Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process. The endothelial cells of capillaries and postcapillary venules form a semi-permeable barrier that is crucial for normal blood-tissue exchange and tissue homeostasis. Compromised endothelial barrier function which occurs during inflammation  significContinuous cytoskeleton maintenance is critical for normal endothelial barrier function ,8, and sRho family small GTPases strongly influence the actin cytoskeleton and have been shown to be important for controlling endothelial barrier integrity. Several studies have shown that RhoA activation correlates with increased permeability of the endothelium ,15,16. IFor this study we focused on the endothelial barrier enhancement elicited by S1P, an endogenously released, bioactive lipid that is a potent endothelial barrier enhancer at its physiological concentration ,23. Afte® electroporation kit and solution were obtained from Mirus Bio LLC . The pcDNA3-GFP-Rac1  and pcDNA3-GFP-Rac1T17N  plasmids were purchased from Cell Biolabs . Rac1 siRNA (Knockdown: UAAGGAGAUUGGUGCUGUA) and control RNA (Non-Targeting: UGGUUUACAUGUCGACUAA) were purchased from Thermo Scientific ; pTriEx-RhoA FLARE.sc Biosensor WT [Human umbilical vein endothelial cells (HUVEC), human adult dermal microvascular endothelial cells (HDMEC), Endothelial Growth Medium-2MV (EGM2-MV), and Endothelial Basal Medium (EBM) were all purchased from Lonza . The Ingenioensor WT  was a gi5 cells were resuspended in 100 μl electroporation master mix containing either 2 μg plasmids or 2 μM siRNA. This mixture was transferred to a cuvette for transfection using a Nucleofector II system  with program A-034 (HUVEC) or M-003 (HDMEC). Warm EGM2-MV (500 μl) was added into the cuvette immediately after electroporation. Cells were later distributed evenly onto gelatin-coated 35-mm dishes for protein extraction, gelatin-coated MatTek 35-mm #1 glass bottom dishes for time-lapse microscopy, or 96W1E ECIS arrays  for determination of barrier function.HUVEC and HDMEC were routinely grown in EGM2-MV in 1.5% gelatin-coated culture dishes. For all studies, passage 1–5 cells were used. For transfection, cells grown to 80% confluence were trypsinized and pelleted, and 5 X 10® Reducing agent containing NuPAGE® LDS sample buffer , heated at 70°C for 10 min, and loaded into Novex® 4–12% Bis-Tris precast gels  for SDS-PAGE. Proteins were transferred onto 0.45 μm PVDF membrane and blocked with 5% BSA in TBST . Membranes were incubated with primary antibodies at 4°C overnight, and washed three times in TBST. Afterwards, secondary antibodies were applied at room temperature for 1 h, followed by three washes with TBST. Bands were visualized with Supersignal® HRP substrate  and imaged with Bio-Rad Gel Doc™ system .Cell protein lysates were obtained as previously described ,28. ProtTransendothelial electrical resistance (TER), which serves as an index of barrier function of cultured endothelial cell monolayers, was determined with an Electrical Cell-Substrate Impedance Sensor (ECIS) ΖΘ System . Cells were seeded into gelatin-coated wells of ECIS arrays (96W1E) and allowed to attach overnight in EGM2-MV to form a confluent monolayer. The next day, medium was changed to EBM at least 1 h before the experiment. A 1-μA AC signal at 4 kHz was applied. Total impedance was reported by monitoring the voltage across the electrodes and its phase relative to the applied current. The cell-covered electrode unit was treated as an RC circuit, from which impedance data was later converted into monolayer resistance and capacitance, respectively representing barrier function and membrane capacitance .Cells were transfected with the pTriEx-RhoA FLARE.sc biosensor and seeded onto 1.5% gelatin coated MatTek 35 mm #1 glass bottom dishes , and grown overnight to confluence. The medium was changed to EBM 3 h before the experiment. Each MatTek plate containing cells was transferred to a temperature-controlled (37°C) imaging chamber. Time-lapse imaging data was acquired with a Leica SP2 confocal microscope with 63X objective at 30-s intervals, using the YFP and CFP channels  in the USF Lisa Muma Weitz Laboratory for Advanced Microscopy and Cell Imaging. Briefly, images were first cropped, background subtracted and converted to 32-bit images. Further processing was done by smoothing both channels, threshold the FRET channel and finally convert the images to ratio images alter we converted (FRET/CFP) by using the analysis method described by Kardash et al [Immunofluorescence labeling and confocal microscopy was performed as previously described ,28. BrieAll data are shown as mean ± SE. For two group comparisons, student t-tests were used. For comparisons of 3 or more groups, one-way ANOVA was used, with Tukey's multiple comparisons test for post-hoc analysis. For multiple group comparisons over time, repeated measures two-way ANOVA followed by Tukey's multiple comparisons test was used. Significance was accepted as P<0.05.We previously observed that overexpression of Rac1 can reduce permeability of endothelial monolayers . Here, wS1P has a well-known role in the enhancement of endothelial barrier function, although recently it has been reported that high concentrations of S1P can disrupt endothelial barrier integrity . TherefoRac1 has been previously reported to mediate the barrier protective effect of S1P . We testA second approach we used to test the role of Rac1 was to transfect HUVEC or HMDEC with WT and DN Rac1 plasmids , which wA third approach we used was to test the extent to which siRNA-induced Rac1 knockdown would inhibit S1P-induced endothelial barrier enhancement . DespiteWe previously reported that that S1P causes a strong and sustained activation of RhoA in HUVEC . To testAs the RhoA inhibitor produced its maximum inhibitory effect on HUVEC, we focused on HUVEC for further studies of the mechanism. The recent report of differential localized RhoA activation in endothelial cells during endothelial barrier maintenance and disruption  promptedBecause S1P elicited increased phosphorylation of MLC-2 in the vicinity of cortical actin near the cell junctions is thought to confer stronger endothelial barrier function , we evalBoth RhoA activation and MLC-2 phosphorylation has been implicated in the reorganization of the actin cytoskeleton and formation of focal adhesions . We testIn this study, we present new evidence that Rac1 activation is not required for S1P to enhance endothelial barrier function in both HUVEC and HDMEC monolayers. We were initially surprised by these results based on the well-established role of Rac1 in maintaining baseline endothelial barrier function ,18 and ret al reported that 1 μM S1P decreased hydraulic conductivity of single-perfused microvessels at 30 min but not 60 min, while higher concentrations (5 and 10 μM) increased hydraulic conductivity at 60 min but not 30 min [To understand discrepancies between our study and reports in the literature, it is important to discuss the time course of the endothelial barrier response to S1P. Our current results indicate that all concentrations of S1P tested (0.05–10 μM) initially increase barrier function of HUVEC monolayers , but witt 30 min . Other gt 30 min ,31,38. It 30 min . It is ct 30 min .50 of NSC23766 is 50 μM that perhaps only a 20% inhibition of Rac1 activity occurred [50 = 12 μM) that is 4 times more effective than NSC23766 (IC50 = 50 μM), which significantly reduced TER, in a concentration-dependent manner. These data indicate reductions in barrier function elicited by inhibition of Rac1 must be taken into consideration in the overall data analysis. Based on the current data, we think that the rapid, S1P-induced, early rise in TER occurs independently of Rac1 activation. The apparent discrepancy of our data from reports in the literature may be summarized by differences in the time points of data collected, whether baseline changes are reported, and perhaps to some extent the endothelial cell types or other experimental conditions. A current limitation is the lack of studies with Rac1 deletion or specific inhibition in intact postcapillary venules, which represents a future step that will help resolve this issue.We confirmed that Rac1 is critical for baseline barrier integrity, as shown by other investigators ,39. Howeoccurred . An addioccurred . Howeveroccurred . In currPreviously we showed that in addition to a transient Rac1 activation, RhoA activity was greatly increased and sustained for at least 10 min upon S1P treatment . In the Several reports have indicated that RhoA- or ROCK-mediated increases in phosphorylation of MLC-2 is endothelial barrier disruptive, particularly with inflammatory stimuli, such as LPS, signals from activated neutrophils, or VEGF ,16,44–47In summary, our results suggest that S1P is able to enhance barrier function independently of Rac1 in HUVEC and HDMEC monolayers. We also presented evidence that S1P activates RhoA primarily near intercellular junctions. Inhibition of RhoA attenuated S1P-induced phosphorylation of MLC-2 near junctions, F-actin assembly and vinculin recruitment at cell periphery, and S1P-induced endothelial barrier enhancement. Our data demonstrate an important role for RhoA-mediated phosphorylation of MLC-2 in S1P-induced endothelial barrier enhancement. Future studies will be needed to elucidate the role of additional RhoA-mediated downstream signals that may underlie S1P-induced endothelial barrier protection.S1 FigA & C. Time courses of TER changes during pretreatment with 25 μM Rhosin (A) or 25 μM Y16 (C), and subsequent treatment with 2 μM S1P or vehicle (N = 8 for each group). B & D. Mean maximal changes in TER (%) within the first 10 min after S1P. *P<0.05, S1P vs. vehicle treated group, same color bar. †P<0.05, inhibitor vs. vehicle pretreatments.(TIF)Click here for additional data file.S2 FigA & C. Time courses of TER changes during pretreatment with 25 μM Rhosin (A) or 25 μM Y16 (C), and subsequent treatment with 2 μM S1P or vehicle (N = 8 for each group). B & D. Mean maximal changes in TER (%) within the first 10 min after S1P. *P<0.05, S1P vs. vehicle treated group, same color bar. †P<0.05, inhibitor vs. vehicle pretreatments.(TIF)Click here for additional data file.S1 MovieThis movie shows time lapse images of an endothelial cell expressing the pTriEx-RhoA FLARE.sc Biosensor used to detect RhoA in its GTP-bound, active form. The top panel shows the CFP channel, the middle panel shows the YFP channel, and the bottom panel portrays the YFP:CFP ratio (FRET) indicating RhoA activation. The frames are at 1 min intervals. S1P (2 μM) was added at time = 0 min. The addition caused an obvious shift in GTP-bound RhoA toward the cell periphery in the first minute after the addition of S1P.(MOV)Click here for additional data file.


Oil body-associated allergens such as oleosins have been reported for important allergenic foods such as peanut, sesame and hazelnut. Here we investigate whether oil body associated proteins (OAPs) are linked with specific clinical phenotypes and whether they are represented in skin prick test (SPT) reagents.A hazelnut OAP fraction was characterized by mass-spectrometry (MS) to identify its major constituents. Polyclonal rabbit antibodies were generated against hazelnut OAPs. The presence of OAPs in commercially available hazelnut SPTs was studied by immunoblot and spiking experiments. OAP-specific IgE antibodies were measured in sera from patients with a convincing history of hazelnut allergy by RAST (n = 91), immunoblot (n = 22) and basophil histamine release .χ2 6.3, p = 0.012). Immunoblot analysis using OAP-specific rabbit antiserum demonstrated that commercial SPT reagents are virtually devoid of OAPs, sometimes (3/9) resulting in false-negative SPT. Spiking of SPT reagents with OAP restored serum IgE binding of these false-negative patients on immunoblot at mainly 17 kDa.Hazelnut OAPs were analysed by MS and found to be dominated by oleosins at ~14 and ~17 kDa, and a 27 kDa band containing oleosin dimers and unidentified protein. In 36/91 sera specific IgE against hazelnut OAPs was detected, and confirmed to be biologically active by BHR (n = 14). The majority (21/22) recognized the oleosin bands at 17 kDa on immunoblot, of which 11 exclusively. These OAP-specific IgE responses dominated by oleosin were associated with systemic reactions to hazelnut  and negative SPT (Hazelnut allergens found in oil bodies dominated by oleosin are associated with more severe systemic reactions and negative SPT. Defatted diagnostic extracts are virtually devoid of these allergens, resulting in poor sensitivity for detection of IgE antibodies against these clinically relevant molecules. Food allergic reactions after hazelnut ingestion are frequently observed ,2. In CeAlthough sufficient quantities of these water-soluble allergenic storage proteins are present in diagnostic reagents , a substWe hypothesize that patients with convincing (sometimes severe) hazelnut allergy but negative diagnostic tests, may often have specific IgE antibodies against oil body-associated proteins (OAPs) such as oleosins, allergens that are largely removed from diagnostic food extracts during de-fatting steps. The aim of this study was to test commercially available skin prick test (SPT) reagents with respect to the presence of OAPs, and to use purified OAPs from hazelnut to evaluate the clinical importance of OAP-specific IgE antibodies using sera from well-characterized patients with a convincing history and/or positive DBPCFC for hazelnut. Immunoblot analysis in conjunction with mass spectrometry was used to establish the role of oleosins as the major allergenic components of OAPs.We used a retrospectively collected panel of 91 sera of patients with a convincing history of hazelnut ingestion-related symptoms that were recruited via the outpatient clinics of the University Medical Centre Utrecht (n = 53) and the Medical Centre Alkmaar  and the Manchester Royal Infirmary . All patients gave written informed consent for using their serum for research purposes. The patients from Utrecht were all collected in the framework of a study on hazelnut allergy, and in 30/53 this was confirmed by DBPCFC . The patSeverity of recorded allergic reactions was classified into five categories, essentially following the Mueller procedure for venom allergies  and adapSPT were carried out following EAACI guidelines . An SPT Ethical approval for the study was available in each of the participating centres.g before extraction like above. The commercially available SPT extracts used have been characterized and described previously -Fibrinopeptide B . Data were acquired in the Data Directed Analysis (DDA) mode. Survey and fragment spectra were analyzed using the software PLGS version 2.2.5 (Waters) with automatic and manual data verification. For sequence identification, a combined Swiss-Prot/TrEMBL database was used.Qualitative variables  are presented as frequency (percent). For quantitative variables such as hazelnut CAP and oleosin RAST median and interquartile range (IQR) are given. Nonparametric correlations (Spearman’s rho) between severity grade and hazelnut CAP and oleosin RAST were calculated. For univariate and multivariate analysis clinical severity was dichotomised as local (grade 0) or systemic (grades 1 + 2 + 3 + 4). In the univariate analysis the association between severity and the clinical variables, hazelnut CAP and OAP RAST was analysed by chi-square test. Associations with p-values below 0.10 from the univariate analysis were further analyzed in a logistic regression model to predict systemic reactions. Association between SPT (positive/negative) and the OAP RAST (positive/negative) was analysed by chi-square test. Calculations were performed using SPSS . P-values <0.05 were considered significant.The OAP fraction of hazelnut was analyzed by SDS-PAGE Figure A. Three A panel of 91 sera was tested for IgE-reactivity to the OAP fraction in RAST. 36/91 had sIgE > 0.35 kU/L with a mean IgE-reactivity of 6.3 kU/L (ranging from 0.35-35.1 kU/L). To establish which constituents of the OAP fraction were recognized by IgE, all available sera with a sIgE > 1 kU/L n = 21) were also analysed by immunoblot with the OAP fraction. In the OAP fraction, 15 sera clearly recognized the 17 kDa oleosin band  was expressed in E.coli and purified (up to ~80% purity) Figure . IgE-bin679 was eTo test the biological activity of IgE antibodies against OAPs, 14 sera were tested by BHR. All showed significant histamine release up to 50-75% with OAPs, starting from a concentration of 100 pg/ml , Figure Nine commercially available SPT reagents and an in-house defatted hazelnut extract were evaluated by immunoblot with OAP-specific rabbit antiserum for the presence of OAPs Figure . As posiχ2 6.8, p = 0.009), pollen allergy , and allergic rhinitis . However, in the logistic model only the OAP RAST was independently associated with systemic reactions with an OR 4.24 .In order to asses whether OAPs may be associated with more severe clinical phenotypes amongst hazelnut allergic patients n = 91), we performed univariate and multivariate regression analysis, using the clinical characteristics summarized in Table , we perfin vitro and in vivo, to form homo-oligomers.The first oleosin identified as food allergen was peanut oleosin, followeOleosins are highly hydrophobic and consequently, once taken out of their oil body environment, poorly soluble proteins. This was observed when isolating OAPs and when purifying rCor a 12 produced in E.coli. Poor solubility hampers accurate evaluation of the immune-reactivity of oleosins. In the present study, we have nevertheless succeeded in establishing the allergenicity of OAPs, and confirming an important role of oleosin as oil body associated major allergen. The hydrophobic nature of OAPs such as oleosins also explains their virtual absence in commercial SPT reagents, them being extensively defatted during their production. The low sensitivity for detection of OAPs may be at the basis of false-negative SPT in patients with a convincing history of hazelnut allergy. It is of great importance to develop soluble recombinant oleosin reagents that can be used for diagnostic purposes, e.g. for spiking current diagnostic hazelnut extracts used for in vitro diagnosis as has been successfully done with rCor a 1 . We are In this study we have for the first time demonstrated in a group of 91 hazelnut allergic patients that OAPs are significantly and independently associated with systemic reactions to hazelnut, with an OR 4.24 . We can not claim with great certainty that (only) oleosins are at the basis of this association, because minor contaminations with Cor a 9 were detected. Indeed some sera showed clear IgE binding at higher molecular mass, potentially pointing towards Cor a 9 recognition. In this study, in most cases we did not have sufficient serum to also analyze IgE responses to other hazelnut allergens such as Cor a 9, an allergen that has been demonstrated to be associated with more severe hazelnut allergy . AnotherBSA: Bovine serum albumin; PBS: Phosphate-buffered saline; PBS-AT: PBS/0.3% BSA/0.1% Tween-20; RAST: Radio-allergosorbent test; SPT: Skin prick test; DBPCFC: Double-blind placebo-controlled food challenge; BHR: Basophil histamine release assay; HRP: Horseradish peroxidase; kU/L: Kilounits of antibody per liter; OAP: Oil body associated protein.The authors declare that they have no competing interests.LZ-J designed the study, conducted the histamine release experiments, immunoblot experiments, OAP purifications and wrote the manuscript. MF-R analysed and classified the patients according to medical history and performed the statistical analyses. MGTW performed the molecular cloning experiments and produced the recombinant oleosin. JHA isolated the first oleosins and performed molecular cloning experiments. CS, AL, ACK and PS collected all the patient serum, provided detailed medical histories and diagnostic data. PB and GG performed the mass spectrometry analysis and analysed the data, and took part in writing the manuscript. RvR helped to design the study and read and corrected the manuscript. All authors read and approved the final manuscript.


The long term radiological status of screw fixation following scoliosis surgery with all pedicle screw construct is not previously studied.To evaluate the incidence of loosening (implant failure) evaluated with low-dose CT two years following scoliosis surgery.Retrospective study.81 consecutive patients with adolescent idiopathic scoliosis (AIS), aged 18 ± 3 years at 2 years follow-up , subjected for scoliosis corrective surgery with all pedicle screw construct  has been examined with plain radiography and with low dose CT 6 weeks and 2 years postoperatively.In 26 out of 81 (32%) patients there were signs of loosening of one or more screws, a maximum 3 screws. 47 out of 1666 (2.8%) screws showed evidence of loosening. Preoperative Cobb angle was 56° among patients with loosening compared with 53° among patients with no evidence of loosening (P = 0.288). In males there were signs of loosening in 8 out of 14 (57%) and in females 18 out of 67 (27%), (P = 0.027). Among cases with loosening, 14% had suboptimal screw placement at the first postoperative CT compared with 11% among patients with no evidence of loosening (P = 0.254). One patient with a loosened L4 screw had neurological deficit and subjected for revision of the construct. Out of 26 patients with evidence of loosening, 5 patients reported minor pain or discomfort, 1 patient had a minor proximal junctional kyphosis of about 15° and 3 patients showed evidence of pull-out of 3–5 mm at the upper end of the construct but no clinical complaint. With plain radiography loosening could be observed only in 11 out of 26 cases, 5 were in the lumbar region.In a consecutive series of 81 cases with AIS who had underwent scoliosis surgery, one third showed, 2 years after the intervention, minor screw loosening. Males were more prone to develop screw loosening. In CT system that enables low-dose protocol, CT is recommended for the evaluation of evidence of screw loosening. Segmental pedicle screw fixation nowadays is a well established and commonly used fixation technique in the correction and posterior fusion of scoliotic deformities. In our institution, we have developed a CT-technique with extremely low radiation dose, which today is used as a routine examination during the preoperative and postoperative work-up of scoliosis surgery -3.Reports have shown that implant loosening is a well-known complication of this widely used operation technique. The etiology of screw loosening is not well known and believed to be multifactorial. Patient’s age, gender, weight bearing forces and probably muscular forces might contribute to the development of screw loosening. However, the biomechanics of screw loosening need further investigation. The evidence of loosening has been previously studied in patients operated on with pedicle screw fixation for lumbar degenerative diseases ,5 and shAll patients with AIS operated on with segmental pedicle screw fixation in our University Hospital between February 2006 and December 2010 have been examined with low dose CT 6 weeks and 2 years, respectively following surgery. All CT-examinations were performed on a 16-slice CT-scanner  according to the low-dose protocols. At 6 weeks control the patients were examined with tube voltage of 80 kV, quality reference mAs of 25, computed tomography dose index (CTDI) of 0.54 mGy and dose length product (DLP) of 20 mGycm (average effective dose 0.38 mSv). At 2 years-control the corresponding parameters were 100 kV, 25 mAs, 0.7 mGy and 30 mGycm (average effective dose 0.54 mSv). The effective dose is the tissue weighted sum of radiation taking into account the type of radiation and the nature of each organ or tissue being exposed for radiation. The slightly higher radiation dose at 2-years control was used to acquire better image quality enabling better detection of the evidence of loosening. At the same day the patients underwent low-dose standing plain radiography. The following findings were evaluated by an experienced senior neuroradiologist with specialized profile in the radiology of spinal deformity: (1) Evidence of screw loosening defined as occurrence of radiolucency around pedicle screws [1- Medial cortical perforation of the pedicle (MCP):Grade 0: Acceptable placement. Screw passes totally within the pedicle medullary canal or with minimal breach of medial pedicular cortex .Grade 1: Partially medialized screw .Grade 2: Totally medialized screw .2- Lateral cortical perforation of the pedicle (LCP):Grade 0: Acceptable placement. Screw passes totally within the pedicle medullary canal or with minimal breach of lateral pedicular cortex .Grade 1: Partially lateralized screw .Grade 2: Totally lateralized screw .3- Anterior cortical perforation of the vertebral body (ACP)Grade 0: Acceptable placement. The screw tip is contained within the vertebral body.Grade 1: Anterior cortical perforation. The screw tip penetrates the anterior cortex of the corresponding vertebral body. The degree of perforation is reported in mm.4- Endplate perforation (EPP)Grade 0: Acceptable placement. The screw tip is contained within the vertebral body.Grade 1: Endplate perforation. The screw tip penetrates the upper or lower endplate into the adjacent disc space.5- Foraminal perforation (FP)Grade 0: Acceptable placement. The screw tip does not penetrate the pedicle border into the overlying or underlying neural foramen.Grade 1: Foraminal perforation. The screw tip penetrates the pedicle border into the overlying or underlying neural foramen.A senior spinal surgeon has thoroughly scrutinized the medical records of all patients included searching for the reports of pain, neurological deficit or any other complaint reported at the 2-years visit planned at the same day as the CT and plain radiography.All operations were performed under general anesthesia with spinal cord monitoring using motor evoked potentials (MEP). The operations were performed according to the technique described by Suk. Self-tapping screws with uniplanar titanium screw head construct were regularly used. Entry points and trajectories for screws were determined by means of metal markers and with aid of fluoroscopy. Screw tracts were prepared by a hand driven drill. The over- contoured rod was then inserted on the concave side of the deformity followed by simple rod rotation and direct vertebral rotation (DVR). Finally, decortication of the posterior element and local bone graft were done to enhance fusion.The use of low-dose spine CT in the work-up of patients with AIS was approved by the Regional Radiation Protection Committee.P value ≤ 0.05.All statistical analyses were performed by means of SPSS  version 21. Data are presented as proportions (%) or as mean ± with standard deviations (SD). Chi square test was performed when studying association between categorical variables whereas Mann–Whitney U test was used when studying association between categorical and continuous variables. The agreement between the occurrence of evidence of loosening on plain radiography and on CT was evaluated by cross tabulation and kappa statistics. Kappa statistics were interpreted according to the method proposed by Landis . StatistA total of 81 patients with AIS operated on with segmental pedicle screw fixation were examined with plain radiography and low dose CT 2 years after surgery . Sixty-seven patients were females (83%). The mean age at the 2-years control was 18 ± 3 years (16 years when operated). A total of 1666 screws were evaluated. Signs of loosening of one or more screws (a maximum 3 screws) were found in 26 out of 81 (32%) patients . Patients with residual lumbar/thoracolumbar curves hade larger residual Cobb angle than those with residual thoracic curve .With plain radiography loosening could be observed only in 11 out of 26 cases (42%) with evidence of loosening on CT; 5 were in the lumbar region. This result in kappa value of 0 i.e. poor agreement, Table The distribution of Lenke types is shown in Table Segmental pedicle screw fixation has the advantage of allowing correction of the 3-dimensional deformity and providing three-column fixation. Implant loosening is a well-known complication of pedicle screw fixation especially in elderly and among patients with osteoporosis. Evidence of implant loosening has been widely studied among patients with lumbar degenerative diseases operated with pedicle screw fixation. Loosening in lumbar surgery 12 months following surgery reported to amount to 7–15% evaluated on plain radiography and 13–26% evaluated on CT . With coIn concordance with Ohtori et al., our study showed great discrepancy between the rate of loosening evaluated on plain radiography and CT . In our Although we did not evaluate bony union in our study, pedicle screw loosening may theoretically negatively influence bone fusion. However, successful bone fusion has previously been shown to be unrelated to the clinical outcome . Wu JC eOne of the limitations of this study is that only one reader has done the evaluation of screw loosening without taking into consideration a possible inter-rater variability. However, all evaluations of CT and plain radiography included in this study were performed by a neuroradiologist with a long clinical and scientific experience and with special profile and interest in spinal deformities. In cases where there is any doubt, a consensus was reached at a joined panel with an experienced spinal surgeon.This study has shown that CT is more sensitive to detect evidence of screw loosening than plain radiography. One of the drawbacks of using CT for evaluating evidence of screw loosening is the possible impact of the artefacts from the metal implant on the accuracy of such evaluation. The increase image noise might have had a negative impact on the detection of the screw loosening. Low radiation dose CT used in our study may induce some halo around the screws resulting in a false positive evidence of screw loosening. On the other hand, image noise might have contributed to miss some cases of true loosening i.e. false negative results. However, we have in previous study reported substantial inter- and intraobserver reliability in the evaluation of screw misplacement on postoperative CT-examinations (6 weeks after surgery) using even a lower radiation dose with subsequently higher image noise . IncludeFinally, the low-dose CT used in this study showed to be more sensitive than plain radiography in the detection of evidence of screw loosening. Furthermore, the effective radiation dose of low-dose CT is significantly lower than that of plain radiography. In one report, plain radiography of whole spine amounted to 6.36 mSv  comparedThis study showed that CT is more sensitive than plain radiography in detecting evidence of implant loosening in patients with AIS operated on with segmental pedicle screw fixation. Evidence of loosening on CT occurred in one third of patients, of which one fifth complained of back pain and only one patient had neurological deficit that required implant revision. The loosening was more than twice as common among boys as girls. There was no association between the radiological evidence of loosening and the degree of correction loss two years following surgery. We recommend including low-dose CT in the postoperative work-up searching for evidence of screw loosening as low-dose CT showed to be more sensitive than plain radiography and means exposure to lower radiation dose.The authors declare that they have no competing interests.KAK has contributed to conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the manuscript and has given his final approval of the version to be published. ACO has contributed to conception and design, acquisition of data, analysis and interpretation of data, and revision of the manuscript critically for important intellectual content, and has given his final approval.


The interface layer between ocean and atmosphere is only a couple of micrometers thick but plays a critical role in climate relevant processes, including the air-sea exchange of gas and heat and the emission of primary organic aerosols (POA). Recent findings suggest that low-level cloud formation above the Arctic Ocean may be linked to organic polymers produced by marine microorganisms. Sea ice harbors high amounts of polymeric substances that are produced by cells growing within the sea-ice brine. Here, we report from a research cruise to the central Arctic Ocean in 2012. Our study shows that microbial polymers accumulate at the air-sea interface when the sea ice melts. Proteinaceous compounds represented the major fraction of polymers supporting the formation of a gelatinous interface microlayer and providing a hitherto unrecognized potential source of marine POA. Our study indicates a novel link between sea ice-ocean and atmosphere that may be sensitive to climate change. The Arctic Ocean is one of the marine environments most sensitive to global warmingIce melting starts with the formation of visible pools, referred to as melt ponds, collecting melt water. As melting proceeds in the summer season, melt ponds penetrate the ice floe and represent an extending new habitat for sea ice microorganisms5Besides warm oceanic currents, freezing or melting of sea ice is also regulated by surface energy fluxes influenced by low-level Arctic clouds917Marine nano- and micro gels form from dissolved exopolymers of polysaccharidic or proteinaceous composition, released by marine microorganisms during exudation, degradation and lytic processes1922232526Sea ice is a large reservoir of organic substances: through the release of exopolymers and antifreeze proteins, sympagic algae and bacteria directly influence the properties of the brine channels increasing the abundance of pores and changing the structure of the ice25272530a concentrations and algal bloomsFYI holds most biomass near the water-air interface32925nd of August and 8th of October, 2012. The study area was characterized by a domination of FYI (>95%) and melt pond coverage of approximately 30–40%In this study, we aimed at understanding how the retreat of Arctic sea ice affects the organic composition of the SML, with implications for POA emission. During a cruise with the RV POLARSTERN (ARK XXVII/3) to the central Arctic Ocean, we sampled the SML from different sites: shallow and open melt ponds and open sea across 7 different ice stations . The cruBased on differences in morphology and physical parameters such as depth, salinity and formation time, we have identified three categories of sampling sites: t1) freshwater melt ponds, t2) open melt ponds and t3) open sea samples . Sea icep < 0.01; MWRS, p > 0.05, n = 21). DOC concentrations were significantly related to salinity both in the SML  and ULW . Enrichment factors (EF) for DOC in the SML differed between the sample types .The concentration of dissolved organic carbon (DOC) increased in the transition from freshwater melt ponds to the open sea  as confile types , indicatle types . At mostle types , yieldinC = 0.52, n = 21, p = 0.02), emphasizing DURA as an important source for TEP formation. However, except for a few sites, DURA were generally depleted in the SML of our study (Dissolved uronic acids (DURA) concentrations ranged from 8.2 nM to 92 nM in the SML and from 6.6 nM to 121 nM in the ULW, with no significant differences. These concentrations represent the lower end of previously reported data from Arctic ice coresur study .C = 0.73, n = 17, p < 0.01), TEP abundance  and TEP size , indicating a general control by substrate availability. Overall, enrichment factors for DOC, DHAA, carbohydrates, bacteria and TEP observed during this study agree well with earlier findings1415Bacterial abundance in the SML of freshwater and open melt ponds was low compared to open sea samples . In gene3 mL−1 to 53 × 103 mL−1 . Higher CSP abundances in the SML with respect to ULW were observed also in the submicron size range (0.4–1 μm) .Arctic surface microlayers were characterized by a high abundance of proteinaceous gels, i.e. CSP , and, as103 mL−1 , signifi3 mL−1 to 48 × 103 mL−1 , along with increasing salinity.TEP abundance ranged from 3 × 10103 mL−1 , with no103 mL−1 . TEP abu2 L−1) than TEP (53 ± 52 mm2 L−1) . Moreover, the submicron fraction contained significantly more CSP than TEP , indicating a higher proportion of larger particles in CSP. The 50% percentile of all CSP fell in the range 0.5–1.5 μm (SML) and 0.3–1.1 μm (ULW). TEP were smaller, mostly in the submicron fraction; the 50% percentile ranged from 0.5 μm to 0.8 μm (SML) and 0.3 μm to 0.6 μm (ULW).Size-frequency distributions of gels may be described by the spectral slope (δ) of the distribution curve. A less negative δ implies that there is a higher fraction of gels in larger size classes. In the SML, average δ for TEP (−3.18 ± 0.2) and CSP (−2.91 ± 0.12) were significantly different , supporting the idea that TEP formation is enhanced when divalent cations such as Ca2+ become increasingly abundant at higher salinityBoth in the SML and ULW the average TEP size increased with increasing salinity  may serve as an indicator of the diagentic state of DOM. A value of ~2% DHAA in DOC has been suggested as threshold separating refractory from labile and semi-labile organic matter2 = 0.66, p < 0.01). As for DHAA-%DOC, the DOC fraction of DURA also showed an inverse relationship with DOC concentrations in the SML . DURA-%DOC was highest in melt ponds, declining towards the open sea  to the ice brine and, thereby, modify sea ice properties such as porosity, salt retention, and sea ice structure252728Where do the organics enriching the Arctic SML come from? One likely source is the sea ice itself. To increase the habitability of sea ice, microorganisms such as the diatom The enrichment of proteinaceous compounds in the SML may also be related to microbial lysis through osmotic shock, or to the direct exposure to UV radiation as soon as the ice melts50522+, Mg2+) bridging between uronic acids, as well as by half-ester sulfate bridging between individual molecules212221DURA are known as important component of TEP that are considered as mucopolysaccharide gels, whose structure is formed due to divalent cation , which also showed higher DOC concentrations, higher bacterial abundance, and higher number of TEP. Open melt ponds are connected to the surrounding ocean, but are not disturbed by waves and usually have lower surface salinity-type, wh2 to the atmosphereWhen temperature drops and melt ponds rapidly freeze up, sea ice brine is expelled upwards to the surface of FYI forming a highly saline skim, which feeds frost flowers, i.e. ice crystals with branched structures at the interface between a warmer surface and a colder atmosphere2 and other gases is controlled by organics in the SML that physically and chemically alter gas exchange coefficients2 through respiration2 during both melting and freeze up.The air-sea exchange of COResuming, during the Arctic summer, the melting of sea ice and the increasing light availability promote a release of organic exopolymers into the newly forming melt ponds, as well as primary production is boosted both in deeper melt ponds as well as in open waters. The summer biological production and release of organic matter from the melting ice may provide the exopolymers for the establishment of an organics-enriched SML. At the end of the summer, when the pack ice forms again, the organics in seawater may get incorporated into the ice and at the same time, microbial survival metabolism provides a new source of polymeric DOM in the ice brine, that will be released during the next melting season.As we observed DHAA in similar concentrations in all stages of ice melting, and CSP dominating gel abundance, we suggest that the transition towards FYI in the central Arctic Ocean and the increasing number of melt ponds during the summer supports proteinaceous compounds in the SML and their contribution to Arctic aerosol mass.2 effects on primary production, DOM accumulation and turnover, in turn affecting exopolymer release and carbon flow61Future changes in the Arctic Ocean affecting sea ice and ocean biology will likely influence organic matter concentration in the SML, with potential consequences for POA composition and air-sea gas exchange. Arctic change does not only account for faster sea ice melting and the loss of MYI, it also implies temperature and CO2 according to the original approach described by Harvey and Burzelld, μm) was on average 54.8 ± 4.8 μm, and was calculated according toSamples were collected at 21 sites across seven different ice stations . These sV is the water volume collected (cm3), (t) the number of dips with the glass plate, and AGP (cm2) the a total area of the glass plate, considering both sides.where Enrichment factors between SML and underlying water were calculated as follows:[x] is the concentration of a given parameter in the SML or in the underlying water (ULW)where Enrichment factors are the ratios of two quantities, and, therefore, include the propagation of the individual standard deviations of these quantities. Standard deviations for replicate analyses for dissolved organic carbon (DOC), dissolved hydrolysable amino acids (DHAA), dissolved uronic acids (DURA) and bacterial abundance in SML and underlying water samples were derived from replicate analyses. The standard deviation of enrichment factors was calculated from standard deviations of the microlayer and underlying water samples according to the Gaussian law of error propagation, and the reference values (SD %) are given in 3PO4). DOC samples were stored for three months at 0–2 °C until analysis. DOC concentrations were determined by high temperature catalytic oxidation modified after Sugimura and SuzukiSamples for dissolved organic carbon (DOC) were prepared by filtering seawater through pre-combusted  GF/F filters  and filling 20 mL into pre-combusted  glass ampoules successively acidified with 80 μL of 85% phosphoric acid (HFor dissolved hydrolysable amino acids (DHAA) analysis, samples were prepared by filtering seawater through 0.45 μm GHP membranes  and sub-sampling 15 mL into pre-combusted  scintillation vials. The analysis was performed according to Dittmar and colleaguesSamples for high molecular weight (>1 kDa) dissolved uronic acids (DURA) were prepared by filtering seawater through 0.45 μm GHP membranes  and sub-sampling 15 mL into pre-combusted  scintillation vials. Samples were kept frozen at −20 °C until analysis. The analysis was conducted according to Engel and HändelFor bacterial abundance, 4 mL of sample were fixed with 200 μL glutaraldehyde (25%) and stored at −20 °C until enumeration within six months from collection. Abundance was determined after staining with SYBR Green I (Invitrogen) and the analysis performed with a flow cytometer . Bacterial cell numbers were estimated by manual gating of the bacterial subpopulation in the cytogram of side scatter versus green fluorescence, with a standard deviation <3% between replicate measurements. Yellow-green fluorescent latex beads (Polyscience) and TruCount beads (Becton Dickinson) were used to normalize the counted events to volumeMarine gels were determined microscopically with the CytoClear slide technique after EngelC), 3rd order polynomial regression, and statistical test like Mann-Whitney Rank Sum (MWRS) test were performed with SigmaPlot (Systat Software Inc.) package and Prism7 (GraphPad Software Inc.). Statistical significance was accepted for p < 0.05.Calculation of Pearson and Spearman Rank Order Correlation Coefficient .


Petrochelidon pyrrhonota), a temperate-latitude migratory songbird in the western Great Plains, USA, typically has a relatively short (eight to nine week) breeding season, with birds rarely nesting late in the summer. Colonies at which ectoparasitic swallow bugs (Oeciacus vicarius) were experimentally removed by fumigation were over 45 times more likely to have birds undertake a second round of nesting than were colonies exposed to parasites. Late nesting approximately doubled the length of the breeding season, with some birds raising two broods. Over a 27 year period the percentage of birds engaging in late nesting each year increased at a colony site where parasites were removed annually. This trend could not be explained by changes in group size, climate or nesting phenology during the study. The results suggest that ectoparasitism shortens the cliff swallow's breeding season and probably prevents many individuals from multiple brooding. When this constraint is removed, selection may rapidly favour late nesting.When blood-feeding parasites increase seasonally, their deleterious effects may prevent some host species, especially those living in large groups where parasites are numerous, from reproducing later in the summer. Yet the role of parasites in regulating the length of a host's breeding season—and thus the host's opportunity for multiple brooding—has not been systematically investigated. The highly colonial cliff swallow ( A numSurprisingly, little is known about how ectoparasites directly affect avian breeding phenology . One stuOeciacus vicarius) from cliff swallow colonies, to investigate the potential role of ectoparasitism in regulating the length of the breeding season. These deleterious blood-feeding parasites are known to affect many aspects of the cliff swallow's social behaviour and ecology [In this study, we take advantage of a long-term experiment, in which we annually removed ectoparasitic swallow bugs  at a colony site after nestlings had fledged there, generally leading to a doubling of the length of time birds were present at a site. We investigated how the frequency of late nesting changed over a period of more than 20 years at a parasite-free site and use the results to gain insight into how ectoparasites potentially constrain the length of the cliff swallow's breeding season and the number of breeding attempts it makes.3.3.1°13′ N, 101°39′ W) in Keith County, southwestern Nebraska, along the North and South Platte rivers and including portions of Deuel, Garden, Lincoln and Morrill counties [n=2318 colonies), with some birds nesting solitarily. The typical phenology (in the absence of late nesting) is for cliff swallows to first arrive in southwestern Nebraska in mid to late April, for most birds to have initiated egg laying by early June, and for nestlings to have mostly fledged by mid-July. Cliff swallows spend the winter in northeastern Argentina, Uruguay and southwestern Brazil [We have studied cliff swallows since 1982 in the western Great Plains, USA, centred near the Cedar Point Biological Station , so by fumigating, we removed only swallow bugs [Swallow bugs were removed from colonies by lightly misting the outside of all cliff swallow nests and adjacent nesting substrate with a dilute solution of the insecticide Dibrom ,8. This Fumigation of colonies began in 1984 and continued at some sites through to 2014. Some sites were fumigated only in certain years, depending on the research question that required parasite removal. Two colony sites, Whitetail and Junkyard, were fumigated each year of the study, beginning in 1984 and 1998, respectively. The fumigated sites were all concrete road culverts, and they did not differ in physical attributes from other culvert sites occupied by cliff swallows in the study area. Non-fumigated colony sites were those with no fumigation of any part and were situated on culverts, bridges and buildings . Only siThroughout this paper, a colony site refers to a physical structure at a particular locale where birds nested, whereas a colony refers to a collection of individuals at a given site . At any 3.3We assessed whether late nesting occurred at a colony site each year by observing the birds' presence at the site after the early round of nesting had been completed (known by fledging of young from most nests). When the birds' presence at nests at a site after about 15 July suggested they might be nesting, we checked nests for eggs using a dental mirror and flashlight inserted through a nest's tubular mud entrance. For many colonies, we ruled out any late nesting (without checking nests) whenever all birds had vacated the site by 25 July . Our definition of late nesting as a temporally distinct second round of breeding at a site meant that all colonies included in our analyses had to have been ones that were active during the early round of nesting in May and June  and thus were available to have a late round of nesting. Late nesting typically occurred in nests that had also been occupied in the early round. The birds in the late round at a site had been there during the early round  and thusAnnual colony sizes were based on first nesting attempts (the early round) and defined as the number of active nests (ones with at least one egg laid) each year. Colony sizes were determined by direct counts of all active nests, known from viewing nest contents, or by estimation based on nest counts of portions of a colony site or from the number of birds present .At Whitetail only, the number of nests active in the late round of nesting was determined each year by either regularly checking all nests in late July and August or estimating the number of active nests from the number of previously banded adults mist-netted at the site after 15 July. The number of late nests was not available for some years. We did not attempt to estimate the number of nests in the late round at any of the other colony sites, where we simply scored the presence or absence of late nesting.We determined a given colony's initiation date, by recording when the first cliff swallows were observed at the site that year and remained daily thereafter . We moniAs a measure of the relative earliness of each nesting season across the study area independent of a given site's phenology, we used the annual date on which the first juvenile cliff swallow was captured in a mist net (at any colony) in the course of our extensive mark-recapture programme ,31 condu3.4As a measure of the quality and condition of individuals initially settling in different colonies, we used the body mass of cliff swallows captured in mist nets at both fumigated and non-fumigated colony sites throughout the study area. We restricted the analysis only to birds captured relatively early in the season when a given colony was engaged in nest-building and egg laying. We thus standardized our body masses temporally with respect to nesting stage, rather than date per se , given t3.5www.ncdc.noaa.gov/cag/time-series/. Division 7 encompasses the centre of our study area, and the regional metrics should be broadly representative of climatic conditions the birds experienced each season. The PDSI is a measure of drought intensity used by NOAA, and it integrates both local temperature and rainfall data into a single index useful in describing soil moisture and extent of runoff [As an overall measure of annual climatic conditions during the study , we used the modified Palmer drought severity index (PDSI) for Nebraska's Climate Division 7 (southwest Nebraska) available from the National Oceanic and Atmospheric Administration (NOAA) at f runoff ,33. Lowe3.6Logistic mixed models were constructed in SAS  using Pr4.F1,548=5.24, p=0.022). Colony size , colony initiation date , date of first juvenile capture , year  and annual PDSI  were not significant predictors of the presence or absence of late nesting. When the analysis was re-run with an additional 612 colonies (that did not have known initiation dates or were from 3 years without first juvenile capture dates), the results were similar . Colony size, colony initiation date, first juvenile capture date, year and PDSI also had no significant effect on whether colonies exhibited late nesting when fumigated and non-fumigated colonies were analysed separately (p≥0.59 for each).Of 1183 non-fumigated colonies, 19 (1.6%) had late nesting activity, compared with 49 (75.4%) of 65 fumigated colonies. Using 636 colonies and controlling for colony site as a random effect, only fumigation status of a colony (yes/no) was a significant predictor of whether late nesting occurred , with Whitetail colony initiation date , annual first juvenile capture date in the study area , Whitetail colony size  and annual PDSI  not explaining any significant variation. Results were similar when colony initiation date and first juvenile capture date were removed from the model.For Whitetail, the percentage of first-round nests that were re-occupied for late nesting increased significantly over time , varyingF1,∼42 000=1621, p<0.0001) and year  but did not vary with a site's fumigation status  or with colony size . Similar results were obtained when each year was analysed separately: in only 2 years  was fumigation status a significant predictor of body mass , and in the other 25 years fumigation status was not significant . For Whitetail, mean body mass of birds at settlement each year did not change significantly over time .When controlling for colony site as a random effect, body mass of cliff swallows soon after initial settlement varied significantly with sex  than were colonies with typical numbers of swallow bugs. Late nesting was unrelated to colony size, phenology of the nesting season or climatic conditions in a given year. That late nesting in Nebraska cliff swallows can be successful and confers potentially higher lifetime fitness than raising only an early single brood  suggestsOur experimental parasite removal establishes the absence of swallow bugs as a causal factor in the initiation of late nesting in cliff swallows. We were able to rule out other possibilities that might have contributed to the observed results. For example, local resources (e.g. the birds' food) could change over time , or earlCould the increased late nesting at fumigated sites reflect non-random sorting of high-quality individuals into those colonies? If so, those individuals might have been more capable of nesting late by virtue of their superior condition. The body masses of cliff swallows soon after settlement in colonies do not support this scenario: individual quality or condition, at least as measured by body mass , did notCliff swallows' responding to ectoparasites by avoiding late nesting is perhaps not surprising, given the highly deleterious effects of swallow bugs and the other ways cliff swallows adjust their behaviour to bug infestations ,8,21,25.While the much greater frequency of late nesting at fumigated colonies compared with non-fumigated ones was striking, perhaps even more surprising was the increase in the extent of late nesting over time at one site . This trThis study demonstrates perhaps the strongest effect yet of a blood-sucking ectoparasite on the length of its host's breeding season and on the host's ability to undertake multiple broods. Our results suggest that parasitism may be an overlooked factor contributing to the wide variability both among and within species in propensity to double-brood in a given year –43. In t


Fmax was measured. Fmax in groups A1-A5 were all significantly higher than group A0. Except between groups A1 and A2, A3 and A4, and A4 and A5, there were significant differences on Fmax between any other two groups. Fmax in groups B1-B5 were all significantly higher than group B0. Except between groups B1 and B2, B2 and B3, and B4 and B5, there were significant differences on Fmax between any other two groups. There was significantly positive correlation between Fmax and volume of PMMA in osteoporotic and severely osteoporotic blocks. PMMA can significantly enhance pedicle screw stability in osteoporosis and severe osteoporosis. There were positive correlations between screw stability and volume of PMMA. In this study, injection of 3 mL and 4 mL PMMA was preferred in osteoporotic and severely osteoporotic blocks, respectively.This study was designed to compare screw stabilities augmented with different volumes of PMMA and analyze relationship between screw stability and volume of PMMA and optimum volume of PMMA in different bone condition. Osteoporotic and severely osteoporotic synthetic bone blocks were divided into groups A0-A5 and B0-B5, respectively. Different volumes of PMMA were injected in groups A0 to A5 and B0 to B5. Axial pullout tests were performed and  Transpedicular screw fixation has been widely used in treating degenerative disorders, unstable fractures, and deformities and tumors of the spine –4. HowevTo effectively improve pedicle screw stability in the setting of compromised bone, many researchers have used polymethylmethacrylate (PMMA) to enhance fixation strength –18. TherThis study was designed to compare the pedicle screw stabilities augmented with different volumes of PMMA in different bone conditions which were simulated using synthetic bone blocks with different density. It was also performed to analyze the relationship between screw stability and volume of PMMA and study the preferred volume of injected PMMA in different degree of osteoporosis.3 and 0.09 g/cm3, respectively, which was used to simulate human vertebral cancellous bone with osteoporosis and severe osteoporosis [Synthetic bone blocks  were used as a substitute for cadaveric spinal bone in this study because of its consistent and homogeneous structural properties , 20. Theoporosis , 16–18. oporosis , 16–20. n = 8). Forty-eight 1522-505# synthetic bone blocks were randomly divided into group B0 to group B5 (n = 8). Each hole was tapped using a 4.5 mm tap into depth with 45 mm . Different volumes of PMMA  were retrograde injected into the pilot hole in groups A0 and B0, groups A1 and B1, groups A2 and B2, groups A3 and B3, groups A4 and B4, and groups A5 and B5, respectively. The bone cement powder was mixed with the cement solution at a ratio of 2 : 1 according to manufacturer's recommendations. Then we connected the syringe and injection sheath and injected the PMMA until it appeared at the tip of the sheath. We used visual observation and palpation to ensure that the PMMA had pushed out the air in the sheath cavity and to evaluate the viscosity of the PMMA. When the PMMA reached a toothpaste-like consistency, we inserted the sheath into the hole. PMMA was injected through the sheath into the hole and slowly diffused towards surrounding synthetic bone material under the continue pressure. Screw was inserted into the hole immediately after injection of PMMA. All screws were inserted by hand using a ratcheted screwdriver until the hub of the screw was firmly seated against the surface of test blocks. After complete solidification of PMMA, all vertebrae were taken for X-ray examination and the distribution of PMMA around screw was observed.To carry out the test, firstly, test samples of 13 cm × 9 cm × 4 cm were extracted from the whole blocks. Forty-eight 1522-507# synthetic bone blocks were randomly divided into group A0 to group A5 (Fmax) was defined as the highest point which was also the inflection point between buildup curve and descending curve [After X-ray examination, the axial pullout test was performed with MTS 858 Material Testing System . A tensile load was gradually applied to the head of the screw at a constant speed of 5 mm/min until the screw was pulled out from the test block. The load and displacement data collected in real time at 50 Hz were used to obtain the load-displacement curve. On the curve, the maximum pullout strength . The data were expressed as mean ± standard deviation. The two-way ANOVA and LSD test were used to detect the differences in The distribution of PMMA and screw was clearly shown in X-ray examination . The denFmax in group A0 to group A5 were 48.75 ± 12.54 N, 252.12 ± 37.59 N, 290.50 ± 46.91 N, 359.25 ± 48.95 N, 402.38 ± 59.50 N, and 434.62 ± 67.11 N, respectively. Fmax in groups A1–A5 were all significantly higher than that in group A0 (P < 0.05). There were 417.2%, 495.9%, 636.9%, 725.4%, and 791.5% increments on Fmax in groups A1–A5 compared with that in group A0, respectively. Except the no significant differences on Fmax between groups A1 and A2, between groups A3 and A4, and between groups A4 and A5 , there were significant differences on Fmax between any other two groups (P < 0.05). As shown in Fmax and the volume of injected PMMA .Fmax in group B0 to group B5 were 30.63 ± 10.45 N, 131.12 ± 34.06 N, 172.38 ± 43.41 N, 202.75 ± 61.71 N, 289.62 ± 51.00 N, and 320.88 ± 46.22 N, respectively. Fmax in groups B1–B5 were all significantly higher than that in group B0 (P < 0.05). There were 328.08%, 462.78%, 561.93%, 845.54%, and 947.60% increments on Fmax in groups B1–B5 compared with that in group B0, respectively. Except the no significant differences on Fmax between groups B1 and B2, between groups B2 and B3, and between groups B4 and B5 , there were significant differences on Fmax between any other two groups (P < 0.05). As shown in Fmax and the volume of injected PMMA .Fmax in groups B0–B5 in 1522-505# blocks was significantly decreased  compared with the Fmax in groups A0–A5 in 1522-507# blocks. Except no significant difference on Fmax between groups A0 and B0 (P = 0.438), there were significant difference on Fmax between any two groups with same volume of injected PMMA (P < 0.05). The Fmax in group A0 was significantly lower than those in groups B1, B2, B3, B4, and B5  and not significantly different from that in groups B0 (P = 0.438). The Fmax in group A1 was significantly higher than that in groups B0, B1, B2, and B3 , significantly lower than that in group B5 (P = 0.004), and without significant difference compared with that in group B4 (P = 0.110). The Fmax in group A2 was significantly higher than that in groups B0, B1, B2, and B3  and without significant difference compared with that in both group B4 and group B5 . The Fmax in group A3 was significantly higher than that in groups B0, B1, B2, B3, and B4  and without significant difference compared with that in group B5 (P = 0.102). The Fmax in both groups A4 and A5 were significantly higher than that in groups B0, B1, B2, B3, B4, and B5 (P < 0.05).As shown in As shown in As showed in Effectively improving pedicle screw stability in osteoporosis has always been a tough problem for spine surgeons. With the excellent mechanical strength, PMMA was used to augment screw fixation in more and more studies. Earlier, many researchers directly injected PMMA into the pilot to improve screw stability. With the development of study, more and more researchers injected PMMA through different-designed injectable screw to enhance screw fixation. However, there was significant variation in the volumes of PMMA injected and in screw stability across studies. As shown in Fmax of the pedicle screw increased in primary and salvage procedures by 119% and 162%, respectively, but pullout strength did not significantly change with increased cement usage between the low-cement group (≤2.8 mL/pedicle) and the high-cement group (≥5.5 mL/pedicle). Paré et al. [Fmax of the augmented fenestrated screws was significantly increased compared with non-PMMA-augmented pedicle screws for the screws receiving 1.0 mL (186%) and 1.5 mL of PMMA (158%) in the thoracic vertebrae. Statistically significant increases were observed for the screws receiving 1.5 mL (264%), 2.0 mL (221%), and 2.5 mL (198%) of injected cement in the lumbar vertebrae. There was no significant difference, however, at higher volumes of cement. They found that screw stability did not increase with incremental increases in PMMA volume. However, they did not analyze the relationship between screw stability and volume of PMMA in their study. Chen et al. [Frankel et al.  injectedé et al.  injectedg 1.0 mL 6% and 1.All of those studies show that there are significant differences in the screw stability provided by various volumes of PMMA. Nevertheless, most comparative studies of different volumes of PMMA mainly focused on injection through fenestrated screws; few studies have focused on the traditional injection method. In studies that did focus on the traditional method, there was no comparison of screw stability for varying volumes of PMMA and no correlative analysis of screw fixation strength and PMMA volume, especially in different degrees of osteoporosis. Thus, our study was designed to fill that research void. As we knew, it was very difficult to obtain osteoporotic and severe osteoporotic cadaveric spinal samples. In this study, we choose synthetic bone (model 1522-507# and 1522-505#) made from polyurethane foam as experiment samples. It has homogeneous structural properties and over 95% open cell structure, which is suitable for modeling osteoporotic and severe osteoporotic cancellous bone and injection of PMMA and has been used in some biomechanical study , 16–18.Our previous study proved that injection sheath worked well and was easy to use. But we have applied for a patent on this sheath in China, so we cannot publicly share images of the sheath yet in the published papers. In this study, under the continuous pressure from injection PMMA was slowly diffusing toward the surrounding pores along the total length of hole. PMMA was proved nearly evenly distributing around screw in both previous studies , 25–27 aPMMA . As the same, though there were 31.5%, 17.6%, and 10.8% increments on screw stabilities in severe osteoporotic block with increasing volume of injected PMMA from 1 mL to 2 mL, from 2 mL to 3 mL, and from 4 mL to 5 mL, all those increments were not with statistical significance . Chen et al. [Though there was no significant difference on The 1522-507# and 1522-505# synthetic bone blocks were used to simulate human vertebral cancellous bone with osteoporosis and severely osteoporosis, respectively, in this study. The synthetic bone block has consistent and homogeneous structural properties. So that the synthetic bone block was just used to simulate but could not replace the vertebral samples. In order to simulate the real condition in operation in clinic, therefore, it is necessary to perform the comparative study in osteoporotic and severe osteoporotic lumbar vertebrae in future study. In this study, we not only found the relationship between screw stability and volume of PMMA and the preferred volume of injected PMMA in synthetic bone blocks with different density, but also provided the feasibility for the future study in vertebral samples. The result in this study also provided the guideline for the injected volume of PMMA in vertebral body in the future. As we knew, there were different microstructures of bone tissue in different vertebral bodies. There were also different microstructures of cancellous bone in different regions in one vertebral body. For this reason, we cannot ensure the exact same distribution and interdigitation of PMMA to the surrounding bone tissue by vertebra to vertebra. That may bring influence on the fixation strength of pedicle screw. In order to study relationship between the different distribution of PMMA and fixation strength, we also had designed several injection sheaths with different lateral holes. With the same volume of injected PMMA, we found varying distribution of PMMA and varying screw stability. The results of that particular experiment are still being interpreted and have not been submitted to any journal.In this study, PMMA can significantly enhance stability of pedicle screw in both osteoporotic block and severely osteoporotic synthetic bone block. There were strong positive statistically significant correlations between screw stability and the volume of PMMA, and the screw stability was increased with the increment in the volume of PMMA. Within a certain range, nevertheless, increasing the volume of PMMA does not significantly improve screw stability. In this study, injection of 3 mL PMMA and injection of 4 mL PMMA were considered as preferred choice in osteoporosis and severe osteoporosis, respectively.


Our previous study reported that muscle cell enhancement factor 2C (MEF2C) was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK) in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors. PI3K plays a key regulatory role in the proliferation and differentiation of skeletal muscle cells , 2. StudActivation of ILK stimulated by growth factor is normally regulated in a PI3K-dependent manner via phosphatidylinositol -trisphosphate (PIP3) interaction with the central pleckstrin homology- (PH-) like domain of ILK , 8. ILK The MEF2 proteins (MEF2A–D) consist of a family of transcriptional factors that play a central role during the development of skeletal muscle . The DNAIn summary, PI3K plays an important role in the proliferation of skeletal muscle cells. PI3K has a great impact on ILK activation. ILK also controls MEF2C-mediated regulation of skeletal muscle cell proliferation, following inhibition of phosphorylation. However, no report is available on PI3K-dependent activation of ILK directly regulating MEF2C phosphorylation during the proliferation of skeletal muscle cells. The present study explored whether activated ILK directly regulated the phosphorylation of MEF2C during the proliferation of C2C12 cells and regulates the expression of myogenic genes.α-tubulin antibody (ab15246) were purchased from Abcam (USA).DMSO , ILK antibody , ILK phosphorylation antibody (sc-130196), and polyclonal antibody p-MEF-2 (Ser59) (sc-13919-R) were purchased from Santa (USA); MEF2C antibody (ab64644) and  2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002), was used. Applicable cultures were supplemented with LY294002 at indicated concentrations, 30 min prior to treatment -4-yl)-1H-pyrazol-3-yl)propanamide (22) , facilitated the dephosphorylation of Akt at Ser473 and other ILK targets [A pharmacological inhibitor specific for the PI3K pathway, namely,reatment , 21. A ppanamide   and maintained at 37°C and 5% CO2 in a humidified atmosphere and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 12% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Sigma Aldrich).C2C12 cells were planted in 10 cm culture dish .Subsequent to different treatments, supernatants were discarded and MTT solution (5 mg MTT/mL PBS) was added to each well containing the cell monolayer and incubated for different times at 37°C in a humidified 5% COThree ILK short hairpin RNA (shRNA) sequences were designed as follows, with nucleotide start positions based on the NCBI entry for ILK (accession NM_001161724.1): ILK-810, 5′-GGCAGGGCAATGATATTGTTG-3′; ILK-999, 5′-GATCTCTCTACAATGTTCTAC-3′; ILK-1048, 5′-GAGCCAAGCTGTAAAGTTTGC-3′; and ILK-1763, 5′-CCCGCCTGTCACAATAAAGTT-3′. And shRNA negative control (NC-shRNA) is 5′-GTTCTCCGAACGTGTCACGT-3′. The sequences were subcloned into PGPU6/GFP/Neo vectors  and transiently transfected into C2C12 cultures.Following treatment, cells were immediately transferred to ice and rinsed in 0.5 mL of RIPA lysis buffer . Lysates were diluted in 5 × SDS-PAGE sample loading buffer  and heated in a boiling water bath for 3–5 minutes in order to fully denature protein, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gels. Gels were run for 30 min at 90 V and 60 min at 120 V. Following SDS-PAGE, proteins were transferred to polyvinylidene fluoride (PVDF) membranes  using a semidry electrotransfer system  for 60 min at 0.3 A (constant current). Membranes were then incubated with specific primary antibodies diluted as 1 : 500–1 : 1000 in TBS-T (1 : 1000), overnight at 4°C. The next day, membranes were incubated in anti-rabbit (or anti-goat) horseradish peroxidase-conjugated secondary antibody  for 1 h at room temperature. Antibodies were detected with the Pierce ECL Western Blotting Substrate as per the manufacturer's instructions, and bands were exposed to autoradiography film . Exposed bands were visualized and then quantified by densitometry using Image J . All bands were expressed as optical density values relative to a control on the same blot.−ΔΔCt) method (n = 3). GAPDH was used to normalize the values.Following treatment, the total RNA of cells was immediately extracted. Reverse transcription using 500 ng total RNA was performed with PrimeScript RT Master Mix (Takara). The MCK (GenBank Acc.: NM_007710.2) primer sequences were designed by GenePharma (China): forward primer (F): 5′-GAGGCAATATGAAGGAGGTTTTCC-3′; reverse primer (R): 5′-GGTGCTCGTTCCACATGAAGG-3′. The GAPDH (GenBank Acc.: NM 001289726.1) primer sequences were designed by Beijing Genomics Institute (BGI) (China): forward primer (F): 5′-TGTGTCCGTCGTGGATCTGA-3′; reverse primer (R): 5′-TTGCTGTTGAAGTCGCAGGAG-3′. The qPCR experiments used SYBR Premix Ex Taq (Takara) and the real-time fluorescent quantitative PCR of JENA . PCR conditions were 95°C for 30 s for the initial denaturation step, followed by an appropriate number of cycles at 95°C for 10 s and 60°C for 30 s. Threshold cycles (Ct) were automatically derived from the amplification plots constructed from the ROX-normalized fluorescence signals by MxPro qPCR software. Relative quantification was performed by the comparative Ct (2n = 3 for each group). The significance of differences between experimental or treated groups and control groups was determined by analysis of variance; ∗p < 0.05, ∗∗p < 0.01 by the t-test were considered statistically significant.Data are presented as means ± SD .We used 20 p < 0.05). MEF2C interacted with the enhancer region of MCK and thus enhanced the expression of MCK, and the expression of MCK was decreased significantly .As demonstrated in Figures μM Cpd22, ILK expression did not change . As shown in Figures p < 0.01) to 1.41 times that of the control group. As shown in p < 0.05).After treatment of C2C12 with 10  Figures , while Ip > 0.05), but four ILK-shRNA had different levels of ILK mRNA (p < 0.05); ILK-999 ; ILK-1048 ; ILK-1763 . The similarity of the western blot results  and NC-shRNA were transfected and untransfected control cells (control) were transiently transfected into C2C12 for 48 h, control (1.00177 ± 0.073) and NC-shRNA (1.005488 ± 0.060) had similar levels of ILK mRNA (ILK mRNA : ILK-810 results  showed tp < 0.05) compared with those of sh-NC  was increased distinctly (p < 0.01) were significantly higher than that of the control .Using Cpd22, C2C12 was further treated with LY294002. As illustrated in Figures p > 0.05). As illustrated in p > 0.05), but it was lower than that of the experimental group following a single treatment with Cpd22 (3.55 times). Moreover, it was significantly lower than that of the experimental group following single treatment using Cpd22, which is consistent with the MEF2C protein analysis.As shown in Figures The C2C12 cell line is an immortalized rat skeletal muscle satellite cell primarily isolated from the thigh bone muscle of a 2-month-old C3H female mouse after crush injury . This cePI3K plays an important role in the proliferation, growth, differentiation, and apoptosis of skeletal muscle cells , 24. TheILK contains three domains to support a variety of functions, including the N-terminal domain composed of four ankyrin repeats and a PH domain and the C terminal kinase domain. The unique protein structure enables ILK to play an important role in the coordination of extracellular matrix (ECM) and the signal transduction of growth factor , 29. EviMEF2C directly regulates gene transcription of myomesin. MEF2C loss in skeletal muscle results in improper sarcomere organization. These results reveal a key role for MEF2C in maintenance of sarcomere integrity and postnatal maturation of skeletal muscle . In the ILK is activated in PI3K-dependent manner and regulates the related proteins or genes in the classical signal transduction pathway. In the present study, the ILK phosphorylation was inhibited after the inhibition of PI3K activity, when both MEF2C phosphorylation and transcription exhibited significantly decreasing trends. In order to further determine the regulatory effect of ILK on MEF2C, both the phosphorylation and the transcriptional activity of MEF2C displayed significantly increasing trends after direct inhibition of ILK phosphorylation and mRNA expression. Two different experimental results explain similar inhibition of ILK phosphorylation activity. According to previous studies, ILK is directly related to MEF2C, and ILK negatively regulates the MEF2C phosphorylation and transcription activity. When two kinds of inhibitors were used, both the phosphorylation and transcription activities of MEF2C were increased, but lower than in the group treated with a single ILK inhibitor, which suggests that although the addition of PI3K inhibitor inhibited ILK activity, it did not negatively modulate MEF2C. Thus, we conclude that ILK activation independent of PI3K negatively regulates MEF2C phosphorylation and transcription in C2C12 cells.As one of the key molecules linking important signaling pathways inside and outside cells, ILK plays a stellar role in the study of skeletal muscle proliferation and tumor therapy. In this study, the relationship of the PI3K/ILK/MEF2C/MCK signaling pathway with muscle proliferation was investigated using C2C12 cell line. A key finding: ILK activation was not entirely PI3K-dependent, especially in the regulation of MEF2C phosphorylation and transcription. Our next study will investigate how ILK regulates MEF2C in detail. We hope that the results of this study will provide a new impetus for the treatment of tumors and improvement of meat quality in domestic animals.Different concentrations of the PI3K inhibitor LY294002 were used to treat C2C12 cells for different times. With increase in the treatment time and drug concentration, the inhibition efficiency showed an increasing trend. From the result, we chose 20µM LY294002 to treat C2C12 for 3h to inhibit the activity of PI3K.


Dichelobacter nodosus cause the majority of lameness in sheep in the UK. Lame sheep often have overgrown hoof horn but recent evidence has indicated that trimming overgrown hoof horn increases recovery time, and that routine foot trimming of the flock does not reduce the prevalence or incidence of lameness. The objectives of this study were to investigate the temporal associations between hoof horn length, footrot and climate. Fifty multiparous ewes were monitored for 10 months. On eight occasions hoof horn length, foot lesions and body condition were recorded. At the first examination, ewes were assigned to one of two treatment groups. All ewes that became lame with footrot were treated at one time point per week, either by trimming hoof horn and applying a topical antibiotic spray or with parenteral antibiotic and topical antibiotic spray.Footrot, including interdigital dermatitis, is caused by Hoof horn length in ewes at pasture varied over the year and was associated with temperature and rainfall. New cases of footrot occurred all year round and were associated with prior prevalence of footrot in the flock and prior temperature and rainfall. Overgrown hoof horn did not precede lameness but occurred once the sheep were lame. One year of prompt treatment of footrot reduced the range in hoof horn length in the sheep in both treatment groups. At the end of the study the hoof lengths of ewes in both groups were not significantly different. On this farm, hoof horn length was self-regulating in both non-lame and treated lame sheep whether trimming was part of the treatment or not and there would have been no benefit from routine foot trimming of this flock. Lameness in sheep causes pain  and reduDichelobacter nodosus , including interdigital dermatitis (ID), is responsible for approximately 90% of all lameness in sheep in Great Britain (GB)  and both nodosus . The occ nodosus . When thad of FR . In contD. nodosus between sheep through close contact and dirty equipment and so directly increases the incidence of lameness, especially if sensitive tissue is exposed when hoof horn is trimmed. It is also possible that the statistical association between prevalence of lameness and routine foot trimming is an indirect association i.e. routine foot trimming is correlated with other management procedures that increase the prevalence of lameness but is itself not causally associated with increased lameness.Routine foot trimming  is a flock management tool for lameness control that has been practised for many years in GB . Those sIrrespective of this association, routine foot trimming does not reduce the prevalence of lameness in subsequent weeks  or in thApproximately 25% of English farmers do not practise routine foot trimming  and anecHoof horn protects the sensitive internal structures of the foot and allows animals to walk on hard and irregular surfaces without pain. Very little is known about the nature of sheep hoof horn but several studies on hoof horn in dairy cows provide some basic principles of its likely nature. In dairy cows, horn grows continuously; it is moderated by the environment e.g. it absorbs moisture and so is softer and more flexible in animals kept in wet conditions  and hardInvestigation of the dynamics of sheep hoof horn length over time and its relationship to FR is therefore timely. In the current study we investigated the temporal patterns of hoof horn length and horn length associations with FR, therapeutic foot trimming and temperature and rainfall, and evaluated how these results can inform the likely usefulness of routine foot trimming.This study was granted approval by the University of Bristol Ethics Committee, UIN number UB/09/020.A flock of 99 Mule and Suffolk-cross ewes was selected for study based on a history of lameness caused by FR, and likely compliance of the farm staff with study protocols. The flock was mated to five Texel and Suffolk rams in November 2010. Barren ewes were separated from the main flock in early February 2011, and pregnant ewes were offered supplementary feed after this date. Pregnant ewes were housed for lambing in late March, and lambing occurred over a 3-week period in March and April 2011. Ewes were housed for between 3 and 21 days. Ewes and lambs were turned out to pasture approximately 24 h postpartum. The flock was maintained as a single population of ewes with lambs at foot until the lambs were weaned in August at approximately 12 weeks of age. Orphan lambs were kept separate from the main flock, and barren ewes were re-introduced into the flock in May 2011.The study ran from October 2010 to August 2011. Fifty ewes were selected for detailed study. Ewes were assigned to one of two groups  by stratified random sampling on body condition, using a scale from 0 to 5 , age de.Ewes were examined during four non-consecutive 4-week periods . Foot lesions were recorded each week, the body condition score (BCS) of each ewe was recorded at week 1, and foot conformation and hoof horn length were recorded in weeks 1 and 4. Hoof length was estimated visually at the mid-point of the abaxial wall and point of the toe of each digit, by one researcher. This was scored as: 0, level with the sole; −0.5, −1 to −5 mm; −1.0 = −6 to −10 mm etc. for wall or toe horn length proximal to the sole, and; 0.5, 1 to 5 mm, 1.0, 6 to 10 mm etc. for wall and toe horn length distal to the sole.In addition to the 4-week periods of routine observation, the locomotion of the flock was scored each week by a trained researcher (EMS), using a validated 0–6 scoring scheme . When a FR was defined as ID with or without separation of the hoof horn from the sensitive tissue beneath together with a characteristic smell. Ewes in Inject with ‘FR and locomotion score >2’ (FR >2) were treated with 2000 mg oxytetracycline  by intramuscular injection and topical application of oxytetracycline  to all feet. Ewes in Trim with FR >2 were treated by trimming all feet of the lame ewe to remove areas of overgrowth and under-running horn and topical application of oxytetracycline to all feet.All treated ewes were monitored for 2 weeks and, with the exception of the first 8 weeks of pregnancy, were locomotion scored and their feet examined each week. If a ewe had FR >2 for 2 consecutive weeks, treatment was repeated and the ewe monitored for a further 2 weeks. Ewes in Trim that had FR >2 for 4 weeks consecutively were given 2000 mg oxytetracycline and their feet were sprayed with topical oxytetracycline.doi:10.1016/j.tvjl.2014.05.021). Each week the data were loaded into an Access database (Microsoft) located on a secure server at The University of Warwick. Weather data for the farm site were downloaded each month from a private weather station websiteAll data were recorded on custom-designed paper recording sheets , level 2 (jk), and level 3 (k), k, vjkf and ijku are the level 3, 2 and 1 residual variances, respectively.Correlations between the wall and toe horn lengths were investigated within digit, foot and ewe. The patterns of wall and toe lengths were plotted over time. Differences in the variation in measures at the start and end of the study were assessed using F-tests of variance. The impact of 2-week mean  daily temperature in the 2 weeks preceding the observation, 2-week mean daily rainfall, age, body condition, locomotion score, FR and treatment for FR on hoof horn length were examined using a mixed effect continuous outcome model that took the form:ijk = a binary variable for whether a FR-affected foot came from a sheep with locomotion score >2 or not, with a logit link with β0 = intercept, βx is a vector of fixed effects varying at level 1 (ijk), level 2 (jk), and level 3 (k) where i is observations, j is foot and k is ewe, kf, and jkv are the level 3 and 2 residual variances respectively, level one was constrained to a binomial distribution. For this model, hoof horn length was categorised into a binary variable overgrown 0/1 where 0 = horn proximal to, or level with, the sole and 1 = horn distal to the sole. Model fit was tested using the Hosmer Lemeshow test.The associations between the occurrence of FR >2 and temperature, rainfall, force of infection (number of sheep lame with FR or ID in previous weeks), length of hoof horn and ewe body condition in the preceding and current weeks were explored for all 50 ewes. A mixed effect logistic binomial regression model was built that took the form:doi:10.1016/j.tvjl.2014.05.021). Four ewes were lost to follow up during the trial. Over the 46 weeks of the study, there were 9920 foot length observations  observation i at time t was associated with significantly longer hoof horn length in the affected foot. All sheep with locomotion score >2 had longer hoof horn before treatment. Compared with untreated sheep, sheep in the Trim group 1 and 2 weeks after treatment had significantly shorter hoof horn length while sheep in the Inject group had significantly longer hoof horn length (doi:10.1016/j.tvjl.2014.05.021).The maximum and minimum temperatures were highly correlated and so only 2-week rolling mean minimum temperature was left in the final model. A locomotion score of 2 (nodding of the head and a shortened stride) 1 week before . There was no significant difference in the number of treatments by group . Sheep wP > 0.05).There was no association between BCS or hoof horn length and FR >2 in the week preceding its development. However, once the ewes were lame, they were significantly more likely than non-lame ewes to have a BCS = 1–1.5 and significantly less likely to have a BCS = 5 . In addiThis is the first prospective study of the relationships between climate, hoof horn length and FR in ewes. One purpose of this research was to elucidate whether sheep with overgrown hoof horn were more likely to become lame with FR or vice versa.Horn overgrowth was modelled at foot level and once feet were affected with FR horn was overgrown. Explanations for overgrown hoof horn on lame feet include: (1) not weight-bearing on an affected limb and so hoof horn is not worn away but continues to grow; (2) horn in diseased feet growing more rapidly; or (3) a combination of both. Once a sheep with FR was treated, the hoof horn on the affected limb reduced in length. Immediate trimming created an iatrogenic shortening of hoof horn length while ewes treated with an antibiotic injection and not trimmed did not have significantly longer hoof horn by the end of the study . This isIt has been suggested that animal weight affects pressure on the digits; in the current study thin sheep (BCS = 1 or 2) had longer hoof horn than ewes with BCS = 5. However, this relationship was only present once ewes were lame so the association between low BCS and long hoof horn might be due to lameness rather than weight of the sheep. BCS was also lower in lame sheep, but only once lame. We did not study BCS for sufficiently long after treatment to see whether and when ewes returned to higher BCS but The occurrence of FR >2 was driven by the force of infection, with increasing treatments following a week with increasing numbers of sheep with FR, and possibly with increasing stocking density (from April lambs were running with ewes). The significantly higher odds ratio for development of FR in April compared with other months might be explained by the altered environment of housing. Ewes were housed for 3–21 days in March/April during lambing and so experienced different underfoot conditions for this short period of time. There was also increased stocking density in pastures in April as lambs were born . The negIn the current study, horn length was measured in relation to the sole, not the rate of growth, which requires detailed marking on the hoof horn . MeasuriD. nodosus did not spread when mean daily temperature was <10 °C: in the current study FR spread at mean daily temperatures <10 °C  the effIn this study, hoof horn length in ewes at pasture varied through the year; this can be explained in part by temperature, rainfall and FR. New cases of FR were related to the prior higher prevalence of FR and lower prevalence of ID and also prior higher temperature and greater rainfall. Overgrown hoof horn was not a precursor to lameness but occurred once sheep were lame with FR. Once lame, sheep were also at greater risk of low body condition. Treatment of FR led to a return of hoof horn length to that of non-lame sheep whether hoof horn was trimmed immediately or left untrimmed. One year of prompt treatment of all lame sheep led to more uniform hoof horn lengths and on this farm hoof horn length was self-regulating so there was no benefit from routine foot trimming.None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.