the inhibitory effect varied depending on the fungal species (B. cinerea was most sensitive to reBnCPI. Concentrations of reBnCPI of 20 μg/mL (1.81 μM) slowed the growth rate of B. cinerea to 54% of that exhibited by B. cinerea cultivated in medium without reBnCPI; at concentrations of 80 μg/mL (7.23 μM), the B. cinerea growth rate was only 21% of that cultivated in medium without reBnCPI. Growth rates of F. oxysporum and A. alternata were reduced by about 50% at a concentration of reBnCPI of 80 μg/mL; that is, the 50% growth inhibition (EC50) for these two pathogens was 80 μg/mL. Moreover, the inhibition efficiency of reBnCPI (at 80 μg/mL) was slightly higher than that of jinggangmycin (50 μg/mL). In the case of the root rot pathogen P. vexans, growth rates fell by 50% at reBnCPI concentrations of 20 μg/mL and 35% at concentrations of 80 μg/mL, indicating that P. vexans is not very sensitive to BnCPI. For all four fungi, however, the growth rate declined as the concentrations of reBnCPI increased.Antifungal activity assays were performed with different concentrations of purified reBnCPI on four plant pathogenic fungi  of ramie for the first time. We also compared BnCPI sequences of three ramie cultivars with different nematode resistance. Interestingly, BnCPI genes of different ramie cultivars with different nematode resistance have identical AA sequences, although there were very few nucleotide differences in the cDNA sequence. Supposing BnCPI plays an important role in the ramie–nematode interaction, the resistance difference of cultivars may be due to different transcriptional regulation or expression regulation of this protein. For instance, expression of two cystatins (WC4 and WC5) in resistant and susceptible wheat cultivars were differently induced during Tilletia indica infection; and the different expressions were regulated by Jasmonic acid (JA) signal [In this study, we have cloned the full-length cDNA encoding a cystatin gene  signal .An accurate classification of BnCPI will help readers to understand it comprehensively. Based on the molecular mass (11.4 kDa) and other molecular characteristics of BnCPI (described above), we concluded that it should be clustered into group I. Subgroups  of the phytocystatin family were generated on the basis of phylogenetic analysis . IntronsThe recombinant BnCPI exhibited inhibition activity toward both papain and ficin, but not bromelain. Plant cystatins are generally poor inhibitors of bromelain ,38, but Phytocystatins are apparently reversible, competitive inhibitors of papain . HoweverF.oxysporum, B. cinerea, A. alternata and P. vexans. Differential inhibitory effect of a phytocystatin on different fungi is common for phytocystatins. Cystatins from chestnut (CSC), wheat (TaMDC1), taro (CeCPI), sugarcane (canecystatin), barley (HvCPI‒1 to HvCPI‒13), strawberry (FaCPI-1), kiwifruit (KCPI1 or rCPI), amaranth (AhCPI), cacao (TcCys), Siam tulip (CaCPI) and sesame (SiCYS) have all been shown to have toxic effects on mycelium growth of various species of fungi [B. cinerea occurred at concentrations of 1.5 μM, 1.9 μM and 2.7 μM, respectively [F. oxysporum, 50% growth inhibition of HvCPI-1, AhCPI and CaCPI occurred at concentrations of 2.14 μM, 2.28 μM and 13 μM, respectively [F. oxysporum was about 7.23 μM, which is higher than that reported for other phytocystatins. To our knowledge, the maximum EC50 reported for phytocystatins on F. oxysporum are those of HvCPI-9 (5.94 μM) and HvCPI-11 (6.0 μM) [A. alternata and P. vexans has not been studied; however, KCPI1 inhibits the growth of A. radicina almost completely at a concentration of 23.1 μM, and displays 50% growth inhibition at a concentration of 2.2 μM [P. aphanidermatum at concentrations of 150 μg/mL (~5.2 μM) to 200 μg/mL (≈6.9 μM) [Pythium spp. and Alternaria spp. However, the mechanisms responsible for the growth inhibition activity of reBnCPI on different fungi require further, detailed elucidation.Recombinant BnCPI exhibited varied growth inhibition on of fungi . For Hv-ectively ,42,43, wectively ,18,44. T(6.0 μM) . The toxBnCPI, a gene encoding a phytocystatin in ramie, was cloned successfully with the RACE technique. The full-length cDNA of BnCPI (accession No. KT438742) contained an ORF of 303 bp, encoding a protein of 100 amino acids with deduced molecular mass of 11.06 kDa and a theoretical pI of 6.0. The corresponding genome DNA consisted of an intron of 104 bp in the coding region. The ORF of BnCPI was subcloned into an expression vector and was expressed successfully in E. coli. The recombinant expressed protein, reBnCPI, exhibited strong cysteine proteinase inhibition activity on papain and ficin, as well as on the growth of some phytopathogenic fungi. These results further contribute to our understanding of the molecular functions of BnCPI in ramie growth and development, and resistance to biotic and abiotic stresses.In summary,


Penetration of surface meltwater to the bed of the Greenland Ice Sheet each summer causes an initial increase in ice speed due to elevated basal water pressure, followed by slowdown in late summer that continues into fall and winter. While this seasonal pattern is commonly explained by an evolution of the subglacial drainage system from an inefficient distributed to efficient channelized configuration, mounting evidence indicates that subglacial channels are unable to explain important aspects of hydrodynamic coupling in late summer and fall. Here we use numerical models of subglacial drainage and ice flow to show that limited, gradual leakage of water and lowering of water pressure in weakly connected regions of the bed can explain the dominant features in late and post melt season ice dynamics. These results suggest that a third weakly connected drainage component should be included in the conceptual model of subglacial hydrology. Surface meltwater draining to the bed of the Greenland Ice Sheet each summer causes ice flow changes inconsistent with the prevailing theory of channelizing subglacial drainage. Here, the authors show this is caused by limited, gradual leakage of water from previously ignored weakly connected regions of the bed. In the ablation zone of the Greenland Ice Sheet (GrIS), the drainage of surface melt to the ice sheet bed via moulins and crevasses causes ice flow acceleration every summer123123423This seasonal evolution of subglacial drainage beneath the GrIS is currently interpreted in the context of traditional theory of a two-component subglacial drainage system consisting of distributed and channelized drainage1234789105891151112et al.414202122Observations and modelling suggest that the current conceptual model of the subglacial hydrologic system overemphasizes the role of channelization in controlling GrIS subglacial drainage system capacity and water pressure. Depressed ice speeds persist through fall and much of winter16143616Here, we use a subglacial hydrology model  to demonstrate that the observations at FOXX can be explained by gradual evacuation of water from weakly connected, but spatially extensive, areas of the bed. We suggest that these areas exert the dominant control on the large-scale subglacial water pressure and basal resistance felt by the ice sheet, which we show by using the modelled ice effective pressure to force an ice dynamics model134et al.16Hydraulic head  reproduces the basic features of the ice velocity observations: while higher channel pressure results in higher ice speed on each day, there is a drop in ice speed for the same channel pressure as the summer progresses . To elimIt is notable that the observations and both model versions exhibit variations in the slope of the lines defining the minimum and maximum channel hydraulic head and ice surface speed on each day . These v38394r=+0.36, P=0.01), while the model with the static weakly connected system is not significantly correlated with the observations .In contrast, evolution within the weakly connected system is required to explain the lowering of the lines in Because some observations on mountain glaciers have shown that ‘isolated' boreholes can become connected during periods of high water pressure in the active drainage system34et al.4et al.42Tedstone While subglacial drainage has generally been categorized into distributed and channelized components51011420343536374Our modelling results confirm that these isolated or weakly connected regions of the bed can play an important role where present by virtue of their large area fraction; modest changes in effective pressure in these extensive regions will have a large impact on basal traction. Importantly, ice dynamics respond to the integrated basal traction over both well-connected and poorly connected regions of the bed, and poorly connected regions therefore moderate active drainage regions40454643435Based on this new understanding, we propose a three-component conceptual model for subglacial hydrology that adds a ‘weakly connected system' to the traditional distributed and channelized forms of subglacial drainage . We findWe have envisaged the weakly connected system being composed of cavities formed in the lee of bedrock bumps or clasts in lodged till. The low hydraulic connectivity that differentiates these regions from the rest of the distributed system may be due to local bed geometry causing smaller cavity orifices or the presence of low hydraulic conductivity till. Indeed, extensive subglacial till has been identified at FOXX48211011The impact of weakly connected drainage on the seasonal evolution of hydrodynamic coupling of the GrIS explains apparent contradictions in a less complex conceptual model. First, our model can explain recent observations36615While the annual cycle of dewatering and recharge of weakly connected regions of the bed suggests a resilience of GrIS ice dynamics to increasing melt2 m) rather than attempting to model every basal conduit explicitly. The three modes of drainage, distributed, channelized and weakly connected, are modelled as separate components, each with appropriate physics, that are coupled together by exchanges of water driven by gradients in hydropotential between the systems. Distributed drainage is modelled as a continuous macroporous sheet in two dimensions on the primary model grid, while channelized drainage is represented by a single one-dimensional channel located along a line of grid cell edges of the primary model grid taking up no area within the primary model grid  is described by the balance between locally generated melt (mw) and exchange of water with the sheet (γw),Using a macroporous sheet continuum formulation for the weakly connected system analogous to that used for the distributed system , mass coρw is the density of water, Aw is the area of the weakly connected system within each grid cell, defined as fwΔxΔy.where Evolution of cavity space within the weakly connected component is the same as for the distributed system, a balance of cavity opening by sliding of the ice over bedrock bumps and close by creep of their ice roof:ub is the sliding velocity, A is the temperature-dependent rate factor for ice deformation, Nw is the effective pressure in the weakly connected system, and hr and lr are parameters describing the height and wavelength, respectively, of bumps on the bed. The value for A for the basal layer , zb is the bed elevation (m), p is water pressure (Pa) and pice is the ice overburden pressure (Pa). For the distributed and channel systems the ice overburden pressure is calculated using a hydrostatic assumption, pice=ρigH, where ρi is the ice density (kg m−3) and H is the ice thickness (m). To allow the weakly connected system to attain water pressure greater than floatation, as observed, we include a simple representation for normal stress transfer34where the subscript rw to a constant value of 0.13, because this corresponds to the maximum value of borehole water pressure observed at our study siteNw=0. Note that this simple representation does not include temporal variations in normal stress transfer.We set γw, between the surrounding distributed system and the weakly connected system within each grid cell based on a Darcy flow law:Similar to the coupling between the channel and distributed system , the weak0w is the permeability between the weakly connected and distributed systems, Pw represents the perimeter between the two systems within each grid cell and s is a characteristic spacing between the two systems.where Pw and Δs. Specifically, we assume each weakly connected patch is a circle of radius 10 m  and grid spacing (Δx=Δy=200 m) we can calculate Pw≈5,355 m from basic geometry. While other choices could be made here, these details are not important from a practical standpoint without detailed knowledge of the bed, as these parameters, along with k0w, are free variables in γc; While in reality the boundary between the two systems is likely to be complex, for simplicity in our parameterization we assume a simple geometry for the weakly connected patches within each grid cell for the purposes of generating reasonable values for k0w, is many times smaller than the permeability within the distributed system itself, k0 . Additionally, the bedrock geometry for the weakly connected system may have different characteristics  than for the distributed system. Though we acknowledge the inherent complexity of the subglacial system, we have chosen the simplest formulation that includes the dominant processes inferred to occur. Our parameterization of the weakly connected system is meant to represent the mean conditions of the bed and thus will not necessarily directly reflect unique measurements from specific boreholes.Our parsimonious parameterization of the weakly connected system leaves room for additional complexity as empirical knowledge of the system increases. We have assumed in our model that all parts of the weakly connected system have changing permeability during summer , while b34−1 . The icudy site . The subudy site ; the larWe choose this simplified geometry over the more complex geometry of the field site to simplify our interpretation of model results, given uncertainty in the true ice sheet geometry. Previous work in our study area has shown that complex topography can affect sliding and deformation over short distances2628hr, lr and k0 . We scale the point ablation rate by the width of our model domain and apply it as a linear source term along the length of the channel model (ω). As discrete moulins can result in the generation of kinematic waves within our channel model, this linear distribution of melt input improves model stability, while reproducing synchronous changes in moulin water level as observed by Andrews et al.58We base the melt forcing  off of 6et al.5 Pa for the entire simulation; though it should vary in reality, we do not have the information to provide a more accurate value, and, in any case, the results are not sensitive to this choice as it only affects frictional melting, which is orders of magnitude smaller than summer surface melt input.The ice sliding speed forcing  comes fr2 at the margin decreasing linearly to zero at 55 km inland, which yields an area of 5.45 m2 at the study site. This value was chosen to allow water pressure to drop below floatation and diurnal variations to develop within days of melt onset, as seen in the ice velocity record. The channel roughness parameter F is chosen to yield diurnal hydraulic head minima in late summer comparable to that measured in moulinsUsing this spun-up state as an initial condition, we model the 2012 summer for days 150 (onset of summer speedup in the GPS record) to 250 (end of GPS and melt forcing observation time-series). For the summer simulation we add a single active channel along the centreline of the domain. While this precludes the formation of a network of channels 4which is slightly different at each measurement site see ref.  and in tτb, is defined by a physically based basal friction law for sliding over hard beds that allows for cavitation and bounded basal drag39Ice velocity for 606162C is a Coulomb friction constant, λmax and mmax are the wavelength (m) and maximum slope, respectively, of the dominant bedrock bumps, and n is the exponent in Glen's flow law. where y domain , chosen With widespread cavitation , the friction law becomes a Coulomb friction law of the form  the friction law takes a power law formThe basal traction appropriate for ice dynamics is the integrated basal traction of both connected and isolated regions of the bedwhere Nint, of the effective pressure in the distributed and weakly connected systems,We approximate N).This simplification is justified by the fact that for the effective pressure and parameter values in the simulations, Nd and Nw calculated in the summer subglacial hydrology simulation. This simulation demonstrates the impact of weakly connected drainage on ice dynamics , diurnal variations in ice speed are almost entirely absent . Model code, processing scripts, input datasets and model output are all available on request from the corresponding author.Model code is development code based off of the Community Ice Sheet Model (CISM) version 2.0.4 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Methods and Supplementary References.


P < 0.01). Hs-CRP  was an independent risk factor of short-term death determined by univariate and multivariate Cox analyses. No impact of cystatin C was observed on the short-term mortality. For long-term mortality, cystatin C  was identified as an independent predictor at above the cut-off value ≥ 1.10 mg/L. ROC analysis showed the AUC values of cystatin C and hs-CRP were 0.772  and 0.640 , respectively, in the prediction of long-term death. The combined AUC value of cystatin C and hs-CRP was 0.883 . Taken together, high cystatin C levels (≥ 1.10 mg/L) on admission are independently associated with the long-term mortality in patients with ATAAD.Increased serum cystatin C levels are related to the prognosis of cardiovascular diseases. This study aims to investigate the effect of admission serum cystatin C levels on short- and long-term mortality in patients with acute type A aortic dissection (ATAAD). From 2010 to 2014, 136 consecutive patients with ATAAD were enrolled and followed up. Clinical data and laboratory assays including were measured. During a median follow-up of 198.7 days, the short-term mortality (30-days) was 20.6%, whereas the long-term death rate was 10.2%. We identified that the expression of cystatin C and high-sensitivity C-reactive protein (hs-CRP) in the dying patients was higher than in the surviving patients ( Acute aortic dissection (AAD) is a devastating cardiovascular condition with an incidence of about 2.6–3.6 cases per 100,000 people per year , C-reactCystatin C is a type of cysteine protease inhibitor whose levels increase much earlier than those of urea and creatinine when renal function declines , 10. PreP = 0.034). Patients who received surgical treatment were more likely to survive (P < 0.001). No significant differences in patients’ age, sex, body mass index, and cardiovascular risks such as hypertension and diabetes mellitus were observed between the death and survival patient groups.A total of 136 patients were enrolled in this study, including 77 males and 59 females, with an average age of 53.7 ± 10.3 years. Patient demographics and baseline characteristics are shown in Table 9 vs 12.8 ± 4.0 × 109 cells/L, P = 0.019), hs-CRP , D-dimer , high-sensitivity troponin I (hs-cTnI) , BNP , and cystatin C  concentrations were significantly higher in the death group than in the survival group. The comparison of cystatin C and hs-CRP levels between the two groups are shown in Figure With respect to laboratory findings, WBCc (14.7 ± 4.8 × 10r = 0.4675 (P < 0.001), which were analyzed by the spearman correlation; serum cystatin C was also correlated with WBCc , but not D-dimer .As shown in Figure P = 0.3917). Interestingly, the log rank test identified a significant difference in long-term survival among the patients stratified by serum cystatin C .As shown in Figure P = 0.037) was an independent risk factor, whereas surgical treatment  was an independent protective factor for short-term mortality (Table P = 0.013) and surgical treatment  were independent predictors of long-term mortality  as shown in Figure Receiver operating characteristic (ROC) curves were used to further investigate the predictive power of cystatin C in evaluating long-term mortality. The results indicated that the area under the curve (AUC) value of cystatin C was 0.772 , as well as WBCc. One potential reason for this correlation is that like many other markers of inflammation, its serum concentration may be higher in patients with decreased renal clearance. However, few patients in our cohort experienced severe renal impairment. Another reason may be that cystatin C is a marker more than for renal function and a marker of inflammation . Althougtic wall . This diIt is likely that no single biomarker will be perfectly predictive, but identifying the optimum combinations to develop risk stratification may determine whether relevant therapies affecting these biomarkers have the potential to reduce mortality. We finally found that the combined use of both cystatin C and hs-CRP levels could increase the sensitivity in assessing long-term mortality up to 97.44%. Therefore, according to our data, it may be reasonable to take both cystatin C and hs-CRP into account as the prognostic markers in patients with ATAAD because of the high sensitivity demonstrated. The data suggesting that if both admission cystatin C and hs-CRP levels were above the cut-off levels in ATAAD patients, the risk of death was significantly increased.Some limitations of the present study need to be addressed. First, we did not obtain the cystatin C profiles at regular intervals during the follow up because of high test costs. As a prognostic marker, it needs more information about dynamic alteration of cystatin C. Second, no patients with end-stage renal disease were included in our cohort, and other studies are needed to evaluate how informative this biomarker will be in individuals with renal dysfunction, as it always occurs in progression of this disease . FurtherIn conclusion, our study preliminary demonstrated that serum cystatin C ≥ 1.10 mg/L and hs-CRP ≥ 5.76 mg/L were important risk factors and independently associated with ATAAD long-term mortality. Furthermore, the combination of serum cystatin C and hs-CRP is a potentially useful biomarker with high sensitivity in predicting long-term death in patients with ATAAD in clinical settings.We enrolled 136 consecutive patients with ATAAD at Shaoxing Hospital between February 2010 and December 2014. Each patient was diagnosed with ATAAD according to the standard guideline  assistinVenous blood samples were drawn into ethylenediaminetetra-acetic acid (EDTA)-containing tubes from all patients at the first day upon admission before administration of any medication. Supernatants were obtained within 1 h after centrifugation  and the serum and plasma were immediately stored at −80°C until further analysis. The serum levels of cystatin C were measured using enzyme-linked immunosorbent assay kits  as previously described . Plasma t-test or Mann-Whitney test was used for the analysis of continuous variables and χ2-test for categorical variables. The correlation between cystatin C and hs-CRP was determined by the spearman rank correlation. Short- and long-term outcomes were determined using the Kaplan-Meier method and compared using the log-rank test. A logistic regression model was constructed for the multivariable analysis for markers, and cystatin C + hs-CRP (cystatin C + hs-CRP = −8.40 + 0.62* hs-CRP + 0.45* cystatin C) was then generated. Receiver operating characteristic (ROC) analysis was used to compare the prognostic value of cystatin C and D-dimer. SPSS 19.0  and GraphPad Prism 6.5  were used to perform statistical analysis. P value < 0.05 was considered statistically significant.Continuous variables were presented as mean ± standard deviation (SD) or median and interquartile range, and categorical variables were displayed as counts or percentages. Student's


New fossil footprints excavated at the famous Laetoli site in Tanzania suggest that our bipedal ancestors had a wide range of body sizes. Related research article Masao FT, Ichumbaki EB, Cherin M, Barili A, Boschian G, Iurino DA, Menconero S, Moggi-Cecchi J, Manzi G. 2016. New footprints from Laetoli (Tanzania) provide evidence for marked body size variation in early hominins. eLife5:e19568. doi: 10.7554/eLife.19568Walking on two hind limbs, or bipedalism, is one of the defining characteristics of the evolutionary lineage that gave rise to modern humans. Though fragments of fossilized bones suggest that this adaptation might date as far back as 7 million years ago , this inAustralopithecus afarensis  of the individuals who left the footprints at the two sites. The prints at Site S were most likely left by individuals who were taller and heavier than any of the three that left prints at Site G. Of the five individuals, the lightest one left footprints at Site G and likely weighed 28.5 kg, while the heaviest walked at Site S and is estimated to have weighed as much as 48.1 kg.A. afarensis individuals might have had very different body sizes. Variation of this magnitude could imply big differences between males and females – a phenomenon referred to as "sexual dimorphism". However, this would only be the case if we assume that all the footprints are from adults, and not if younger individuals made some of the smaller ones.These estimates for body mass fit comfortably within the wide interval calculated for this species based upon fossils of its limb bones . The preHomo floresiensis – a species of ancient hominid from Indonesia (which have been commonly referred to as "hobbits"; A. afarensis.Nevertheless, and as Masao et al. acknowledge, the height estimates depend on a previously reported relationship between foot length and stature . It is iLooking back, 2016 has been a banner year for trace fossils in human evolution e.g., , and the


In humans, the amount of spinal homonymous recurrent inhibition during voluntary contraction is usually assessed by using a peripheral nerve stimulation paradigm. This method consists of conditioning the maximal M-wave (SM stimulus) with prior reflex stimulation (S1), with 10 ms inter-stimulus interval (ISI). The decrease observed between unconditioned (S1 only) and conditioned (S1+SM) reflex size is then attributed to recurrent inhibition. However, during a voluntary contraction, a superimposed SM stimulation leads to a maximal M-wave followed by a voluntary (V) wave at similar latency than the H-reflex. This wave can therefore interfere with the conditioned H-reflex when two different stimulation intensities are used (S1 and SM), leading to misinterpretation of the data. The aim of the present study was to assess if conditioning V-wave response instead of H-reflex, by applying SM for both stimuli (test and conditioning), can be used as an index of recurrent inhibition. Conditioned and unconditioned responses of soleus and medial gastrocnemius muscles were recorded in twelve subjects at 25% and at 50% of maximal voluntary contraction at the usual ISI of 10 ms and an optimal inter-stimulus of 15 ms determined upon M- and V-wave latencies. Conditioned H-reflex (obtained with S1+SM paradigm) was significantly lower than the unconditioned by ~30% on average, meaning that the amount of inhibition was 70%. This amount of recurrent inhibition was significantly lower at higher force level with both methods. Regardless of the level of force or the conditioning ISI, results obtained with V-wave conditioning (SM+SM) were similar at both force levels, linearly correlated and proportional to those obtained with H conditioning. Then, V-wave conditioning appears to be a reliable index of homonymous recurrent inhibition during voluntary contraction. During voluntary contraction, the motoneuronal output can be auto-regulated by a post-synaptic recurrent pathway involving the Renshaw cells. These cells receive among others a cholinergic motor axonal projection originating from motor neurons upon which they are projected –3. The mmax  over the mixed peripheral nerve –9. Fig 1max . With anmax . The decTo specifically investigate recurrent inhibition, Pierrot-Deseilligny et al. in 1976  have sugThe validity of this method is even more dependent on the stimulation intensity used to evoke S1 . InitialThe aim of this study was to assess whether the experimental approach using V-wave conditioning can be a valuable method to analyze recurrent inhibition during voluntary contraction, at two different force levels of the plantar flexor muscles. The use of several levels of force would be useful to identify and compare the evolution of conditioned responses with both methods, as it is well known that recurrent inhibition levels decrease when the force level increase . We hypoExperiments were performed on twelve young healthy subjects . None of them reported neurological or physical disorders. After being fully informed about the investigation and possible related risks and discomfort, they all gave written informed consent to participate in the study. The experimental protocol was approved by the regional ethics committee (CPPGE—Comité de Protection des Personnes de la region Grand-Est) and was carried out in agreement with legal requirements and international norms (latest version of the Declaration of Helsinki). All experiments were carried out in one single session of about 2 hours.Experiments were performed on the right leg in a sitting position using an isokinetic dynamometer , with hip and knee joints at 90° (0° = full extension) and ankle joint at 90° . The ankle was firmly strapped to the dynamometer with the motor axis aligned with the external malleolus of the ankle. During all experiments, particular care was taken in monitoring the subjects’ posture during the test and avoiding head rotations to maintain constant cortico-vestibular influences on the excitability of the motor pool . The trugastrocnemii over the Achille’s tendon; for the MG electrodes were placed over the mid belly of the muscle; and for the TA electrodes were positioned at 1/3 of the distance on the line between the fibula and the tip of the medial malleolus  for each condition (level of force and ISI) and muscle (SOL and MG). The conditioned responses were expressed as a percentage of unconditioned to give the level of recurrent inhibition, using the following calculation: [(H’/H1)-1] x100 and [(V’/V1)-1] x100, for recurrent inhibition level obtained on H1 and V1 respectively.All data are presented as the mean ± standard deviation. The normality of the data was tested using the Shapiro-Wilks test.“level of force” (25% and 50% MVC) and “stimulus intensity”  for SOL and MG muscles. For each muscle, separate three-ways repeated measures ANOVA were performed on normalized responses (H/Mmax or V/Mmax) to assess the effects of factors “inter stimuli interval” , “conditioning intensity” (S1 and SM) and “level of force” (25% and 50% MVC). The same ANOVA was performed on percentages of inhibition.Latencies of unconditioned H-reflex responses were compared through a repeated measures ANOVA with factor “level of force” (25% and 50% MVC) and “conditioning”  on normalized responses (H/M ratios).Reference H-reflexes were compared to H’ by using a two-way repeated measures ANOVA with factors vs. SM conditioning intensity), with P obtained in the Bravais-Pearson table (degree of freedom = 10). Coefficients of variation were determined as the ratio of standard deviation over the mean.Main effects or interactions were followed-up by HSD Tukey’s tests. Statistical analysis was performed using STATISTICA . The level of significance was set at P < 0.05. The size effect was calculated by the partial eta-squared method as recommended by Levine and Hullett in 2002 . Pearsonmax) of the other tested muscles (SOL and MG) were significantly greater at 50% MVC than at 25% MVC  and were on average 4.01 ± 0.64% at 25% MVC and 4.38 ± 0.98% at 50% MVC. As expected, EMG activities . As expected, at 25% MVC normalized reference H-reflex was not statistically different from conditioned H’/Mmax (P = 0.99 and P = 0.97 for SOL and MG respectively). However, H’/Mmax was statistically greater than reference H/Mmax at 50% MVC (P = 0.006 and P = 0.04 for SOL and MG respectively). Then, no statistical differences were found between normalized reference H-reflex at 25% MVC and at 50% MVC, for both muscles , and no effect of force level was found in either of the latencies . On the contrary, V1/Mmax was significantly greater at 50% than at 25% MVC for both SOL  and MG .Then, both SOL and MG H1/Mmax and V/Mmax between factors “level of force”, “conditioning intensity” and “inter-stimuli interval” for SOL  and MG . Post-hoc analysis then revealed that regardless of the condition , a significant decrease was found between conditioned (H'/Mmax and V'/Mmax) and unconditioned responses .An interaction effect was found on H/M1,10 = 16.25, P = 0.002, ηP2 = 0.64) and MG , i.e. the recurrent inhibition observed was statistically lesser at 50% than at 25% MVC. However, no statistical effect of the factor “conditioning intensity” (S1 vs. SM) was found on percentages of H and V inhibition , i.e. the inhibition observed was similar with both methods. The most significant correlation between inhibition induced by the S1 conditioning and the one induced by SM conditioning was found at 15 ms ISI for both levels of force, r2 coefficient ranging from 0.29 to 0.56 at 10 ms ISI and from 0.75 to 0.98 at 15 ms ISI  and -0.71 ± 5.41% ; for MG between– 4.73 ± 2.14% Mmax  and– 0.34 ± 3.38% Mmax .The classical method consisting of subtracting V-wave amplitude from H’ showed negative values close to zero in nearly all conditions (ISI and force level). A great variability (coefficient of variation from 0.66 to 7.61) was found in H’-V1 differences; for SOL between– 9.00 ± 3.69% MFinally, it can be noticed that for some subjects (4 out of 12), a prior response to H’ or V’ appeared  and V conditioning, at different levels of muscle contraction. The main finding was a similar inhibition induced in conditioned responses between both methods, whatever the muscle and the level of force tested.max were also observed between both level of force, showing that the use of reference H-reflex might not be necessary when the analysis is performed from normalized ratios. Therefore, the fact that normalized responses were not increased from 25% MVC to 50% MVC emphasizes the assumption that the increase in raw responses can be attributed to a rightward shift of the recruitment curve and not necessarily to changes in spinal excitability background. According to previous methodological reviews and studies [As recurrent inhibition is one of the main post-synaptic mechanisms that modulate the output of alpha motoneurons, its evaluation is of particular interest during voluntary contraction . Neverth studies ,24, the max or reference H/Mmax, conditioned H’/Mmax was statistically greater at 50% than at 25% MVC. These results, in line with those of Hultborn and Pierrot-Deseilligny [Contrary to H1/Meilligny , corroboeilligny  hypotheseilligny ,12,26 toeilligny . HoweverHowever, in these 4 subjects despite an incomplete collision a decrease is still observed in conditioned response compared to unconditioned, showing that other mechanisms than recurrent inhibition may be involved, such as after-hyper-polarisation mechanisms . Others max), while another study subtracted the V-wave regardless the size [max always exceeded 0.05 (max (S1 stimulus), whereas a significant increase of V1/Mmax (SM stimulus) was found from 25% to 50% MVC. We recently found that V-wave was well correlated to motor evoked potential amplitude elicited by transcranial magnetic stimulation and thus represents a reliable index of corticospinal neural drive [Contrary to rest, the descending neural drive onto the motoneuronal pool leads to the recording of a reflex response (V1) following maximal M wave. This response, which amplitude is commonly used as a marker to quantify the descending command, may widely influence recurrent inhibition. In fact, the link between V-wave response and conditioned H’ responses, both recorded during voluntary contraction, remains unclear. Hultborn and Pierrot-Deseilligny  suggestethe size . Matchinded 0.05 , the limal drive . Thus, cal drive  suggesteal drive ,11. Thismax can also reflect recurrent inhibition mechanisms.The high correlations observed between the inhibition levels induced by V-wave conditioning and those induced by H-reflex conditioning  suggestemax has reached a plateau in both muscles by the use of supra-maximal intensity. This helped recording conditioned response simultaneously in all triceps surae muscles, contrary to H1 recording which necessitated two different intensities between SOL and MG muscles.To summarize, the present study showed that results obtained with V-wave conditioning, i.e. the use of SM intensity for both conditioning and conditioned stimuli, were consistent and well correlated with those obtained with the classical method. This consideration was particularly relevant when using the theoretically optimal ISI of 15 ms, calculated according to response latencies and subjects’ height. Thus, V-wave conditioning method, easy to implement with H-reflex measurement, could be of interest when studying transitional phenomena and allows assessing both recurrent inhibition and voluntary neural drive through V1 amplitude. In addition, this method allowed using the same stimulation for SOL and MG muscles since the M