Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn. Treated and control samples at each time point were processed in parallel from start to finish. Five biological replicates for each condition were included for statistical analyses. Generally, the coefficients of variation between biological replicates were less than 20%. The signal intensity of fragment ions depends on the amino acids constituting that fragment. The charged amino acids histidine, lysine, arginine and proline result in the best (highest intensity) fragment ions for detection, but these ions are not always the most informative. Often, there are several possible sites of phosphorylation within a tryptic fragment. Precursor mass and highest intensity fragment masses may be the same regardless of the location of the phosphorylation. Only fragmentation between two possible sites will enable the localization of the phosphorylated residue, and the closer the two sites, the more challenging it is to make a confident determination. The different sites of phosphorylation usually result in a chromatographic retention time shift, but the precursor and monitored product ion masses can be identical. For several peptides in this study, multiple endogenous phosphorylation sites were measured simultaneously, for the reasons described above- precursor and product ion masses are constant, but the different phosphopeptides are chromatographically resolved. In these cases, one endogenous peak co-eluted with the isotopic standard while the other shifted within the chromatographic window. One such case, MtAHA4, was detected as two chromatographically resolved endogenous phosphopeptides. The width of the scheduling window was fortuitous, because a smaller window for this peptide would have prevented detection of the alternately phosphorylated form. Similarly, two distinct phosphorylation sites were observed on a single tryptic peptide for SNF1-related kinase Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Mycobacterium tuberculosis (MTB) synchronizes a number of processes and controls a series of events to subvert host defense mechanisms for the sake of residing inside macrophages. Besides these, MTB also possesses a wide range of signal enzyme systems, including eleven serine threonine protein kinases (STPKs). The present study describes STPK modulated modification in one of the hypothetical proteins of the RD1 region; EspJ (ESX-1 secretion associated protein), which is predicted to be involved in virulence of MTB. We have employed knock-out MTB, and M. bovis BCG as a surrogate strain to elaborate the consequence of the phosphorylation of EspJ. The molecular and mass spectrometric analyses in this study, confirmed EspJ as one of the substrates of STPKs. The ectopic expression of phosphoablative mutants of espJ in M. bovis BCG also articulated the effect of phosphorylation on the growth and in survival of mycobacteria. Importantly, the level of phosphorylation of EspJ also differed between pathogenic H37 Rv (Rv) and non pathogenic H37 Ra (Ra) strains of MTB. This further suggested that to a certain extent, the STPKs mediated phosphorylation may be accountable, in determining the growth and in intra-cellular survival of mycobacteria. This is important for maintaining cell wall integritymym promoter DNACharacteristically, the phosphorylation engages a wide range of proteins in mycobacteria and most of them regulate cell wall biosynthesis. A number of findings have suggested that phosphorylation in the proteins which are associated with the virulence factors affect the mycobacterial physiologyBesides STPKs; signaling in mycobacteria is also controlled by two component system. The putative transcription regulator factors PhoP/PhoR, in MTB, are the part of this system which control transcription of key virulence genes essential for survivalExpressions of STPKs as proteins involved in pathogenesis, have also been reported earlierM. bovis BCG. Herein, we have elucidated the role of phosphorylated and un-phosphorylated EspJ in the growth of mycobacteria. Surprisingly, a higher degree of phosphorylation in Rv was observed over Ra which may imply, the distinctive behavior of this protein in pathogenic and in non-pathogenic strains. Further, in order to identify the key residues undergoing phosphorylation, we used LC/MS/MS which are potentially being used for the identification of phosphorylation sites at several instancesin vitro kinase assay, we have identified phosphorylation sites in EspJ. Generation of phosphoablative mutants by site directed mutagenesis, followed by the transfer of these phosphoablative alleles in M. bovis BCG; we have deciphered its role in the growth and in persistence of mycobacteria. This phenotype was also confirmed by knocking-out the gene from MTB and then complementing with wild type and phosphoablative genes.We have investigated the interrelationships between EspJ (encoded by Rv3878) with STPKs of mycobacteria and their differential behavior in pathogenic Rv and in non-pathogenic Ra strains. EspJ, so far known as a hypothetical protein, has been putatively categorized as a regulatory protein70, Ser85 and Thr144 as other most probable phosphorylation sites in EspJ protein.Web based bioinformatics tools like Kinasephos 2.0, Disphos 1.3, Netphos 2.0 and NetPhosBac1 predicted putative phosphorylation sites in RD1 encoded proteins . Based oespJ and pknG ORFs were amplified by PCR from the genomic DNA obtained from different mycobacterial strains, using gene specific primers. The presence of espJ in Rv and Ra, and its absence in BCG and in MS were confirmed by PCR. Likewise, it was observed that the MTB orthologous pknG is present only in Rv, Ra and in BCG  as well as increment in growth units for BCG, containing EspJ and its mutants  increases the survival of M. bovis BCG inside macrophages as compared to rBCG expressing wild type allele  were determined to enumerate the intracellular survival of recombinant BCG. Results showed that infection of macrophages with rBCG expressing phosphoablative mutants at Sere allele . Other mespJ as well as phosphoablative allele espJ_S70A) demonstrate involvement of this protein in growth of mycobacteria  domain get phosphorylated by STPKs of MTB and hence protein containing this motif has been identified as a substrate1920proteins . All thebacteria .in vitro kinase assay with PknG (one of the MTB STPKs). Although we could see the partial phosphorylation of EspJ with other STPKs .Phosphorylation may occur at several positions in a protein. We used the bioinformatics tool to identify several putative phosphorylation sites in EspJ protein, which are characteristic features of phospho-residue of MTB STPK, like presence of RXXS/T motifs. We made mutants, at all such positions, but didn’t get significant difference in the phosphorylation level by M. smegmatis) has been annotated as translation initiation factor IF-2. Alignment of MTB EspJ (Rv3878) with MSMEG_0069 demonstrates large similarity between these two proteins  motif found in most of the phosphorylated residues in MTBTo corroborate the biological effect of this protein we used e strain . RecombiThe role of EspJ phosphorylation in mycobacterial growth has also been established by gene knockout strategy in MTB . Overallin vivo phosphorylation of EspJ by STPKs. As expected, we found a higher level of phosphorylation in pathogenic strain compared to non pathogenic strain  and nonpathogenic (Ra) mycobacteria can be distinguished based on the variable levels of phosphorylation of EspJ. Study with a surrogate strain, –3) culture was transferred to BBL MGIT tube and incubated at 37 °C, and monitored for the change in fluorescence. The BACTEC MGIT 960 system monitors fluorescence (in Growth units) every hour. The growth obtained was recorded about every 24 h during the first 120 h. The prepared dilutions were also plated on MB7H10 agar and incubated at 37 °C to detect CFU and to eliminate the possibility of any other bacterial contaminations.Mycobacterial strains  were culpknG and RD1 encoded espJ were amplified from MTB Rv genomic DNA using flanking HindIII anchored primers (E. coli BL21 (DE3). E. coli strains DH5α and BL21 (DE3) were cultured in Luria–Bertani medium. Over-expressions of His-tag recombinant proteins were done using 0.2 mM IPTG concentration at 18 °C for overnight and purification was done with Ni-NTA column chromatography. The protein showed a relative molecular mass of 43.0 kDa in E coli, while in mycobacteria it is expressed as 27.4 kDa monomeric protein. In addition, espJ was also sub cloned downstream of the hsp60 promoter into pMV361 vector. This vector contains an E. coli origin of replication (oriE), the attP and int genes of mycobacteriophage L5  and a kanamycin resistance marker (Kanr). The resulting plasmid was used for complementation in M. bovis BCG to explore the role of espJ and generation of phosphoablative mutants by site directed mutagenesis at T68V, S70A, S85A and T144V positions by PCR using overlapping primers  were obtained from the animal colony of the institute. The animals were maintained in an animal house accredited by the National Accreditation Board for Testing and Calibration Laboratories guidelines controlled by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). CSIR-Central Drug Research Institute constituted ethics committee for use of laboratory animals has approved all the protocols for the study. The animals were maintained in standard conditions of temperature and humidity  and given proper pellet diet and water ad libitum. Antibodies for hsp70 (Sigma) and pSer/Thr (Qiagen) were procured commercially.http://www.cbs.dtu.dk/services/NetPhos/), NetPhosBac1 (http://www.cbs.dtu.dk/services/NetPhosBac-1.0/), DISHPHOS 1.3 (http://www.dabi.temple.edu/disphos/pred.html) and, Kinasephos 2.0 (http://kinasephos2.mbc.nctu.edu.tw/). Alignment of protein sequence of closely related orthologs of EspJ was done by MUSCLEPhosphorylation potential as well as putative phosphorylation sites in RD1 encoded proteins were predicted by publicly available online analysis tool ; Netphosin vitro kinase assays were performed with either using ProQ Diamond (Invitrogen) or ADP-Glo™ Kinase Assay (Promega) according to manufacturer’s instruction. For ADP-Glo assay, PknG (25 ng) was used as a kinase and incubated either with EspJ or with the mutants (100 ng) in buffer containing 40 mM Tris (pH 7.5), 2 mM MnCl2, 20 mM MgCl2, 2 mM DTT, 0.5 mg/ml BSA, and 0.1 mM ATP for 1 h at 26 °C. The assay was also standardized using different concentrations of substrate .To validate the MS-MS data for phosphorylation sites; quantitative and qualitative in vitro kinase assay as described above, and 2DE was done to resolve these two proteins, using a standard protocol. Phosphorylated as well as un-phosphorylated EspJ spots from two different gels were excised and digested with Trypsin and processed further for LC/MS/MS analyses by HCT Ultra PTM Discovery System  with 1100 series HPLC (Agilent) to identify phosphorylated sites. Briefly, the protein spots were excised from the coomassie stained gel, diced into small pieces in microcentrifuge tubes. The gel pieces were destained by washing them twice with 200 μl of 25 mM ammonium bicarbonate .The gel pieces were dehydrated thrice with 50 μl of a 2:1 mixture of acetonitrile  and 50 mM ABC for 5 minutes and twice with 25 mM ABC for 2 minutes. The proteins were reduced with 10 mM DTT in 100 mM ABC at 60 °C for 1 hr, washed with 25 mM ABC and alkylated with 100 mM iodoacetamide at room temperature for 20 minutes to alkylate the cysteine residues. Gel slices were dehydrated twice with two washes of acetonitrile followed by a final wash with 25 mM ABC. The gel pieces were dried in speed vac for 15 minutes and rehydrated with 30 μl of 20 μg/ml solution of trypsin  overnight at 37 °C. The supernatant was acidified with 0.1% trifluoroacetic acid  and a 1:1 (v/v) mixture of the sample and α-Cyano-4-hydroxycinnamic acid (CHCA) was spotted on the MALDI plate prior to analysis. The mgf file generated after the sample run was used for MTB UniProt database search using a Mascot software package (Matrix Science London). The peptide precursor mass tolerance was set to 1.2 Da, and fragment ion mass tolerance was set to 0.6 Da. Carbamidomethylation on cysteine residues was used as fixed modification and oxidation of methionine and phosphorylations of serine, threonine, and tyrosine were used as variable modifications. Phosphopeptides were detected in phosphorylated sample.Purified recombinant protein EspJ was subjected to 2 in RPMI-1640 medium , supplemented with 10% heat inactivated fetal calf serum (FCS). The cells were differentiated into macrophage-like cells by treatment with phorbol myristate acetate (PMA) before infection. The cells having a density of 5 × 105 cells/ml were seeded in tissue culture plates. The single cell suspension of log-phase mycobacteria was prepared by vortexing in presence of sterile glass beads and passing through a 26 gauge needle and was used for macrophage infection (MOI = 1:10) as describedThe macrophage cell line THP-1 was cultured at 37 °C and 5% COg for 15 minutes. Cell pellets were washed twice with PBS. Cell pellets were re-suspended in protein extraction buffer  and disrupted by bead beating. The protein loading buffer was added to cell lysates, and samples were boiled for 15 min before loading onto SDS-PAG. Since albumin confounded analysis of secreted bacterial proteins we have used Sauton medium supplemented with 0.05% Tween 80 instead of 7H9 medium. To analyze secretory proteins by SDS-PAGE, mid-log phase mycobacterial strains were resuspended in fresh medium and cultured overnight at 37 °C. Culture supernatants obtained after centrifugation at 2500g for 15 minutes and sterilized by double filtration through 0.2-μm filters were treated with trichloroacetic acid  , to finally obtain a final concentration of 10% (w/v). The resulting mixture was kept at −20 °C for 5 h and centrifuged at 18,000 g for 30 min at 4 °C. Pellets thus obtained were washed thrice with pre-chilled acetone  and re-suspended in Laemelli buffer for western analysis.Mid-log phase cultures were harvested by centrifugation at 2500t) of the test gene was normalized to the Ct of the internal control (sigA and 16S) genes, before calculating the fold change in the test samples.RNA was isolated from log and stationary phase cultures of mycobacterial strains Rv and Ra, using the RNeasy Mini kit (Qiagen) according to manufacturer’s instructions. DNase treated RNA was used for cDNA synthesis using first-strand cDNA synthesis kit (Fermentas) and random hexamer primers. Quantitative real time PCR was performed in 96 well plate on Applied Biosystems StepOne™ Real-Time PCR Systems using Power SYBR®Green PCR master mix (Applied biosystem) with 5 pM primers . The calHow to cite this article: Singh, P. K. et al. RD-1 encoded EspJ protein gets phosphorylated prior to affect the growth and intracellular survival of mycobacteria. Sci. Rep.5, 12717; doi: 10.1038/srep12717 (2015).


Botulinum toxin A (BoNT-A) is a new treatment modality in various causes of bladder dysfunction; like neurogenic detrusor overactivity and overactive bladder. The best technique of administrating BoNT-A in patients is unknown. A validated in vitro model could be used to investigate newer intravesical administration techniques of BoNT-A. In this study, we describe the development and validation of in vitro model to measure inhibitory effects of BoNT-A on bladder strip contractions.Rat bladder strips were mounted in organ baths filled with Krebs’ solution. The strips were stimulated chemically  and electrically  100 shocks, 50 V, 20 Hz, every 3 minutes). The viability of the strips was measured by carbachol stimulation at the beginning and at the end of the experiments. The strips were incubated in various concentrations of BoNT-A . Controls were incubated in Krebs’ solution only. The inhibition of strip contraction induced by EFS was measured. These measurements were statistically analyzed with a log-logistic model representing diffusion.All strips remained viable during the experiments. Inhibition of strip contraction was observed after incubation with 0.3 nM BoNT-A. The measurements fitted to a log-logistic model describing diffusion of BoNT-A in the bladder strip. The parameters of the log-logistic model representing diffusion were significant for 0.3 nM BoNT-A. Incubation with 0.2 nM BoNT-A showed insignificant results for 2 out of 3 runs. Incubation with 0.03 nM BoNT-A did not result in significant inhibition of strip contractions.An in vitro model was developed and validated in which the inhibitory effect of low concentrations of BoNT-A on bladder strip contractions can be measured. Botulinum neurotoxin A (BoNT-A) was introduced for the treatment of overactive bladder (OAB) by Schurch et al. at the beginning of this century. Since tTo gain insight into the mechanism of spread of BoNT-A after injection in the bladder wall, we would need a reliable in vitro model with reproducible results. Smith et al. were the first to measure bioactivity of BoNT-A in an in vitro model with rat bladder strips. Howles 4 1.2, KH2PO4 1.2, CaCl2*2H2O 2.5, NaHCO3 25, glucose 11.5, bubbled with 95% O2 and 5% CO2 to attain a pH of 7.4) at 35 degrees Celsius.Eight adult male Wistar rats  were used. All experiments were performed in accordance with institutional guidelines and were approved by the ethics committee for animal experiments of the VU University Amsterdam. The urinary bladder was removed following isoflurane anaesthesia and heart dissection of the rat. The bladder was immediately placed in Krebs’ solution   during continuous EFS to verify that the EFS protocol induced primarily nerve cell depolarization. Subsequ2 and 5% CO2 (pH 7.4 at 35°C).KCl, CCh, TTX and BoNT-A were obtained from Sigma-Aldrich, Zwijndrecht, The Netherlands. The BoNT-A used was pure BoNT-A (molecular weight of 150 kDa), i.e. toxin without any enveloping proteins. KCL, CCh, TTX and BoNT-A were all dissolved in Krebs’ solution. All solutions were gassed with a mixture of 95% OTo establish the viability of the bladder strips after preparation and during the stimulation protocol, the strips were excited chemically by adding 80 mM KCl and 1 μM CCh to the organ baths. The organ baths were flushed with Krebs’ solution after chemical stimulation. KCl induces smooth muscle membrane depolarization without the need of activation of any receptor or second messenger system. CCh indKCl and CCh were used to verify viability, while EFS provided baseline contractions during phase 1. Organ baths were perfused with 80 mM KCl for 10 seconds, followed by flushing the organ baths with Krebs’ solution for 3 minutes. This cycle was performed three times. Subsequently, organ baths were perfused 3 times with 1 μM CCh solution followed by flushing the organ baths with Krebs’ solution for 3 minutes. Finally, contractions were induced by EFS  for five times, each with an interval of 3 minutes.In phase 2, strips were incubated in various concentrations BoNT-A . Control strips were perfused by Krebs’ solution only. Repetitive EFS was performed.Phase 3 consisted of perfusion of Krebs’ solution in the organ baths for at least 5 minutes. Subsequently, repetitive EFS and CCh stimulations with the same settings and the same concentrations as used in phase 1 were performed. We refer further to a completed protocol of stimulations as a ‘run’.Each BoNT-A concentration was tested 3 times. Two strips of the same bladder were incubated in 2 parallel organ baths. One strip was incubated in 0.3 nM BoNT-A  whereas the other strip was perfused with Krebs’ solution . To establish whether there was a minimum concentration necessary to measure an inhibitory effect of BoNT-A in bladder strip contractions, run 3a, 4b and 5a contained strips incubated in 0.2 nM BoNT-A. Run 5b, 6a, 6b contained strips incubated in BoNT-A 0.03 nM.mPF CCh). The viability of the strip was established by the equation: Contractility = mPF CCh phase III/mPF CCh phase I. A strip is viable if contractility equals 1.Three peak forces of strip contraction induced by CCh stimulation were measured and averaged (Inhibition = 1 – (mPF incub/mPF max), in which mPF incub is the mean of five repetitive peak forces by EFS measured every 30 minutes during incubation, and mPF max is the mean of five highest repetitive peak forces of strip contractions induced by EFS at the start of phase II.Inhibition of bladder strip contraction by BoNT-A was measured by comparing the mean peak force induced by EFS before and during BoNT-A incubation. Inhibition is defined by: I = Imax/(1 + exp (- b*ln (t/m))), where I = measured inhibition of contraction of bladder strip, Imax = calculated maximum of inhibition, b = shape parameter of the curve, t = time after highest peak forces of EFS at start of incubation (hours), and m = mid inhibition time, at which I = 0.5 Imax (hours). R2 is predictive accuracy of the model, so the closer R2 is to 1.0, the better the data fit to the curve. The curve fits if R2 is more than 0.95, and if the 95% confidence intervals around b and m are significant. Data analysis and curve fitting by non-linear regression was done with SPSS 20®.We analyzed the data by a mathematical model representing diffusion, as we hypothesized that BoNT-A spreads in the bladder strip by diffusion. The model is a log-logistic model: I = ImStrip contraction induced by EFS decreased by more than 90% after 1 μM TTX incubation. The oxygen tension in the organ bath was about 700 mmHg (pO2 95%) and the critical oxygen tension during EFS was about 600 mmHg (pO2 80%) with a diameter of the strip of maximally 0.7 mm.(mPF CCh phase III/mPF CCh phase I) of all strips was 1.1 (range 0.9 – 1.4). One strip (run 6b) was lost due to technical problems.All bladder strips contracted during perfusion of 80 mM KCl at the start of the runs. Median contractility 2 was 0.99. Respective mid inhibition times (m) were 2.33 (95% C.I. = 2.22-2.44), 1.49 (95% C.I. = 1.18-1.80) and 1.80 (95% C.I. = 1.31-2.28) hours. Shape parameters of the curve (b) were 6.44 (95% C.I. = 5.33-7.54), 3.35 (95% C.I. = 1.90-4.79) and 3.23 (95% C.I. = 1.55-4.90).After incubation of strips in 0.3 nM BoNT-A, the Imax was 0.42, 0.62 and 1.00 for run 1a, 2a and 4a respectively  and b = 2.44 (95% C.I. = 1.54-3.33). However, run 4b and 5a had an insignificant 95% C.I. of m and b although R2 was good. BoNT-A concentration of 0.03 nM did not induce inhibition of strip contraction during run 5b and 6a, Imax was respectively 0.06 (95% C.I. = 0.05-0.08) and 0.83 (95% C.I. = min 8.04 – 9.71). No inhibition of strip contractions was observed in the control runs  using Krebs’ solution.Incubation of strips in 0.2 nM BoNT-A solution during run 3a, 4b and 5a showed incompatible results. Run 3a demonstrated a good fit of the curve with RIn this study, an in vitro bladder strip model was validated to measure the spread and the inhibitory effects of low concentrations of BoNT-A on bladder muscle contractions. The spread of BoNT-A in bladder strips could be reproduced and confirmed using a statistical model representing diffusion at a concentration of 0.3 nM. A lower concentration of 0.2 nM showed inhibition, however insignificant results for 2 out of 3 runs. The inhibitory effects of BoNT-A on bladder strip contractions could be demonstrated at lower concentrations than previously described.Smith et al. were the first to demonstrate inhibition of bladder strips by BoNT-A in an in vitro model. The modTo be sure that the EFS protocol primarily induces nerve cell depolarization, strips were incubated in TTX before applying EFS. TTX selWe demonstrated that the inhibitory effect of BoNT-A at a concentration of 0.3 nM is the result of diffusion. Incubation of strips with 0.2 nM BoNT-A demonstrated equivocal diffusion results. Runs with lower concentrations (0.03 nM) demonstrated no inhibition. This suggested that there is a minimum BoNT-A concentration needed for bladder muscle strip inhibition in vitro. We demonstrated that the lowest effective concentration in this setting lies between 0.2 and 0.3 nM.It is self-evident that continuous perfusion of BoNT-A in a bladder strip model is not directly comparable to intravesical injections in patients. The much higher concentration needed in vitro 0.3 nM) compared to the concentration needed in patients (onabotulinumtoxinA 10 U/mL = 0.001 nM) might be explained by the technique of administration. Moreover, the effect in vitro is measured within a few hours after administration due to susceptibility of the strip, whereas the effect of intravesical injections in patients becomes apparent after days or weeks[ nM compaOur study completed the model of Smith et al. and Takahashi et al.,8 by conIn this study we describe an in vitro model in which the inhibitory effect of low concentration of BoNT-A on bladder strip contractions can be measured. This model can be used in the future to investigate newer intravesical administration techniques of BoNT-A.OAB: Overactive bladder; BoNT-A: Botulinum neurotoxin A; KCl: Potassium chloride; CCh: Carbachol; EFS: Electrical field stimulation; TTX: Tetrodotoxin.JIMU, EJHM, AAG, GMAB and WJL have no competing interests. JAN is a consultant for Astellas and performed teaching courses for Coloplast, Allergan and AstraTech.JIMU, EJHM and AAG participated in the conception of the study. The study is designed by JIMU, WJL and AAG. The data are collected by JIMU and WJL. The data are analyzed by JIMU, GMAB and JAN. The manuscript writing is done by JIMU, GMAB and JAN. The manuscript is critical revised by WJL, EJHM and AAG. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/14/37/prepub


The study diagnoses the relative impacts of the four known tropical Indo-Pacific drivers, namely, El Niño Southern Oscillation (ENSO), ENSO Modoki, Indian Ocean Dipole (IOD), and Indian Ocean Basin-wide mode (IOBM) on African seasonal rainfall variability. The canonical El Niño and El Niño Modoki are in general associated with anomalous reduction (enhancement) of rainfall in southern (northern) hemispheric regions during March-May season. However, both the El Niño flavours anomalously reduce the northern hemispheric rainfall during June-September. Interestingly, during boreal spring and summer, in many regions, the Indian Ocean drivers have influences opposite to those from tropical Pacific drivers. On the other hand, during the October-December season, the canonical El Niño and/or positive IOD are associated with an anomalous enhancement of rainfall in the Eastern Africa, while the El Niño Modoki events are associated with an opposite impact. In addition to the Walker circulation changes, the Indo-Pacific drivers influence the African rainfall through modulating jet streams. During boreal summer, the El Niño Modoki and canonical El Niño (positive IOD) tend to weaken (strengthen) the tropical easterly jet, and result in strengthening (weakening) and southward shift of African easterly jet. This anomalously reduces (enhances) rainfall in the tropical north, including Sahelian Africa. Africa experiences large spatial variability in rainfall, with relatively high mean annual rainfall along equatorial region, poleward extremes, and also over the highland areas such as eastern Africa, Cameroon and Nigeria and coastal sectors of Liberia, Sierra Leone and Guinea; lower annual rainfall is, on the other hand, seen in the arid regions of Sahara desert in subtropical latitudes, Namib desert in the coastal areas of southern Africa and desert areas in equatorial eastern Africa1Oceanic forcing from different basins, such as the tropical Atlantic357911Among the tropical Indo-Pacific climate drivers, the canonical El Niño Southern Oscillation [ENSO]151619823The ENSO Modoki has distinct signatures in SST anomaly (SSTA) and its evolution as compared the canonical ENSO. El Niño (La Niña) Modoki events are characterised with warm (cold) SSTA in the central Pacific flanked on the east and west by cooler (warmer) SSTA2323252627282930313223Given the interference between teleconnections of the various tropical Indo-Pacific drivers, this study is focused on assessing the relative impact of the known tropical Indo-Pacific drivers, namely ENSO Modoki, canonical ENSO, and IOD/IOBM (depending on the seasons they mainly prevail in), on African rainfall variability during different seasons, and in particular, to bring out the hitherto unexplored potential influence of ENSO Modoki on African rainfall. Further, as the rainfall variability of the tropical West Africa and Sahelian region has been a subject of critical research interest161222Briefly, we focus on the March-April-May (MAM) and October-November-December (OND) seasons, as prominent rainfall in the central and east African region mainly occurs during these seasons362.Also, January-February (JF) period is selected considering that the austral summer rains with January maximum are common mainly in north eastern parts of Southern Africa, southeast Angola and Eastern Africa south of 10 °SAs shown in −1 can be seen during January and February.The tropical west and central portions of the continent experience so called ‘long rains’ during MAM. These are associated with the seasonal migration of the solar irradiance into northern hemisphere and shift of the ITCZ to the seasonal mean position of ~13 °N . In asso1634The relative impacts of Indo-Pacific drivers on the African seasonal rainfall during various seasons are explored using the partial correlation analysis between the local rainfall and SST indices . Figure During the ‘long rain’ season, the El Niño Modoki , A2a eveAssociated with the occurrence of El Niño Modoki events, a significant below-average rainfall during the boreal summer season occurs mainly over northern latitudes of Africa, over central and east African regions especially Ethiopia, parts of Kenya, Uganda, Chad, Central African Republic, and Sahel , A2b. Ca122225During the ‘short rain’ season, several broad regions of eastern and southern African regions like Sudan, Ethiopia, Uganda, Kenya, north eastern regions of Dem. Rep. of the Congo, exhibit significant below-average rainfall in association with El Niño Modoki events , A2c. In21A reduction in rainfall activity in the southern latitudes of Africa, especially, the east African coastal region (between 15 °S to 30 °S) can be observed during the JF season in association with the El Niño Modoki events , A2d, siThe ENSO Modoki events influence the tropical and subtropical African rainfall significantly by inducing anomalous changes in Walker Circulation. The partial correlations of the EMI with divergent wind at 850 hPa and those with velocity potential at 850 hPa are presented in During the boreal summer, the occurrence of El Niño Modoki are associated with anomalous subsiding limb of the Walker Circulation in the tropical Africa, indicated by significant large-scale low level subsidence  and reduFurther, an anomalous enhancement of rainfall along the tropical central African region  and a reduction in rainfall along the southern and eastern part of Africa are associated with the El Niño Modoki events during the OND season , A2c, anTropical west and Sahelian regions are highly sensitive to the SST variability in all tropical basins, both remote (Pacific) and neighbouring (Atlantic and Indian). However, much of the interannual variations can be accounted for by the remote effect of the tropical Pacific SSTs onlyTo describe the possible mechanisms of the impacts of the tropical Indo-Pacific drivers, it is important to understand the influence of the drivers on African easterly jet (AEJ) and TEJ, in addition to studying the relevant Walker circulation changes with the variability of these drivers. The boreal summer rainfall over Sahel exhibits significant anomalous reduction (enhancement) in association with weakening (strengthening) of TEJ and strengthening (weakening) of AEJTo get more insight into the Modoki impacts, we carry out a composite analyses of various anomalous fields of rainfall, zonal winds at 200 hPa and 700 hPa (where the TEJ and AEJ respectively have their maximum intensity) over seven El Niño Modoki summers . As a reA similar composite analysis over the canonical El Niño events also show an anomalous weakening of TEJ , consistA composite analysis over the positive IOD events, reveals an anomalously positive rainfall signal over the tropical northern Africa  in assoc2225To confirm that such an attributed change in zonal winds during the boreal summer is indeed due to the forcing from Indo-Pacific events, we perform the following several sensitivity experiments using the LMDZ (Laboratoire de Meteorologie Dynamique and Z stands for Zoom) Atmospheric General Circulation Model (AGCM), (a) a control experiment with climatological SST, and experiments in which the AGCM is forced with SSTs representative of a typical (b) El Niño Modoki, (c) canonical El Niño and (d) positive IOD. Before analysing the results, we ascertain the ability of the LMDZ model in simulating the climatological rainfall and zonal winds over Africa. A comparison between the observations  and modeThe simulated seasonal mean differences in climate variables between the El Niño Modoki experiment and the control experiment  can be iThe simulated difference between the El Niño experiment and control experiment also show a weakening of TEJ , strengtDifference in the rainfall between positive IOD and control experiment  confirmsThe study suggests that the ENSO Modoki events significantly, and distinctly, affect the seasonal rainfall Africa. During JJAS season, both the canonical ENSO and ENSO Modoki exhibit similar signatures on seasonal rainfall variability in Africa. On the other hand, the impacts are opposite in east African region during the OND season. We also show that a co-occurrence of an ENSO flavour with the tropical Indian Ocean drivers, namely, the IOD or the IOBM results in interference of signals, leading to either enhancement or reduction in the impact of the Pacific drivers on African rainfall.During the MAM season, the El Niño Modoki is associated with anomalous surplus rainfall in the northern tropical Africa. However, in the southern parts of the Africa, either type of the El Niño anomalously reduces the rainfall. During the JJAS season, the El Niño Modoki events, just as the canonical El Niño events, cause an anomalous reduction of rainfall in the tropical northern latitudes of Africa, however with relatively weaker amplitude as compared to the canonical flavour. On the contrary, an anomalously enhanced rainfall signal is noted during a positive IOD event during the season, especially in the Sahel and tropical Africa. Hence, a co-occurrence of a positive IOD event, in a linear sense, could reduce the impact from the tropical Pacific drivers.The El Niño Modoki and canonical El Niño are associated with opposite impacts on the east African rainfall during the OND season, with below (above) average rainfall during the El Niño Modoki .The positive IOD events are associated with above normal rainfall activity over east Africa. Thus a co-occurrence of an El Niño Modoki and positive IOD events can be expected to reduce each other’s impact on the east African rainfall. In contrast, the co-occurrence of canonical El Niño and positive IOD events will result in a mutual reinforcement of the individual impacts, and thereby result in a relatively strong anomalous enhancement in rainfall in east Africa. On the contrary, in central Africa, we find a destructive interference of individual impacts between the canonical El Niño and positive IOD events. From the above discussion, we can, in a linear sense, visualize the combined impacts of the co-occurring tropical Indo-Pacific drivers of various phases.During the JF season, both the Pacific drivers have similar impact on rainfall over southern parts of Africa. Below normal rainfall occurs in association with El Niño Modoki and canonical El Niño, while above normal rainfall activities occur in association with Indian Ocean Basin-wide warming. However, in Madagascar, below (above) average rainfall activity is associated with El Niño Modoki , whereas Indian Ocean Basin-wide warming could result in below normal rainfall activity.We find that the El Niño Modoki events, just as the other Indo-Pacific drivers, influence the seasonal rainfall variability in the tropical Africa through anomalous anomalous baroclinic changes such as a displaced Walker circulation. We also find from our observational analysis and AGCM experiments that, in addition to this mechanism, all the tropical Indo-Pacific drivers influence the boreal summer rainfall in tropical Africa by modulating the position and intensity of the AEJ and TEJ, which play an important role in governing the instability mechanisms for the rain producing systems in northern parts of Africa. Specifically, the El Niño Modoki and canonical El Niño are associated with an anomalous strengthening and a southward shift of AEJ and weakening of TEJ, thereby an anomalous reduction of rainfall in northern tropical Africa including Sahel region. Our results suggest that the positive IOD events, in contrast to the El Niño flavours, anomalously weaken (strengthen) the AEJ (TEJ), and enhance the rainfall in northern tropical African region.The study is subject to somewhat subjective definitions of seasons such as JF, which are of course, based on earlier studies. Further, given the data quality issues, some noisy correlations, despite being statistically significant, may be irrelevant. Therefore, further verification of these results using several other AGCMs and GCMs will be helpful. Further, while an examination of asymmetric response of the rainfall in different African regions to the opposite phases of the tropical Indo-Pacific drivers will be also an interesting issue, it is beyond the scope of the current work owing to our linear analysis methodology and limited number of the AGCM experiments. Notwithstanding these issues, the work provides, for the first-time, a comprehensive appraisal of the potential impacts of the ENSO Modoki on the African climate with emphasis on equatorial east Africa and the West African monsoon region. Thus, the work provides background information for applications such as seasonal prediction.We use the Climate Research Unit (CRU) time series (TS) version 3.20 of high resolution monthly precipitation dataC − 0.5 [SSTA]E − 0.5 [SSTA]W, where the square bracket with a subscript represents the area mean SSTA, averaged over one of the three regions specified as the central , eastern , and western  regions of the tropical Pacific. The IODMI51To represent the canonical ENSO, ENSO Modoki, IOD and Indian Ocean Basin Mode events, we use the NINO3 index, ENSO Modoki Index (EMI), Indian Ocean Dipole Mode Index (IODMI) and the Indian Ocean Basin Mode Index (IOBMI), respectively. The NINO3 index is defined as the area-averaged sea surface temperature anomalies (SSTA) over the region bounded by 5 °N-5 °S and 150 °W-90 °W. The EMIImportantly, while the IOD events occur during boreal summer and fall, the IOBM events are mainly prominent during the boreal winter and spring. We therefore consider only one of these two as the main driver from the tropical Indian Ocean depending on the season under consideration. It is to be noted, that IOD are largely independent of ENSO1819205556In this study, to isolate the individual impacts of the tropical Indo-Pacific drivers on the African rainfall variability, we employ the well-accepted partial correlation method.23252325We also implement a composite analysis technique for an understanding of the impact of the tropical Indo-Pacific drivers on African rainfall during boreal summer season. Seven El Niño Modoki years , four El Niño years , four positive IOD years namely 1999, 2003, 2007 and 2008, and three positive IOBM years  were selected for the composite analysis. Out of the four positive IOD events selected, two of them (1999 and 2007) occurred during the development of La Niña events. The SST indices and basis for the selection of years are narrated in our earlier papers23To understand the influence of TEJ and AEJ on boreal summer rainfall of Africa, correlations between a zonal wind index for TEJ and AEJ with the JJAS rainfall have been performed. An index for the TEJ variability is obtained by area-averaging of Zonal winds at 200 hPa in the TEJ core region, bounded by. The corresponding index for the AEJ is obtained by area-averaging of the 700 hPa zonal winds over the region bounded by [15 °W-40 °E; 10 °N-20 °N. The statistical significance of the results obtained from the linear and partial correlation, and composite analysis are assessed by using a one tailed Student’s t-test. Results significant at 85%, 90% and 95% confidence level are displayed in the figures.during boreal summer season. Four sets of experiments were carried out, each with ten ensemble members with perturbed initial conditions of 1 January, and integrated for 9 months. In the first experiment, henceforth referred to as the control experiment, the AGCM is forced with monthly varying climatological (1979–2010) SST, derived from the HADISST, as the lower boundary conditions. In the second experiment, what can be referred to as the El Niño Modoki experiment, the AGCM is forced with El Niño Modoki type of SST, obtained by imposing monthly June to September El Niño Modoki type of SST anomalies on the climatological SSTs only in the tropical Pacific region , following previous studies26The LMDZ AGCMHow to cite this article: Preethi, B. et al. Impacts of the ENSO Modoki and other Tropical Indo-Pacific Climate-Drivers on African Rainfall. Sci. Rep.5, 16653; doi: 10.1038/srep16653 (2015).


A single iliosacral screw placed into the S1 vertebral body has been shown to be clinically unreliable for certain type C pelvic ring injuries. Insertion of a second supplemental iliosacral screw into the S1 or S2 vertebral body has been widely used. However, clinical fixation failures have been reported using this technique, and a supplemental long iliosacral or transsacral screw has been used. The purpose of this study was to compare the biomechanical effect of a supplemental S1 long iliosacral screw versus a transsacral screw in an unstable type C vertically oriented sacral fracture model.A type C pelvic ring injury was created in ten osteopenic/osteoporotic cadaver pelves by performing vertical osteotomies through zone 2 of the sacrum and the ipsilateral pubic rami. The sacrum was reduced maintaining a 2-mm fracture gap to simulate a closed-reduction model. All specimens were fixed using one 7.0-mm iliosacral screw into the S1 body. A supplemental long iliosacral screw was placed into the S1 body in five specimens. A supplemental transsacral S1 screw was placed in the other five. Each pelvis underwent 100,000 cycles at 250 N, followed by loading to failure. Vertical displacements at 25,000, 50,000, 75,000, and 100,000 cycles and failure force were recorded.p < 0.05) within each group with each increase in the number of cycles. However, there was no statistically significant difference between groups in displacement or load to failure.Vertical displacement increased significantly (Although intuitively a transsacral screw may seem to be better than a long iliosacral screw in conveying additional stability to an unstable sacral fracture fixation construct, we were not able to identify any biomechanical advantage of one method over the other.Does not apply—biomechanical study. Pelvic fractures account for 1–3 % of all skeletal fractures and comprise a broad spectrum of injuries: from low-energy fractures in osteoporotic patients to high-energy disruptions of the pelvic ring . Type C Many surgical techniques have been described for fixation of the posterior pelvic ring injury, with iliosacral screw fixation into the first sacral body being in common practice , 23–26. The purpose of this study was to biomechanically compare the effect of a supplemental S1 long iliosacral screw versus a transsacral screw in an unstable type C vertically oriented zone 2 sacral fracture model , 5.T score of ≤−1 [The right superior and inferior pubic rami were osteotomized in a vertical fashion using an oscillating surgical power saw with a thin blade.An ipsilateral vertical zone 2 sacral fracture was created by making a unilateral cut through the sacral neuroforamina using an oscillating surgical power saw with a thin blade.The ipsilateral sacrospinous and sacrotuberous ligaments were transected to ensure complete disruption of the sacroiliac complex.Ten embalmed cadaver pelves with intact, attached 4th and 5th lumbar vertebrae were harvested with ligamentous structures  and sacroiliac joint capsules kept intact. Using a GE Lunar Scanner , dual-emission X-ray absorptiometry was performed on each specimen. All specimens were osteopenic, with a e of ≤−1 . SubsequT-score values. The pelves in the long iliosacral and transsacral groups had T scores that were not significantly different, with means of −2.28 (range −1.4 to −3.4) and −2.38 (range −1.1 to −4.1), respectively . The osteotomized ipsilateral superior and inferior pubic rami were not fixed in any of the pelvis specimens. After fluoroscopic imaging confirmed appropriate sacral fracture reduction and screw placement  , 33. TheSubsequently, each pelvis was loaded at 250 N and cycled 100,000 times  at U test was used to compare bone density (as noted above), displacement, and load to failure of the two fixation groups. Freidman test was used for displacement comparisons within each group. The level of statistical significance was defined as p < 0.05.Statistics were calculated using SPSS software . The Mann–Whitney p < 0.05) within both groups (Tables All specimens completed 100,000 cycles with no gross evidence of construct failure. The progressive increase in displacement between each of these measured intervals was significantly different (s Tables . In the s Tables . After us Tables .Table 1Pp < 0.05.Despite the fact that mean displacement values for the long iliosacral group were almost twice that of the transsacral group, analysis of displacements at 25,000, 50,000, 75,000, and 100,000 cycles showed no significant difference between groups Table . In addiStabilization of posterior pelvic ring injuries with iliosacral screws inserted into the first sacral body is a commonly used technique , 39–41. Matta and Tornetta suggested that longer iliosacral screws might provide better fixation because they have greater resistance to toggle and are more resistant to vertical shear stress . However The purpose of our study was to assess the potential improvement of fixation using one of two alternative long-screw fixation options: a transsacral or a long iliosacral implant. In order to create an “extreme” condition, osteopenic/osteoporotic pelvic specimens were used, and the anterior fractures were not fixed. This allowed us to focus directly on the posterior fixation in a model at the greatest risk for fixation failure. In addition, to maximize vertical shear and minimize compression across the posterior pelvic arch, a single-limb-stance model was used .Our study has a number of limitations. First, despite the use of nonparametric statistics, our failure to show a difference between the two groups may be type 2 error due to the relatively small sample size and lack of sufficient statistical power. Our selection of five specimens in each group was based on the findings of Tabaie et al. . HoweverAlthough there have been a number of studies comparing standard iliosacral screws with longer screw constructs, we know of no study directly comparing these longer screw methods. Tornetta et al. described the concept of different modes of failure , reportiAlthough intuitively a transsacral screw may seem to be more advantageous than a long iliosacral screw in conveying additional stability to a type C, zone 2, vertically unstable sacral fracture fixation construct, we were not able to identify any biomechanical advantage of one fixation method over the other. Further study with a larger number of samples may be required to more accurately compare screw configuration in these injuries.


Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown.Monitoring nucleotide exchange of Ras in permeabilized cells we find, unexpectedly, that the decline of growth factor-induced Ras-GTP levels proceeds in the presence of unabated high nucleotide exchange, pointing to GAP activation as a major mechanism of signal termination. Experiments with non-hydrolysable GTP analogues and mathematical modeling confirmed and rationalized the presence of high GAP activity as Ras-GTP levels decline in a background of high nucleotide exchange. Using pharmacological and genetic approaches we document a raised activity of the neurofibromatosis type I tumor suppressor Ras-GAP neurofibromin and an involvement of Rsk1 and Rsk2 in the down-regulation of Ras-GTP levels.Our findings show that, in addition to feedback inhibition of Sos, feedback stimulation of the RasGAP neurofibromin enforces termination of the Ras signal in the context of growth-factor signaling. These findings ascribe a precise role to neurofibromin in growth factor-dependent control of Ras activity and illustrate how, by engaging Ras-GAP activity, mitogen-challenged cells play safe to ensure a timely termination of the Ras signal irrespectively of the reigning rate of nucleotide exchange.The online version of this article (doi:10.1186/s12964-016-0128-z) contains supplementary material, which is available to authorized users. Ras activation in response to growth factor stimulation is a central mitogenic signaling pathway. Extensive work has shown that the duration of the Ras signal is a key determinant of cell fate in the sense that growth factor activation of Ras must be transient to promote a proper proliferative response. It is well established that growth factors stimulate the nucleotide exchange factor (GEF) Sos to promote Ras activation via Ras-GTP loading but it is not known how Ras activation is terminated to ensure a short-lived signal. We document here a new mechanism for Ras signal termination, namely activation of a RasGAP activity in the context of a feedback signal propagated via Rsk1 and Rsk2, the kinase mutated in Coffin-Lowry syndrome. We provide evidence that neurofibromin, the product of the tumor suppressor of neurofibromatosis type 1 (NF1), is the RasGAP species mediating the deactivation of Ras. In summary our findings disclose a positive feedback loop leading to the stimulation of neurofibromin as a mechanism that restricts the duration of growth factor-induced Ras activation.Cells emerging from quiescence upon growth factor encounter feature a pronounced activation of Ras which is characteristically short-lived. Extensive work has elucidated that the duration of the signal elicited by Ras is decisive for cell fate decision taking. For example, seminal studies in PC12 phaechromocytoma cells illustrate that the duration of the signal provided by Ras and its downstream effector kinase Erk is the key event determining whether these cells will enter the cell cycle or cease proliferation and differentiate in response to a given stimulus . Accordideactivation. Current models invoke feedback inhibition of Sos as a critical step GTP leads to the time-dependent incorporation of radioactivity into Ras-immunoprecipitates (IPs). Radioactivity was specifically associated to Ras because it required the input of the permeabilizing agent Streptolysin O (SLO) and the Ras-IP antibody and was chased off by excess GTP  in HeLa Fig.  or MEF cion Fig. , provingTo collect more evidence we combined the permeabilization assay with the Ras-GTP affinity pulldown. We reasoned that Ras-GTP loading driven by the uptake of the non-hydrolyzable GTP analogue GppNHp should accurately mirror Sos activity, because Ras-GppNHp is insensitive to GAP action. HeLa cells were permeabilized in the presence of GTP or GppNHp before or after EGF administration followed by the analysis of Ras activation Fig. . As expeNumerous studies have described a feedback loop acting via the Ras/Raf/MEK/Erk pathway through Sos inhibition to terminate Ras activation –18, 43. To determine the signal path downstream of MEK, we inhibited Erk and Rsk, two downstream kinases that reportedly mediate feedback inhibition of the pathway , 26, 29.These findings evidenced that a negative feedback operating via Erk and Rsk1/2 mediates Ras deactivation. Since we did not observe an ostensible reduction in GEF activity during the decline of Ras-GTP levels Fig.  the feedIn order to identify the Ras-GAP species involved in Ras deactivation we performed single knockdowns of RASA1 Fig. , DAB2IP In 2004 Markevich et al. predicted on purely theoretical grounds that Sos downregulation was insufficient to effectively deactivate Ras  but manyFigure Extensive work suggests that the life-time of Ras activation has dramatic consequences on cell fate. In most investigated settings prolonged activation of the Ras/Erk pathway prohibits proliferation and shifts the balance to differentiation , 2, 56. Inactivating mutations in Rsk2 are causative of the Coffin-Lowry syndrome . The invThis study shows that transient Ras activation in response to growth factors is ensured by the sequential stimulation of Sos and the ensuing activation of the tumor suppressor Ras-GAP protein neurofibromin in the context of a feedback mechanism that involves also Rsk1/2. Hence, transient Ras signal termination does not result solely from feedback inhibition of Sos-driven nucleotide exchange but involves additionally feedback stimulation of neurofibromin RasGAP activity. This model of Ras activation represents a new paradigm and assigns for the first a time a precise role to neurofibromin in growth-factor-dependent control of Ras activity.-/-, N-Ras-/-, K-Raslox/lox MEFs  and Human Embryonic Kidney 293 (HEK293T) cells were cultured at 37 °C and 5 % CO2 atmosphere in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10 % (v/v) fetal calf serum. Inhibitor treatments: all inhibitors were applied for 30 min at the following concentrations: U0126 (10 μM), FR108204 (50 μM) BI-D1870 (10 μM), Wortmannin (100 nM). EGF was added at a final concentration of 10 ng/ml.Cervical cancer (HeLa) cells, Mouse embryonic fibroblasts (MEF), H-RasStreptolysin O was purchased from AaltoBio Reagents . Glutathione–Sepharose and all nucleotides were from JenaBioscience . [α-32P]GTP  was from Hartmann Analytic . GammaBind–Sepharose was purchased from Amersham Biosciences . U0126 and BI-D1870 were purchased from Enzo Life Science . The Erk1 and Erk2 selective inhibitor FR108204  was fromAntibodies were obtained from the following sources: SOS1 (clone 25/SOS1), p120RasGAP (clone 13/RAS-GAP), MEK1 (no. 610121), MEK2 (no. 610235) were from BD Transduction Laboratories; K-Ras F234 (sc-30), N-Ras F155 (sc-31), pan-Ras C-4 (sc-166691), p-ERK1/2(Y204) (sc-101761), and Neurofibromin (sc-67) from Santa Cruz Biotechnology ; Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) (no. 2325), p44/42 MAPK (ERK1/2) (no. 4695), Akt (no. 9272), p-Akt(S473) (no. 4060), EGFR (no. 4267), p-EGFR (Y1068) (no. 2236), Phospho-p90RSK (Ser380) (no. 9341), RSK1/RSK2/RSK3 (32D7) (no. 9355), p-GSK-3β (Ser9) (D85E12) (no. 5558); GSK-3β (27C10) (no. 9315), Phospho-S6 Ribosomal Protein (Ser235/236) (no. 2211), S6 Ribosomal Protein (5G10) (no .2217), p70 S6 Kinase (49D7) (no. 2708) were from Cell Signaling Technology . Anti-DAB2IP (ab87811) was from Abcam . Y13-259 rat monoclonal anti-Ras IP-antibody was purified from hybridoma supernatant .Total RNA isolation and purification was performed using an RNA isolation kit from Macherey-Nagel  according to the manufacturer’s protocol. cDNA synthesis was performed using First Strand cDNA Synthesis Kit  with 50 ng/μl of total RNA per sample and Oligo-dT-based priming. QRT-PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (2X)  using primers for all four RSK isoforms reported in . RelativGFP-neurofibromin (type 1 isoform) was cloned in pCDH-EF1a-EGFP-C2-IRES-Puro, a customized vector based on the parental vector pCDH-EF1-EGFP-C2-IRES-Puro from System Biosciences, with expression driven by the EF1a promoter. Cloning details will be presented elsewhere. An expression construct for HA-tagged RASA1  was kindE. coli by standard procedures). GDP and GST-RBD were included in the lysis buffer to quench post-lytic GTP-loading and GAP-dependent Ras-bound GTP hydrolysis, respectively. Cell material was scraped off and lysates were cleared by centrifugation. GST-Raf-1-RBD/Ras-GTP complexes were collected on glutathione-sepharose (30 min at 4 °C on a rotating wheel), washed once with 750 μl lysis buffer lacking GDP and GST–Raf-1-RBD and processed for Western Blotting.Cells seeded in 6 well plates were deprived of serum overnight, challenged or treated as appropriate and lyzed in 0.5 ml ice-cold lysis solution  supplemented with protease and phosphatase inhibitors, 100 μM GDP and 25 μg/ml soluble recombinant GST-RBD  supplemented with freshly thawed 15 unit/ml SLO and 9 MBq [α-32P]GTP. For treated cells, this solution was supplemented with the relevant drug. Kinetics were started at this point and reactions were quenched by aspirating the solution and lysing cells in 1 ml/well ice-cold lysis buffer  supplemented with 2,5 μg/ml Y13-259 Ras-antibody for IP. Cells were scraped off and extracts were placed on ice. Lysates were cleared by centrifugation and supernatants were made up to 500 mM NaCl, 0.5 %sodium deoxycholate and 0.05 % SDS. Immunocomplexes were collected on GammaBind–Sepharose by 45 min incubation at 4 °C under rotation. After six rounds of washing with 1 ml of ice-cold washing solution , immunoprecipitates were subjected to Cerenkov counting. Ras nucleotides were eluted from the same samples and analyzed by Thin Layer Chromatography [Cell permeabilization was performed essentially as described previously . Serum-stography . GDP andThe cervical carcinoma HeLa cells gene expression datasets reporting on the transcriptome of EGF-stimulated HeLa cells  were ideLive-cell imaging was performed on a Zeiss LSM 510 axiovert confocal microscope equipped with a thermostated stage chamber  as previously described . BrieflyA toy ODE model was built for two alternative network structures that represent regulation through GEF only Fig. , or GAP The data sets supporting the results of this article are included within the article and its additional file(s).


Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance.  Leishmania genus. In Brazil, the infection is caused by L. (L.) infantum chagasi. Human VL is the most severe form of the disease and is lethal if not treated early after the onset of clinic and pathological signs  / Km + [S] + Km[I] / Ki),, where vi is the initial rate in the presence of the inhibitor, v0 is the initial rate in the absence of the inhibitor, Km is the Michaelis constant for inosine, [S] and [I] are inosine and inhibitor concentrations, respectively, and Ki is the inhibition constant.The inhibition of NH36 activity by each one of the eight immucillins  was condL. (L.) infantum chagasi and L. (L.) amazonensis (5 × 104 viable promastigotes/mL) were incubated with 0.05, 0.1, 1.0, 5.0, 10, 25, 50, 250 and 500 μM of each immucillin and, when needed, also with 0.01, 0.1, 0.5, 1, 5, 10, 25 and 50 nM in FBS supplemented Schneider’s insect medium. Promastigotes were recorded in a Neubauer chamber after 24, 48, 72 and 96 hours of incubation at 26°C. The 50% inhibitory concentration (IC50) was evaluated after 48 hours [A + [(B-CA) x ((N control/2)-NA))/ (NA-NB)], where C is concentration, N is the number of promastigotes and A and B are the points between which 50% of inhibition occurs. Alternatively, we assessed the inhibitory effect of IA, IH and SMIH on L. (L.) infantum chagasi growth in a daily addition protocol , and of IA using the chemically defined RPMI liquid medium (Sigma Co.) which lacks nucleotides, nucleosides or nucleobases. We also assayed the possibility of reversing the inhibitory effect of IA in RPMI medium by the addition of adenosine to the culture medium. The concentration of adenosine in Schneider´s insect serum supplemented medium was assessed by HPLC using a C-18 reverse-phase column as described [Stock solutions (20 mM) of immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG) SerMe-ImmG and SerMe-ImmA  were pre48 hours , 33 by lescribed .L. (L.) infantum chagasi were incubated in the absence or presence of the immucillins IA, IH and SMIH at a 10 μM concentration, for 72 h, fixed in 2.5% glutaraldehyde, formaldehyde 4% in 0.1 M cacodylate buffer, pH 7.2, washed with cacodylate buffer, and post fixed for 1 h in 0.1 M cacodylate buffer containing 1% osmium tetroxide and 1.25% potassium ferrocyanide. The cells were washed with Milli Q water, dehydrated in increasing concentration of acetone and embedded in Epon (1: 1) (PolyBed 812). The ultrathin sections were obtained, contrasted with 5% uranyl acetate and lead citrate and observed under a Joel 1200 electron microscope.Promastigotes of L. (L.) infantum chagasi during 2 h, washed, incubated for additional 24 h and treated with a single 10 μM dose of IA, IH and SMIH (0 h), or with daily doses added at 0, 24 and 48h. The amastigote growth was assessed 24 h after the last drug addition, on 200 cells in Giemsa stained coverslips and expressed as phagocytic index = percent of infected macrophages x average number of intracellular amastigotes. The effective concentration of drugs at which intracellular amastigote replication is inhibited by 50 percent (EC50) was determined by linear interpolation, after addition of the drugs (0.5–10 μM) at 0h and incubation for 24 h. All experiments with mice were reviewed and approved by the Animal Care and Use Committee of the Instituto de Biofísica Carlos Chagas Fo.-UFRJ  and were performed according to the guidelines of the National Institutes of Health, USA. We made all efforts in order to minimize animal suffering.Peritoneal macrophages  were obtFor comparison of means we used the non-parametrical Kruskall Wallis and Mann Whitney tests and IC95% (GraphPad Prism6 program)L. (L.) donovani demonstrates the characteristics expected for nonspecific nucleoside hydrolases (Leishmania (L.) donovani is therefore a nonspecific nucleoside hydrolase.The nucleoside hydrolase of drolases , includiL. (L.) infantum chagasi and L. (L.) amazonensis  infantum chagasi, while SMIA induced stronger inhibition on L. (L.) amazonensis. Furthermore, DIA was active against L. (L.) infantum chagasi (L. (L.) amazonensis. Only SMIH reached its IC50 on L. (L.) infantum chagasi and L. (L.) amazonesis promastigotes, at nanomolar concentration (L. (L.) infantum chagasi promastigote growth was 78% for IA, 69% for DIA, 89% for DIH, 99% for IH, 68% for DIG, 70% for SerMe-ImmG and 71% for SerMe-ImmA. SMIH induced 99% of inhibition at 50 nM concentration (L. (L.) amazonensis replication was 76% for IA, 16% for DIA, 89% for DIH, 71% for IH, 67% for DIG and 71% for Serme-ImmH and for SerMe-ImmA while SMIH induced a 61% of growth inhibition at 50 nM.All parasite growth inhibitions were determined in a time- and dose-dependent manner Figs  and 3. Ssis Figs . DIH, IH chagasi  but not ntration  while DIntration . After 9ntration . Also atin vitro were IA and IH . In spite of the low IC50 values for SMIH, DIH, DIG and SerMe-ImmG on both Leishmania species, their relatively high Ki values (1.80 or >5 μM) indicate the inhibition of growth is not related to inhibition of NH36 enzymatic activity. Finally, the DIA (DADMe-ImmA) and SerMe-ImmA, which did not impair the parasite growth at low concentrations, also did not inhibit the NH36 function (Ki >5 μM)  .L. (L.) infantum chagasi promastigote growth after the successive addition of the drugs at 0, 24 and 48 h  and SMIH (most potent inhibitor of promastigote replication) on and 48 h . Using tand 48 h , the daiL. (L.) infantum chagasi in the chemically defined RPMI medium, which lacks nucleosides or nucleobases  infantum chagasi promastigotes, by transmission electronic microscopy. We also assessed the impact effect of IA, IH and SMIH, at 10 μM concentration, on the ultrastructure of in vitro culture with IA, IH or SMIH at a 10 μM concentration. We used 10 μM of Glucantime as control. We show the percent of viable macrophages, after in vitro culture for 48 h, with immucillins added at 0 h (L. (L.) infantum chagasi followed by treatment with the immucillins. When compared to untreated controls, a 74% reduction in the number of intracellular amastigotes was detected in macrophages treated with IA at 0h  donovani, cloned in the pET-22 expression vector, was described before by Cui et al. [kcat = 9.5 s-1) and inosine (kcat = 7.6 s-1), respectively [L. (L.) major [L. (L.) donovani, shows an 11 fold decreased catalytic turnover for inosine (kcat = 119 s-1), similar levels for uridine (kcat = 32 s-1) and 6 and 36 fold increased catalytic rates for adenosine (kcat = 0.57) and cytidine (kcat = 0.36), respectively [Leishmania (L.) donovani NH36 displays a wider range of activity on naturally occurring nucleosides.The nucleoside hydrolase from i et al.  as a nonectively . When co.) major , also anectively . This coin vitro multiplication of promastigotes of L. (L.) infantum chagasi and L. (L.) amazonensis induced by micromolar concentrations of IA, IH, SerMe-ImmG [L. (L.) amazonensis [L. (L.) amazonensis [L. (L.) donovani/ L. (L.) infantum complex [We described the inhibitory effect of immucillins on the rMe-ImmG  and DIG rMe-ImmG  and was zonensis , which ezonensis , to our  complex –31, 39.We demonstrated the inhibitory effect of immucillins on promastigote replication in Schneider’s supplemented insect medium, which contains serum in its composition. Inhibition of multiplication was obtained, in spite the presence of adenosine, a favored substrate of NH36. Other purines or pyrimidines are probably also present in this complex medium. Additionally, IA was able to inhibit promastigote replication in the RPMI medium which completely lacks nucleobases, nucleotides or nucleosides, and this inhibition was bypassed by the addition of adenosine, suggesting that immucillin IA might act on the parasite nucleoside hydrolase activity.L. (L.) infantum chagasi promastigotes with IA, IH and SMIH induced alterations in vital parasite structures that suggest irreparable metabolic damage that could lead to the parasite death.In the sand fly vector, promastigotes inhabit the insect gut and transmit the initial infection. In the host, amastigotes replicate inside the macrophages maintaining the infection, causing the disease and are the real target of chemotherapy. Therefore, the inhibition of replication of intracellular amastigotes is the most important target for chemotherapeutic purposes. Our results show strong chemotherapeutic effects of IA, IH and SMIH, that decrease the intracellular amastigote parasite load by 74–95% when used at low micromolar concentrations. Treatments of Leishmania to survive inside insect or mammalian hosts depends of its capability to sense and adapt to environmental changes [L. (L.) donovani can survive in a purine deprived media for three months because it acquires purine-transporters and surface enzymes that undergo “up-regulation”, as a metabolic reprogramming during purine starvation [The ability of  changes . L. (L.)arvation . Our resL. (L.) infantum chagasi at 1000 fold greater concentrations (0.43–5.76 mM) and showed even higher values against Leishmania strains isolated from dogs (19.97–32.27 mM) [The inhibitory effect of immucillins in the context of leishmaniasis is impressive considering that Glucantime, the recommended standard drug, reached the IC50 for human strains of 2.27 mM) .Leishmania parasite stage responsible for the development of leishmaniasis in humans, rodents and dogs and are therefore the important drug target. While a 74% inhibition of intracellular multiplication was achieved using a single IA 10 μM dose, the daily addition of IA, IH and SMIH was extremely effective, reducing by 95% the parasite load. Using a single dose, the treatment with IA showed the highest potency while no significant differences were found between IH, SMIH and Glucantime, which gave 70% growth inhibition. An important physiological finding is that either a single dose or the daily addition of three doses of each drug at 10 μM did not cause any decrease in macrophage viability but did give potential anti-parasite activity. Thus, the result indicates a new potential use of immucillins in therapy of leishmaniasis. Leishmania therapy. A recent study on the effect of Palladacycle complex on L. (L.) amazonensis replication showed that this drug destroyed axenic promastigotes and intracellular amastigotes in vitro at nanomolar concentrations while displaying 10-fold less toxicity than Glucantime to macrophages [A crucial result of this investigation is the characterization of the inhibitory effect of immucillins on the amastigote replication inside the macrophages of a mammalian host. Amastigotes are the rophages .L. (L.) infantum chagasi and 62.58 μM to inhibit amastigotes of L. (L.) amazonensis. Morais-Teixeira et al. [L. (L.) infantum chagasi. The efficacies of immucillins are more compatible to the ones described for Miltefosine (4.4 μM) and Amphotericin (0.04 μM) also against amastigotes of L. (L.) infantum chagasi [L. (L.) amazonensis intracellular amastigotes [Leishmania infantum chagasi at a 31.1 μM Glucantime concentration.Our results gain impact if compared to other reports showing that much higher concentrations of the presently commercial available drugs, of known toxicity, are required in order to achieve the IC50 against intracellular amastigotes. Morais-Teixeira et al.,  describea et al.  reported chagasi . Similarstigotes . Ramirezstigotes , on the L. (L.) major, Crithidia fasciculata and T. brucei brucei [Leishmania replication. Other immucillins assayed in this investigation were developed as inhibitory ligands of purine nucleoside phosphorylases [Leishmania parasites. Therefore, this study provides the first evidence of in vitro studies on the potential use of immucillins in leishmaniasis therapy.In the present study, IA showed a slight advantage in therapeutic efficacy above that of IH in inhibition of promastigote and amastigote replication. Although the immucillin IA was first described as an inhibitor of the nucleoside hydrolases of i brucei , it was orylases , 28 and Plasmodium parasites. Our study expands the potential use of this inhibitor family to the potential therapeutic efficacy and low toxicity against visceral leishmaniasis. Although additional in vivo studies are needed, to elucidate the impact of Forodesine on T and B cells of patients with visceral leishmaniasis, our results are encouraging considering that IH (Forodesine) is orally available, had already undergone safety trials in humans, and thereby shortens the time required until trials could begin for use against leishmaniasis.IH (Forodesine) and DADMe-ImmH (Ulodesine) are potent inhibitors of human PNP that have been used in human clinical trials against leukemia and gout . Low nucTaken as a whole, these results are promising, considering the increased incidence of human VL cases caused by parasites resistant to Glucantime  and the The development of new drugs to treat leishmaniasis is urgent, mainly because the currently existing drugs were developed for other indications, have invasive routes of administration, and have many adverse effects and therapeutic unresponsiveness. The immucillins should be the target of therapeutic studies, since it is reasonable to assume that effects seen at the cellular level might be recapitulated in humans.S1 Fig4K2, 300 mM Na Cl, pH: 8.0) (EF), 10 μL of the fractions obtained with elution buffer containing 50–300 mM imidazol, 10 μL of whole supernatant and 10 μL molecular weight standards (M). Elution of the purified NH36 is starts with 150 mM (*).Electrophoresis was performed in 12% polyacrylamide gels stained with Commassie blue. In (A) from left to right: BioRad molecular weight standards  (Mw), 10 μL of bacterial supernatant loaded in the column (BS), 10 μL of the fraction eluted with elution buffer 50 (mM PO(TIF)Click here for additional data file.S2 Fig2PO4, 4mM tetra-n-butylammonium bromide (TBAB), 10% methanol solution, pH 6.0, and injected into a C-18 reverse-phase column  coupled to an LC10AS-HPLC model (Shimadzu) through a 50-mL loop. Adenosine was separated using a 1ml/min flow rate  and its concentration was calculated by peak integration in comparison with the standard concentration of adenosine.(A) An aliquot of 300 μl of Schneider´s insect medium supplemented with FBS was concentrated to dryness, resuspended in 0.1mL of a 50mM KH(TIF)Click here for additional data file.


Infectious bronchitis virus (IBV), a major pathogen of commercial poultry flocks, circulates in the form of several serotypes/genotypes. Only a few amino-acid changes in the S1 subunit of wild-type IBVS proteins may result in mutants unaffected by current vaccines.Partial S1 gene sequences of 3 IBV isolates of the Moroccan Italy 02 genotype from vaccinated and unvaccinated broiler chicken flocks, located in southern and central regions of Morocco, were amplified by RT-PCR, sequenced, and aligned for phylogenetic and amino-acid similarity analyses.The three isolates were found genetically highly distant from known avian IBV based on partial sequences of their S1 genes: gammaCoV/chicken/Morocco/I01/2011(IBV/Morocco/01), gammaCoV/chicken/Morocco/I30/2010 (IBV/Morocco/30), and gammaCoV/chicken/Morocco/I38/2013 (IBV/Morocco/38), nucleotide sequence identities reached 89.5 % to 90.9 % among the three isolates. The deduced protein sequence identities ranged from 29.7 % (between IBV/Morocco/38 and Egypt SCU-14/2013-1) to 78.2 % (between IBV/Morocco/01 and Spain/05/866). Amino acid sequence comparison and phylogenetic analysis indicated the emergence of a new Moroccan genotype, clustering with regionally related isolates from Spain (Spain/05/866) and belonging to a new sub-genotype.Our sequencing results demonstrate a co-circulation of wild-type infectious bronchitis viruses in broiler chickens. These results justify permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the evolving field situation. Infectious bronchitis (IB) is one of the most contagious diseases that affect poultry worldwide and is responsible for severe economic losses. Clinically, the disease causes respiratory distress, drop in egg production and quality in layers. Some strains of IBVare associated with nephritis –3.Avian coronavirus, genus Gammacoronavirus  [Infectious bronchitis virus (IBV) is a member of the species virinae) . IBV hasvirinae) , 6. S1 mvirinae) , 8. Hencvirinae) , 10.Different IBV variants are distributed globally. Some of these variant are endemic only in particular regions, while others circulate worldwide . More thThe first isolation and characterization of IBV from poultry flocks in Morocco was reported by El-Houadfi et al. in 1986 . Six iso) under the objective of promoting poultry product export in Africa, including chicks, hatching eggs, and compound feed for poultry. In 2012, 6.7 million hatching eggs and 1.9-million–d-old chicks were exported to Mauritania, Mali, Cameroon, Côte d'Ivoire, and Senegal, and 25,000 tons of compound feed were exported to Mauritania. Breeder chicks  were imported in January 2014 (an increase of 28 % compared to 2013). However, imports of layer and broiler breeder and turkey poults declined in recent years, mainly due the development of local production.The Moroccan poultry industry has developed significantly under the framework "Plan Green Morocco" and meets the growing domestic consumer demand for poultry products, including meat and eggs The industry generates a turnover of around 30 billion dirhams/year, totaling mean investments of around 8.7 billion dirhams/year. The official data regarding the general poultry rearing systems in Morocco are: broiler production, 450 million/year ; layers: 20 million/year (250 farms); turkeys: 12 million/year (25 % imported d-old chicks); broiler breeders: 3.2 million/year (70 % imported from Spain and 30 % from other European countries); 48 hatcheries. The Interprofessional Federation of Poultry Sector (FISA) aims to develop exports of poultry products in particular to Northern and Western Africa. FISA is affiliated with Association of Moroccan Exporters , whereas 793B (4/91 and CR88 strains) and Arkansas vaccines were introduced in 2000 and 2013, respectively. New IBV serotypes and genotypes can emerge as a result of only very few changes in the amino acid sequence of the S protein. Vaccination programs largely rely on the use of the IBV Massachusetts strain, which is also the most commonly used IBV vaccine strain in Morocco. However, despite the use of this vaccine, the common presences of IB in vaccinated chickens continue to have a major negative economic impact . In thisInfectious bronchitis was diagnosed between January 2010 and December 2013 in southern and central regions of Morocco. Suspicious IBV infections (prominent respiratory disease) were found in broiler chickens from 47 commercial farms. The flocks had been vaccinated against Newcastle disease and infectious bursal disease  3 of their S1 regions (nucleotides 700–1095). Of those isolates, three isolates, classified as the Moroccan Italy 02 genotype, underwent further sequencing to include all three hypervariable S1regions . These three IBV strains were isolated from trachea, lungs, and kidneys of broiler chickens. Between the first and fourth passage, virus propagation from the three suspect samples in specific pathogen-free eggs resulted in characteristic IBV lesions in 90 %–100 % of the embryos. Results of both RT-PCR targeting the IBV N gene and RT-PCR using primers amplifying a partial sequence of the IBV S1 gene were positive, confirming the identity of the three isolates. The amplified partial S1 fragments (approximately 700 bp in length) of the three isolates were sequenced. The sequences were submitted to GenBank and assigned the accession numbers KJ701019, KJ701020 and KM594187 for isolates gammaCoV/chicken/IBV/Morocco/I38/2013 (IBV/Morocco/38), gammaCoV/chicken/IBV/Morocco/I30/2013 (IBV/Morocco/30), and gammaCoV/chicken/IBV/Morocco/I01/2013 (IBV/Morocco/01), respectively  to 90.9 %  when compared to each otherand from 12.9 % (between IBV/Morocco/38 and Egypt SCU-14/2013-1)to 76.8 % ,  when comThe partial S1 protein sequences of the Moroccan isolates were 69.4 % to 76.8 % homologous to those from genotype Italy-02  but were <66.5 % homologous to the remaining reference strains .Phylogenetic analyses comparing the partial S1 gene sequences of the three Moroccan field isolates with those of reference and variant strain sequences revealed that the Moroccan isolates were 69.4 % to 76.8 % homologous with Spanish isolates of the Italian 02 genotype . This siThe deduced amino-acid sequences of Moroccan IBV isolate proteins demonstrated that the Moroccan isolates represent a unique genotype compared too ther reference strains of various countries. These Moroccan isolates are only distantly related to African, European, and American strains. The origin of this novel genotype remains to be identified. In addition to population diversity –22, wideAlignment of the partial S1 glycoprotein sequence of Moroccan IBV isolates with thatof published non-Moroccan IBV strain, H120, identified most sequence variations between residues 62–77 and 117–131  , 27–29. In the phylogenetic tree shown in Fig. This molecular study has revealed that a new genotype can emerge as a result of only a few changes in the amino acid composition in the S1subunit of the virus Sprotein . This isOur data provide evidence of a new emerging genotype in Morocco. Three IBV field isolates were different from published referenced strains in nucleotide and amino acid sequences and clustered into a separate branch based onphylogenetic analysis. All Moroccan IBV variants presented specific nucleotide and amino acid sequence insertions in the S1 gene in comparison to the Massachusetts vaccine strain that might be associated with the occurrence of clinical disease in vaccinated flocks. Further investigation related to cross protection and pathogenicity of this new genotype detected in this country are needed.Tissue samples from trachea, lungs, and kidneys were obtained from 10 birds from each flock, placed on ice, and transferred to the laboratory in sterile transport medium containing 5 % antibiotics . These samples were used for virus isolation.g) for 15 min, and stored at −80 °C. The remaining eggs were further incubated for 5 more d and observed for typical embryonic lesions of IBV infection, such as dwarfing, stunting, and curling. Stored allantoic fluids were further passaged three times in specific pathogen-free eggs; the allantoic fluids were harvested and tested for the presence of IBV using rRT-PCR.Virus isolation was performed by inoculation of 9–11-d-old, embryonated, specific pathogen-free hen’s eggs with 200 μl of 10 % tissue homogenates. Eggs were incubated at 37 °C and candled daily. Embryos that died within 24 h after inoculation were discarded. On d 2 after inoculation, three eggs were chilled at 4 °C for 2 h, and the allantoic fluids were harvested, centrifuged at 8,000 rpm  according to the manufacturer’s instructions. Allantoic fluid samples and reagents were dispensed into X-well microplates prior to magnetic particle processing. After processing, each sample of extracted RNA recovered from the wells of the plates was subjected toRT-PCR amplification.4 (50 mmol/l), 0.5 μl of Rox reference dye , 0.5 μl of Moloneymurine leukemia virus reverse transcriptase (200 U/μl), 0.5 μlof Taq DNA polymerase (Life Technologies), 0.5 μl of primers (0.2 μmol/l), 0.25 μl of probe (0.1 μmol/l), and 5 μl of RNA template to make a final volume of 25 μl.The reaction was carried out using a StepOne Plus real-time PCR system  at 50 °C for 15 min, 95 °C for 5 min, and followed by 40 cycles of 9 °C for 15 s; 60 °C for 45 s; 72 °C for 30 s, and a final extension step of 74 °C for 5 min. Amplifications were recorded and analyzed, and the threshold cycle (Ct) was determined with StepOne software (Smart Cycler).The oligonucleotides and the probe used for initial screening of IBV were described previously . These o2 (25 mmol/l), 2.5 μl of dNTP (10 mmol/l), 0.75 μl of each primer (10 μmol/l), 10.2 μl of sterilized water, 0.5 μl of RNAase inhibitor (20 U/μl), 0.3 μl of RT (50 U/μl), and 0.5 μl of Gold Taq polymerase (5 U/μl). The PCR products were analyzed on a 1 % agarose gel.Amplification of partialS1 gene sequences by RT-PCR was performed using the forward S1S15’ mod (5’-TGA-AAA-CTG-AAC-AAA-AGA-3’) and reverse S1CK2 (5’-CNG-TRT-TRT-AYT-GRC-A-3’) primers . RT-PCR Partial RT-PCR products of the S1 gene (700 bp) containing HVR1, HVR2, and HVR3 were purified using the Gene Clean Kit  according to the manufacturer’s instructions. Purified PCR products were used as templates for sequencing using the BigDye® Terminator v1.1 Cycle Sequencing Kit (Life Technologies). The second purification step was performed using the Big Dye XTerminator Purification Kit (Life Technologies). Purified PCR products were sequenced from both directions using the same primers (S15’ mod and CK2).http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for sequence comparison. Nucleotide sequence and deduced amino acid sequences were aligned using ClustalW and MEGA software Version 6.0 [Assembly and analysis of sequence data were conducted using the BioEdit Software version 5.0.9 . The opesion 6.0 . PhylogeArk/C6d (EU283056); D274 (X15832); Egypt/Beni-Suef/01 (JX174183); Egypt/SCU-14/2013-1 (KF731612); H120 (M21970); Italy-02 (AJ457137); Italy It/497/02-1 (DQ901377); Ma5 (AY561713); QX (AF193423); Spain/04/22-1 (DQ386100); Tunisia TN296/07 (FJ716133); Tunisia TN556/07 (FJ716132); Spain/05/866 (DQ386102); Spain/04/221 (DQ386103); Isolate Q1 (AF286302); SARS (D14056); Strain 04/91 (AF093794);NGA/A116E7/2006 (FN430415).


Herein, we demonstrate that EcBFR mineralization depends on three aromatic residues near the diiron site, Tyr25, Tyr58, and Trp133, and that a transient radical is formed on Tyr25. The data indicate that the aromatic residues, together with a previously identified inner surface iron site, promote mineralization by ensuring the simultaneous delivery of two electrons, derived from Fe2+ oxidation in the BFR cavity, to the di-ferric catalytic site for safe reduction of O2.Ferritins are iron storage proteins that overcome the problems of toxicity and poor bioavailability of iron by catalyzing iron oxidation and mineralization through the activity of a diiron ferroxidase site. Unlike in other ferritins, the oxidized di-Fe Prokaryotic and plant ferritins are composed exclusively of H-chain-like subunits. The H-chain ferroxidase sites of animal ferritins play a central role in the mineralization process, which involves binding of Fe2+, oxidation to Fe3+, and labilization/hydration to generate the final mineral form in the cavity. The  The 3] Tlization., 4 This ron site. The modeentified. Disrupti2+. Site-directed variants, in which each residue was substituted with Phe, were generated and the mineralization activity of the variant proteins was determined. Figure 2+ ions per protein. Substitutions of Tyr25, Tyr58, and Trp133 resulted in significantly slower mineralization, with rates of 15 % (Y25F), 30 % (Y58F), and 25 % (W133F) relative to wild type BFR  that are ∼4.0–6.5 Å away from the nearest iron, and by a Trp residue (Trp133), which is ∼9.7 Å away from site FeB Figure . BFR alsR Figure . Y45F exR Figure .2+ at the ferroxidase site. Stopped-flow absorbance spectroscopy was used to monitor changes following the aerobic addition of Fe2+ to EcBFR and variants. In all of these cases, the variants were very similar to wild type BFR  in each variant, as observed for the wild type protein. demo demo2+ aR Figure  showed c protein.2+ at the ferroxidase site, Tyr25, Tyr58, and Trp133 must have important functional role(s) in mineralization only after the initial formation of the di-ferric ferroxidase site. We note that mineralization was not entirely abolished in the Y25F, Y58F, and W133F variants. Ferritins catalyze Fe2+ oxidation, a thermodynamically favorable reaction, and so inhibition of ferritin function through the substitution of a functionally important residue can never result in complete loss of Fe2+ oxidation.X-ray crystal structures were determined for the Y45F, Y114F, and Y149F variants  and the sample was frozen after ∼10 s. The resulting EPR spectrum contained a signal in the g=2 region that was not present prior to the addition of Fe2+ , a potent inhibitor of mineralization that binds tightly at the ferroxidase sites, did 2+ oxidation when no further Fe2+ is available to quench the remaining Tyr radicals. During mineralization, when Fe2+ is available, quenching of the radical would be expected to occur much more rapidly, at a rate similar to that of radical formation.EPR studies of the EcBFR variants revealed that Tyr25 is the site of radical formation; only in Y25F was the Tyr radical not observed Figure . This agThe inhibition of mineralization observed in the three near-ferroxidase site variants was very similar to that observed previously for inner surface site variants H46A and D50A, indicati3+) and reduced (di-Fe2+) states.[2+ in the central cavity, with resulting electrons channeled to the ferroxidase site causing reduction to the di-Fe2+ form, which is then primed to react again with O2 (or H2O2). Hydrolysis of the accumulating hydrated Fe3+ in the cavity leads to mineral formation. The importance of aromatic residues for mineralization, and the detection of a transient Tyr25 radical, provides important evidence about the origins of the two electrons required to re-reduce the ferroxidase site. We propose that one electron comes from the oxidation of the inner surface site Fe2+, while the other is from Tyr25, generating the radical. This is subsequently quenched by an electron that must be ultimately derived from a second Fe2+ ,[2+ ions are oxidized with the delivery of two electrons to the di-Fe3+ ferroxidase site. This enables near simultaneous arrival of the two electrons at the oxidized ferroxidase site  states., 5 Cycliom Fe2+), most like Figure , minimiz storage.–cE.coli FtnA,[Pyroccocus furiosus Ftn,[2+ oxidation in the related Dps ferritins, pointing to a wider significance of radical chemistry in iron storage/detoxification proteins.[2+.[P.furiosus Ftn, Tyr24 was found to be essential for the ferroxidase site oxidation reaction, and therefore mineralization,[E.coli FtnA, Tyr24 was found to be important for mineralization but not the initial ferroxidase site reaction,[Tyr25 is absolutely conserved in all 24 mer ferritins, and tyrosyl radical formation at this residue has been observed in other ferritins, including human H-chain, E.coli oli FtnA, and Pyroosus Ftn, and it iproteins. The roleeins.[2+. In P.fulization, while inreaction, as foundreaction,E.coli R2 numbering). The radical character is subsequently shuttled back and forth over >30 Å to the site of ribonucleotide reduction in the R1 subunit.[The R2 subunit of ribonucleotide reductase contains a diiron site closely related to that of BFR and generates a stable radical at the nearby Tyr122 ( subunit. The mech2+ in the cavity to the ferroxidase site, through a mechanism that involves transient formation of a Tyr25 radical. Several questions remain to be answered, particularly concerning formation and decay of the radical and the roles of Tyr58 and Trp133 in this, and the nature of the diiron species that oxidizes Tyr25.In summary, we have shown that three aromatic residues surrounding the EcBFR diiron site are crucial for mineralization, consistent with a role in electron transfer from Fe