Ascaris lumbricoides infection and antibody levels to this helminth are associated with polymorphisms in locus 13q33-34. We aimed to explore this and other genomic regions to identify genetic variants associated with the IgE responsiveness in humans. Forty-eight subjects from Cartagena, Colombia, with extreme values of specific IgE to Ascaris and ABA-1, a resistance marker of this nematode, were selected for targeted resequencing. Burden analyses were done comparing extreme groups for IgE values. One-hundred one SNPs were genotyped in 1258 individuals of two well-characterized populations from Colombia and Sweden. Two low-frequency coding variants in the gene encoding the Acidic Mammalian Chitinase  were found enriched in high IgE responders to ABA-1 and confirmed by genetic association analyses. The SNP rs4950928 in the Chitinase 3 Like 1 gene (CHI3L1) was associated with high IgE to ABA-1 in Colombians and with high IgE to Bet v 1 in the Swedish population. CHIA rs10494133 and ABDH13 rs3783118 were associated with IgE responses to Ascaris. SNPs in the Tumor Necrosis Factor Superfamily Member 13b gene (TNFSF13B) encoding the cytokine B cell activating Factor were associated with high levels of total IgE in both populations. This is the first report on the association between low-frequency and common variants in the chitinases-related genes CHIA and CHI3L1 with the intensity of specific IgE to ABA-1 in a population naturally exposed to Ascaris and with Bet v 1 in a Swedish population. Our results add new information about the genetic influences of human IgE responsiveness; since the genes encode for enzymes involved in the immune response to parasitic infections, they could be helpful for understanding helminth immunity and allergic responses. We also confirmed that TNFSF13B has an important and conserved role in the regulation of total IgE levels, which supports potential evolutionary links between helminth immunity and allergic response.Helminth infections and allergic diseases are associated with IgE hyperresponsiveness but the genetics of this phenotype remain to be defined. Susceptibility to  Upon infection with helminths humans synthesize specific IgE antibodies to parasite components as well as high levels of total IgE. The intensity of this response differs among exposed individuals –4, whichAscaris lumbricoides infects about 0.9 billion people worldwide [Ascaris infection (ascariasis) is a good model for analyzing the genetics of IgE responsiveness. In addition, ABA-1  is considered a resistance marker for Ascaris and also an Ascaris-specific component since it has no cross-reactivity with house dust mite (HDM) allergens [Ascaris.The intestinal helminth orldwide , inducinorldwide , high leorldwide . Therefollergens , 14. ABAAscaris have identified several associated loci including the signal transducer and activator of transcription 6 (STAT6) [ADRB2) [TNFSF13B) [LIG4) [Ascaris and Ascaris-specific components such as ABA-1 in humans. Also, it remains to be defined whether the IgE responses to helminth and environmental allergens are under the same genetic control, which is of evolutionary significance given the features that anti-helminth immunity and allergic inflammation have in common [Genetic studies on the susceptibility to  (STAT6) , 16, β2- [ADRB2) , tumor nNFSF13B) –20, and n common , 22. Thin common , 16, 23 n common  that evaAscaris [Ascaris [Ascaris, (2) from these variants to select a panel for genotyping two populations with different genetic and environmental backgrounds and (3) to investigate whether variants influencing IgE response to Ascaris are associated with the IgE response to non-parasite allergens. We here detected significant associations between polymorphisms in chitinase related genes and the intensity of the specific IgE response to the Ascaris resistance marker ABA-1 supporting that genetic factors play an important role in host responses to this parasite. We also add evidence suggesting that genes at 13q.33 locus are involved in the regulation of total and specific IgE response in humans.We previously observed a great inter-individual variation in the IgE antibody response to Ascaris , 4, 25 t[Ascaris . In the This study was conducted following the ethical principles for medical research stated in the Declaration of Helsinki. The Bioethics Committee of the University of Cartagena (Res. 26/06/2009) and the Swedish Regional Ethics Committee (Drn. 2011/1051-31) approved the study. Written informed consent was obtained from all subjects. Parents/guardians provided informed consent on behalf of all child participants.th and ≥75th percentiles) of specific IgE levels to Ascaris and ABA-1 were included . For the genotyping phase, samples from 1258 individuals from two independent cohorts (Colombia and Sweden) were selected to analyze genetic associations with total serum IgE, specific IgE to Ascaris, ABA-1 and other non-parasitic allergenic sources.For resequencing phase, forty eight subjects from Cartagena, Colombia  at the extremes of the distribution  were included to provide more subjects with specific allergen IgE sensitization and to model the effect of genotypes on total IgE levels.. Asthma was defined according to the Global Initiative for Asthma criteria using a standardized questionnaire previously tested in patients with a history of physician-diagnosed asthma. A physician belonging to the research staff confirmed the diagnosis. Subjects meeting the following criteria were recruited: current asthma, ≥8 years old and a history of ≥2 years of asthma, ≥3 episodes of asthma symptoms  in the last 12 months or absence of symptoms due to the use of antiasthmatic medications. Children under 8 years of age were excluded to avoid asthma misdiagnosis due to the high prevalence of transitory wheezing in this age range. Unrelated control subjects without a history of asthma, allergy or other diseases were recruited randomly from the same neighborhoods as the patients, using a questionnaire. All participants lived in an urban, non -industrialized setting, belonging to the lower three (out of six) socio-economic strata in the city, where most people are naturally exposed to HDM [A. lumbricoides and receive periodically anthelminthic treatment. The genetic background of this population resulted from racial admixture between Native Americans, Spaniards, and an important proportion (37.9%) of African ancestry [260/A280 ratio between 1.8 and 2.09 . Both cases and controls had total IgE and specific IgE to Ascaris and HDM and statistical analyses were adjusted by disease status.The Colombian cohort  consists of 988 subjects; 597 non-asthmatic controls and 391 asthmatics d to HDM  and A. lancestry , 28. Theancestry . Each DN We assumed that there is no exposure to Ascaris in this cohort, therefore it was not tested for Ascaris allergens and the analyses on the IgE responses to Ascaris and ABA-1 were done in the CGA cohort.The Swedish cohort (Swedish Eczema Study) comprised 170 atopic-dermatitis (AD) patients and 100 healthy controls. They were recruited from the Stockholm area and examined by a dermatologist at the Dermatology and Venereology Unit, Karolinska University Hospital in Stockholm, Sweden, during September until May to avoid the summer season as previously described , 30. IncAscaris extract and ABA-1  were detected by ELISA as described previously [et al [In the CGA cohort, total IgE was determined in duplicate using an enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions. Specific IgE to yprotein , 32 as weviously . In the y [et al . In briey [et al  and the CHIA, CHI3L1, FCER1A, IL10, TSLP, IL5, RAD50/IL13, IL4, IL33, STAT6, LIG4, ABHD13, TNFSF13B and IRS2) to evaluate their genetic variation and select markers of IgE hyper-responsiveness for further association studies.Targeted resequencing was performed in 14 genes (Genomic coordinates of the coding and non-coding regions of the 14 genes included in the study were extracted via the UCSC browser. A library of RNA baits (120 mer) was designed using e-Array and produced by chemical synthesis (Agilent Technologies). For targeted enrichment, 3 μg genomic DNA from each individual was fragmented by sonication (Covaris S2 instrument) and then linked with specific adaptors and indexes. The samples were incubated overnight with the biotinylated RNA baits . After targeted selection using magnetic streptavidin beads, the enriched regions were eluted according to the manufacturer's instructions. After amplification, the samples were sequenced using a 100 bp sequencing protocol (paired-end). The sequencing runs were performed according to manufacturer’s instructions (Illumina) with a setup aiming for a minimum coverage of 30X in the targeted regions. The production of the libraries and the sequencing procedures were done at Science for Life Laboratory in Stockholm, Sweden. The flow chart for data analysis is presented in www.qiagen.com/ingenuity) from QIAGEN Redwood City.Sequence reads passing Ilumina’s chastity filter were aligned to human genome reference version 19 (hg19) and post-processed for variant calling. Read alignment was done for each sample with BWA version 0.6.2  and sampAscaris and ABA-1 was calculated between groups of high IgE responders  and low IgE responders . Ingenuity Variant Analysis software identifies genes that exhibit significant differences in variants of low frequency (MAF < 0.05) between groups. The statistical test is based on an extension of the Optimal unified Sequence Kernel Association Test (SKAT-O) and can be used to find variants associated with dichotomous and quantitative traits [Ascaris and ABA-1. Variants with a p value less than 0.05 after Bonferroni corrections and with an odds ratio greater than or equal to 1.5 between high and low responders were considered statistically significant. In addition, for quantitative traits the analysis was corrected by age, gender and asthma status.The.vcf files containing all the variants per individual were uploaded to the Ingenuity Variant Analysis software. The burden of variants according to specific IgE levels to e traits . StartinS1 Table. The variants for genotyping were selected based on the following criteria: (1) A statistically significant association in the variants burden analysis with specific IgE levels to Ascaris and ABA-1; (2) the most informative TagSNPs around the regions associated in the burden analysis (3) SNPs with a clinical or functional association with total IgE levels in PubMed and dbSNP (NCBI); (4) SNPs predicted to affect transcription factor-binding sites by different publicly available bioinformatics tools (i.e. F-SNP and Genomatix software suite v 3.1) and/or (5) related to significant promoter or enhancer chromatin state annotations based on ENCODE data (explored using Haploreg2). Ninety SNPs passed the quality criteria and were further analyzed in both populations. Primer for multiplex PCR and extension reactions were designed by the SpectroDesigner software . PCR and extension reactions were performed according to manufacturer’s standard protocols. Concordance analysis with HapMap data was performed. Ten novel variants that were found enriched in high IgE responders in the burden analysis were included in the multiplex PCR reactions (of which seven were successful). The required 50 bp upstream and downstream flanking regions were extracted from the UCSC Genome Browser using Galaxy utilities .One hundred-one SNPs were selected for genotyping by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry . A detailed list of SNPs genotyped is presented in th) were done in PLINK v 1.09 (http://pngu.mgh.harvard.edu/~purcell/plink/). IgE levels according to genotypes were compared using non-parametric tests . The associations with IgE levels as a continuous variable were modeled by using median regression and quantile regression with the package (quantreg) implemented in R. The regression models were adjusted by age, gender and clinical condition (asthma in CGA and atopic eczema in Swedish cohort) considering the confounding effect of these covariates on IgE levels (padj). The significant level was set at p < 0.05.The genetic association analyses between the genotyped variants (n = 91) with the risk of being a high responder (HR) (≥ percentile 75S2 Table. The highest numbers of variants were found in IRS2 (n = 341) and IL-33 (n = 311) and the lowest in IL-5 (n = 17). There were remarkable differences in the distribution of the 75 coding variants among the loci studied, suggesting different degrees of conservation. For instance, the gene CHIA encoding for the acidic mammalian chitinase (AMCase) contained the highest number of exonic variants with 14 non-synonymous, 6 synonymous, 1 stop-gain and 1 frameshift, while the genes encoding the cytokines IL-10 and TSLP had no coding variants. Based on the ratio of the number of observed variants to those expected from the gene size, the most polymorphic genes were LIG4, IRS2, IL13 and CHI3L1.Targeted resequencing in 48 individuals from the CGA cohort resulted in 2423 variants (1851 located on the targeted genes). Of these, 1290 were already known (dbSNP137) and 561 novel (without a reference SNP ID number in dbSNP build 137) including 1663 single nucleotide substitutions and 188 indels. A summary of targeted resequencing metrics by gene region is presented in CHIA, CHI3L1, TSLP, IL13, LIG4, ABHD13, IRS2 and STAT6) were enriched in high IgE responders to whole Ascaris antigen and ABA-1 (IgE level ≥ 75th percentile) and absent in low responders. These included 65 single nucleotide variations (SNVs), three insertions and two deletions, 70% of them being non-coding variants. Coding variants included 11 missense; 8 synonymous, 1 stop gain and 1 causing a frameshift change. The distribution of variants enriched in high IgE responders to Ascaris is shown in We analyzed 1955 variants (out of 2423) with call quality of 100 in the Phred Scale and a read depth of 30x for their enrichment according to the intensity of the IgE levels . Seventy variants, distributed among 8 genes  that were enriched in high IgE responders to Ascaris extract and ABA-1. They were located 508 base pairs apart and in strong linkage disequilibrium (D’ = 0.99). These variants were tagged by an intronic SNP , observed in 14% of the individuals in the CGA dataset and associated with IgE levels to ABA-1 above percentile 75th under additive  and dominant models , Fig 2A.(Table 5).Haplotype analysis revealed a significant increased risk of high IgE levels to ABA-1  in carriers of the minor allele A in the two coding variants and the minor allele C in rs10494133 . Since only one individual was homozygous for the A/A genotype in rs79500525 and rs139812869, dominant code for quantile regression (GG vs. GA + AA) was used, confirming that the allele A was significantly associated with increased IgE levels to ABA-1 (p = 0.03). The tagSNP CHIA rs10494133 T/C was also associated to high IgE responses to Ascaris under additive  and dominant models , driven by a higher frequency of the minor allele C in subjects with IgE levels to Ascaris above percentile 75th. This was confirmed by quantile regression analysis (p = 0.01) but was not significant after adjusting by age, gender and the presence of asthma (padj = 0.2). There was also association between high IgE response to ABA-1 and STAT6 rs73118440 (G/T) under additive model and quantile regression analysis, Fig 2B. Neither CHIA nor STAT6 variants were associated with IgE responses to HDM or other common allergens.We then implemented quantile regression analyses to model the effect of these genetic variants on IgE levels to ABA-1 as a continuous variable. The tagSNP rs10494133 (T/C) was associated with increased IgE levels to ABA-1 independently of age, gender or the presence of asthma  under additive model, dominant model and quantile regression analyses (. In patients from the Swedish cohort (n = 170) the SNP rs17565502 (A/C) located in the gene encoding B cell activating factor (TNFSF13B) was associated with an increased risk of high IgE responses to the cat allergen Fel d 1(>4.52 ISU). This association was significant under the additive model and quantile regression analysis.To address whether loci influencing IgE levels to . In the CGA cohort CHI3L1  was associated with high IgE response to ABA-1 under recessive model, which was confirmed by quantile regression analysis, Fig 3A. Also, in patients from the Swedish cohort two variants in the CHI3L1 gene (rs4950928 C/G and rs880633 C/T) were associated with high IgE response (≥75th percentile) to the birch pollen allergen Bet v 1 (>10.3 ISU) . It is worth mentioning that these SNPs are 3081 base pairs apart, in strong LD (D’ = 0.94).Variants in two genes were associated with both Ascaris and common allergens ABHD13 rs3783118 A/C, located in the Ascaris susceptibility locus (Cr. 13q33.3), was under-represented in the group of high responders to Ascaris under the dominant model. Quantile regression confirmed this finding showing significant association with lower IgE levels (padj = 0.00001). In the CGA dataset we found that, as occurred with the Ascaris extract, ABHD13 rs3783118 was associated with lower levels of IgE to D. pteronyssinus under dominant model. This effect was driven by one or two copies of the minor allele C (padj = 0.03), Fig 3B.The th percentile) in allergic patients from the CGA dataset and the Swedish cohort. In the CGA cohort TNFSF13B rs17565502 A/C was associated with high total IgE levels (≥ 1074.5 IU/ml) under the additive model. In the Swedish cohort TNFSF13B rs8181791 A/G was associated with high total IgE levels (≥852.5 kU/l) under additive and recessive models . Both associations were also observed when total IgE levels were modeled as a continuous variable by quantile regression . Other variants associated with high total IgE were the coding SNP IL13 rs20541 G/A; IRS2 rs12584136 C/A and the IL5 rs2069816 A/C. Fig 5 summarizes the main genetic associations found in the study.We investigate associations with high total IgE levels , however, this study was designed to study QTL for total and specific IgE and is underpowered to address associations with disease phenotypes.CHIA and CHI3L1 (chromosome 1) overrepresented in high IgE responders to Ascaris and ABA-1 (here a purified recombinant IgE binding protein of Ascaris).Tropical settings provide several advantages for investigating the molecular genetics of the IgE responses in helminthiases and allergies. First, due to the high prevalence of these conditions , 41, 42,There is evidence for considering ABA-1 as a resistance marker for ascariasis , but amoCHIA has 13 exons spanning around 29 kb and encoding AMCase, a protein with functional catalytic and chitin-binding domains in humans. This enzyme is produced by epithelial cells and alveolar macrophages, and Th2 cells are potent stimulators of its expression at both mRNA and protein level [CHI3L1 is a gene of 8 Kb, which encodes YKL40, a 40 kDa heparin and chitin-binding glycoprotein. Chitinases and chitinase-like proteins may act directly as chemotactic agents or by inducing other chemokines that attract eosinophils and T cells to the sites of parasitic infection. AMCase activity is required for the increased expression of chemokines involved in the recruitment of monocytes, macrophages, eosinophils and neutrophils. Also, it reduces the expression of the Th1 chemokines interferon gamma-inducible protein 10 (IP-10) and interferon-inducible T-cell alpha chemoattractant (I-TAC), thus contributing to a stronger Th2 response [+ T cells [CHIT1 deficient individuals from South India were more susceptible to Wuchereria bancrofti infection [e.g. rs880633 Arg145Gly), our findings suggest that they are relevant in the regulation of the intensity of the specific IgE response to ABA-1, which is potentially important information for understanding the genetic susceptibility to ascariasis. It is worth adding that CHIA rs10494133 was also associated with the intensity of the IgE response to the Ascaris extract, which can be explained because ABA-1 is abundant in this extract. However, the underlying mechanisms of these associations remain unknown and require functional studies.This is the first report of association between chitinase related genes and the IgE responsiveness to a nematode allergen (ABA-1). in level . CHI3L1 response . Further T cells . These f T cells ; pertinenfection , althougnfection , 50. OurSTAT6 is involved in the susceptibility to Ascaris. Gao et al [Our results also support previous findings suggesting that ao et al  showed tABHD13 with the IgE responsiveness to Ascaris supports previous findings linking the 13q33 locus with the susceptibility to Ascaris-infection and the regulation of IgE responses to the parasite [ABHD13 but we now found that the rs3783118 variant was associated with lower levels of specific IgE to Ascaris and D. pteronyssinus. This variant generates a new binding site for the transcriptional repressor Foxp1 (Fkhd domain) that plays an important role in the differentiation of lung epithelium and is an essential transcriptional regulator of B-cell development [Ascaris and HDM extracts [ABHD13 and the IgE responses to both sources may involve cross reactive components. Interestingly the SNP was not associated with the IgE response to ABA-1, which does not cross-reactive immunologically with HDM [The association of parasite –20. In oelopment . Since textracts  the assowith HDM .Ascaris or the purified allergen ABA-1 are associated with resistance to this nematode and decreased worm burden [Ascaris, it is feasible that genetic regulation of antibody production may play a role on Ascaris susceptibility.We did not include parasitological data in this study; therefore, the associations with IgE levels are not necessarily related to resistance to Ascaris infection. However, immunity to helminthiases involves a wide spectrum of effector mechanisms including the specific IgE response –54. The m burden , 57. Sinm burden  identifiTNFSF13B present in both populations, Swedish [TNFSF13B on this phenotype supports potential evolutionary links between helminth immunity and allergic responses. TNFSF13B encodes B cell-activating factor (BAFF), a well-known major regulator of B cells development that has a critical role on the production of IgA and IgG and the synergic effect with IL-4 on the class-switching to IgE [A. lumbricoides [2 = 0.53) when analyzing a large pedigree from the Jirels population in Nepal [A. lumbricoides. In addition, the fact that the association was also found in subjects from Sweden, where intestinal parasite infections are not endemic, rule out the potential confounding effect of helminthiases. Genomic region 13q33 is evidently of interest for further fine mapping and association studies in larger populations. Recently, another region in the chromosome 13 (13q21.31) has been suggested as locus regulating total IgE levels [Another aspect of the 13q33 locus is its influence on total IgE levels. In this study it was driven by variants in  Swedish  and Colo Swedish . This sug to IgE . In addig to IgE , and in in Nepal , which iE levels .IL13 rs20541, which has been associated with allergic phenotypes and helminth susceptibility in parasite exposed populations [IL5 and total IgE levels in the Colombian population replicating previous findings on the influence of this gene on IgE production [Another variant influencing total IgE levels in the Swedish population was ulations –66 suggeoduction , 68.ABHD13 rs3783118, genetic variants associated with specific IgE reactivity to Ascaris or ABA-1 were not associated with the IgE response to HDM, suggesting that in the CGA dataset specific IgE responses to parasite and HDM allergens are controlled by different genes, which seems to contradict the idea that allergic response is a side effect of helminth immunity; however, in the Swedish cohort the IgE response against two common inhaled allergens (Bet v 1 and Fel d 1) were associated with CHI3L1 and TNFSF13B respectively, which could reflect differences in the regulation of gene expression by environment. Recently, using bioinformatics tools, it was found that Bet v 1 shares an IgE binding epitope with a Bet v 1 like protein (SmBv1L) from Schistosoma mansoni [CHI3L1 suggest that chitinase related genes may have evolved in the context of more primitive immune responses than those elicited by non-parasite allergens.Excepting  mansoni . The autAscaris and its resistance marker ABA-1. Most associations were restricted to the response to this parasite and not to other allergens such as HDM. We also confirm previous associations, especially the relevant role of locus 13q.33 in modulating total and specific IgE levels.In summary, we have uncovered genetic variants strongly associated with the IgE response to the nematode S1 FigSamples from the Colombian Dataset are indicated by the acronym CGA; Individuals were classified as high IgE responders (HR) or low IgE responders (LR) based on the percentile corresponding to their IgE levels; SKAT-O: Optimal unified Sequence Kernel Association Test; HDM: House Dust Mites; AE: atopic eczema. *Adjusted by age, gender and disease status. **For this trait only data from patients was analyzed.(PDF)Click here for additional data file.S1 TableSuccess rate for genotyping (%), the exact p-value for the calculations on Hardy-Weinberg equilibrium in controls (HWE) and the minor allele frequencies in each population are indicated in columns. The variants without rs number correspond to novel single nucleotide substitutions as detected in CGA.(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Translaminar screw fixation (TSF) of the axis is considered as an efficient, safe and simple surgical procedure, however the study of the potential risk of TSF to the transverse foramen in lower cervical spine is lacked. Head-neck CT images of 60 patients were included in this study. Maximum screw length, laminar thickness, the screw angle and the laminar height were measured. The feasibility of 3.5-mm diameter screw fixation and the potential risk of transverse foramen injury was analyzed. The TSF was safe at C3 and C4, but risky to the transverse foraman at a rate of 8.7% at C5 (0% on the left side and 20% on the right side), 33.3% at C6 (24.4% on the left side and 42.9% on the right side). C7 had the highest 77.8% rate (65.5% on the left side and 89.8% on the right side). The safe screw length was 27.7 mm at C3, 27.4 mm at C4, 28.0 mm at C5, 25.6 mm at C6 and 25.5 mm at C7, respectively. The present study showed that translaminar screw could place the transverse foramen of C5–C7 at risk. Preoperative CT scanning was necessary for safe screw placement. The adult patients with cervical spondylotic myelopathy or cervical spondylosis radiculopathy, requiring thin layer CT scan of the cervical spine were included. Patients were excluded if they had any congenital deformities, history of spinal surgery or traumatic. We firstly enrolled 69 cases, and 9 cases did not meet inclusion criteria . A total of 60 adult patients  with mean age of 57.5 ± 15.1 years was available for analysis.Images were obtained from CT scans  with slice thickness of 1.0 to 1.5 mm, collimation of 0.75 to 1.5 mm, pitch of 0.7 mm, 120 kV, 180 mA, 512 × 512 matrix, and reconstruction level of 1 mm. Images of the sagittal and axial planes of the CVJ region were obtained after multiplanar reconstruction on the workstation . Bone windows were used for analysis.The translaminar screw entered the lamina from the junction of the lamina and spinous process, then traversed the lamina and entered the lateral mass. In the axial plane, maximal screw length (SL) was measured from the junction of the lamina and spinous process to the contralateral outer cortex of the lateral mass; The screw angle (SA) was measured between a line dropped from the midpoint of the vertebral body to the spinous process and the line of the screw trajectory; laminar thickness (LT) referred to the measurement of the narrowest portion of the lamina . In the 1324For the lamina which was feasible for a 3.5-mm screw along the axial direction of the lamina, a dotted line extended from the screw was drawn. If the extension dotted line passes into the TF, the lamina was defined as “at risk” as the wall of TF may be broken with a longer screwTwo observers blinded to clinical information performed CT morphometric measurements. The intraobserver and interobserver reliability were calculated, for each parameter in 1 set of 15 patients who were randomly selected at 3-week intervals, using the intraclass correlation coefficient.t-test was used to determine differences between different groups . Pearson chi-square test and Fisher’s exact test were used to compare the virtual screw placement acceptance rates and potential risk rates between different groups. Spearman’s correlation coefficient was calculated to determine the correlation between the risk rates with SA. Differences were considered to be significant at a level of P < 0.05.The statistical analysis was conducted by SPSS 20.0 software . Values were represented as mean ± standard deviation. Single-factor analysis of variance and LSD multiple comparison were used to compare the measurement data among different vertebral levels. The student A total of 60 patients (35 men and 25 women) were available for this study. The mean age was 57.5 ± 15.1 years (range: 21–84 years).There was no significant difference of SL between vertebra levels . The meaUnilaterally, C7 showed acceptance rate above 96%, C3 and C6 showed acceptance rates more than 60%, C4 and C5 showed acceptance rates below 45% . There wThe potential risk for transverse foramen was not noted at C3 or C4 , but theThe minimum screw length at each vertebral level was recommended as the safe screw length. In our study, the recommend safe screw length from C3–C7 was 27.7 mm, 27.4 mm, 28.0 mm, 25.6 mm and 25.5 mm, respectively .The intraobserver reliability ranged from 0.91 to 0.95 for the initial examiner and from 0.90 to 0.96 for the secondary observer. The interobserver reliability ranged from 0.81 to 0.97.in vitro as other techniques, but it may be a suitable salvage technique should pars or pedicle screw fixation failPosterior cervical screw fixation was applied to stabilize the cervical spine in deformity, degenerative, trauma, tuberculous spondylitis and tumor reconstructive surgeries, and it can be accomplished through a variety of screw techniques including transarticular screws, lateral mass screws, pedicle screws, and, more recently, translaminar screws. Lateral mass screws are relatively safe and easy constructs to insert, but screw loosening or avulsion has still been reported as a failure mechanism for lateral mass screws because of the lower pullout strength. Pedicle screw fixation is the most biomechanically stable technique, but they may carry a high risk of neurovascular complications, and can be technically difficult2032However, the present study showed translaminar screws may jeopardize TF in the lower cervical spine depending on individuals and vertebral levels. The risk of translaminar screws was higher at C6 and C7, but free at C3 and C4. Moreover, the highest acceptance rate of the translamiar screws was in C7, while the lowest in C4.et al.2 at C3, 22.48 ± 8.19 mm2 at C4, 26.40 ± 4.05 mm2 at C5, 28.92 ± 8.16 mm2 at C6)Riesenburger RI et al.et al.Feasibility of lower cervical translaminar screw placement based on computerized tomographic measurements was studied by Alvin MD et al.et al.Consequently, our results indicate that the spine surgeon should review CT scan images preoperatively to evaluate the safety and feasibility, and to determine the optimal screw length, angle, ideal entry points and trajectories, especially when a longer screw will be placed at C5–C7. To avoid the injury to the TF, adequate screw length and a larger screw angle were recommended for C5–C7 translaminar screw placement. We recommended that the safe length of the translaminar screw from C3–C7 were 27.7 mm, 27.4 mm, 28.0 mm, 25.6 mm and 25.5 mm, respectively. At C3 and C4, the screw with appropriate length could avoid damage to the outer cortex of the lateral mass, and at C5–C7, to the outer cortex of the lateral mass and the wall of TF. Ventral spinal canal violations have been considered as a major drawback of translaminar screwThere were some limitations of this study. Our study population was comprised of 60 patients all come from China and may not have been sufficiently large to be generalized to other ethnic groups. We only used CT images to evaluate the potential risk to TF of lower cervical translaminar screw, and the measurements may not predict clinical outcomes directly.Lower cervical translaminar screw placement was a reliable alternative to other earlier techniques, but at C5–C7, it could still place the transverse foramen or even the vertebral artery at risk. We suggested that preoperative CT scanning was obligatory for all vertebral levels. Adequate screw length and a larger screw angle were recommended especially during C5–C7 translaminar screw placement.How to cite this article: Kong, G. et al. The risk of translaminar screw fixation to the transverse foramen of the lower cervical spine: a computed tomography study. Sci. Rep.7, 46611; doi: 10.1038/srep46611 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The ability to forecast sea ice (both extent and thickness) and weather conditions are the major factors when it comes to safe marine transportation in the Arctic Ocean. This paper presents findings focusing on sea ice and weather prediction in the Arctic Ocean for navigation purposes, in particular along the Northeast Passage. Based on comparison with the observed sea ice concentrations for validation, the best performing Earth system models from the Intergovernmental Panel on Climate Change (IPCC) program (CMIP5—Coupled Model Intercomparison Project phase 5) were selected to provide ranges of potential future sea ice conditions. Our results showed that, despite a general tendency toward less sea ice cover in summer, internal variability will still be large and shipping along the Northeast Passage might still be hampered by sea ice blocking narrow passages. This will make sea ice forecasts on shorter time and space scales and Arctic weather prediction even more important.The online version of this article (doi:10.1007/s13280-017-0951-5) contains supplementary material, which is available to authorized users. The Arctic climate is subject to a drastic change. A key element in Arctic climate change is the rapid decline in sea ice coverage and thickness seen in observations in particular since 2000. Research is needed to improve understanding of sea ice development and weather forecasting abilities since an increased human use of the Arctic in all kinds of economic sectors is anticipated. Here we report on results with respect to the potential future of large-scale sea ice development in the Arctic as they are relevant for shipping. We also make suggestions on how to improve weather forecasts, which are essential for regional sea ice forecasting on shorter time scales. The results described here also provide relevant input on the environmental conditions to studies of the potential development in other economic sectors such as oil and gas exploration and fisheries  program called Coupled Model Intercomparison Project phase 5 (CMIP5)  and we need to derive information on finer spatial scales. Previous studies which analyzed sea ice along shipping routes based on CMIP5 model results derived projections on finer spatial scales by re-gridding the original coarser CMIP5 model grids , more than 30 global Earth system models were used to project the development of the global climate, assuming a set of different scenarios for the potential future release of greenhouse gases  in March (September)  Fig. . Ensembl3 to about 22 000 km3 corresponding to a sea ice loss of 10 000 km3 at the end of recent winters compared to earlier winters. Remarkably, PIOMAS indicated a similar trend in summer with 16 000 km3 Arctic sea ice volume at the end of the melting season (September) 35 years ago, versus 4000 km3 during more recent years corresponding to a sea ice volume decrease of 12 000 km3 at the end of recent summers compared to earlier summers. According to this simulation, the total sea ice volume at the end of the summer season today is only 25% of what it was 35 years ago and corresponds to a summer sea ice extent half the size as before, combined with an overall thinning of sea ice by half over the same period.PIOMAS simulates the northern hemisphere sea ice volume seasonal cycle showing a similar trend in winter (April) and summer (September) over the past 35 years Fig. . Over th3 over the 35-year period indicative of a stronger seasonal cycle. Still, compared to the sea ice loss both at the end of the winter (10 000 km3) and at the end of the summer (12 000 km3) over the same period of time (past 35 years), this increase for sea ice growth and sea ice melt is 5–6 times less and is simply resulting from the fact that there is more and more open water to freeze up in each fall–winter season.From the winter sea ice volume maximum and the summer sea ice volume minimum, we can easily deduce the ice growth from October to April and the ice melt from May to September, for each year over the past 35 years. It is interesting to compare sea ice growth  corresponds to Arctic warming superimposed to natural interannual variability. There is more likely no precise time prediction when Arctic will be summer ice free because of the interannual fluctuations. The natural variability of the Arctic sea ice cover, superimposed on a long-term trend due to anthropogenic climate change, plus model uncertainties, makes a prediction of a precise timing for a quasi-ice-free Arctic in summer uncertain. This kind of prediction is marginally important compared to the real situation where 75% of the Arctic sea ice has already melted away at the end of the summer. More likely, it will not take very long (one or two decades) to melt away the remaining 25% of Arctic sea ice by the end of the summer season and this is what really matters for a number of critical issues such as shipping activities and other human activities across the whole Arctic Ocean.In addition to helping improve the understanding and prediction of the coupled climate system, model simulations in the framework of ACCESS have been used to provide guidance on what range of future sea ice conditions may be encountered in areas where it affects human activities, such as shipping. Here we make use of the DEXP8.5 experiment with finer spatial resolution, zooming into the regions most relevant for long-distance shipping in the Arctic. We focus on the sea ice conditions in near-coastal areas and along potential shipping routes, in terms of sea ice concentration, thickness, and the length of periods with ice conditions that are favorable for shipping. As different ship types have different limits regarding their capability to navigate in different sea ice thickness and concentration conditions, we analyze the number of days for which sea ice thickness (sit) and sea ice concentration (sic) fall below specific thresholds relevant for shipping. The analysis is based on daily mean sic and sit from experiment DEXP8.5. Figure An alternative view on the ice conditions as they are relevant for shipping is presented in Fig. The model results and the analysis in terms of parameters relevant for shipping suggest that despite an overall increase of navigability in the Arctic, still a lot of variability of sea ice cover needs to be taken into account over the coming decades. This will make a reliable weather forecast, which is also the prerequisite for a short-term sea ice prediction, even more important.The possibilities in the Arctic for increasing activities such as ship traffic and resource exploitation also mean that operators need to deal with risks related to the physical Arctic environment . Operational challenges include risk factors such as low temperature, occurrence of high winds, fog, and darkness during the winter season. Marine operations might additionally be influenced by ocean waves, icing from sea sprays, and the presence of sea ice and icebergs. These environmental factors may occur in combination, thus increasing the operational challenges. In remote areas, the infrastructure and capability to manage difficult situations, hazards, or accidents may be distant or even unavailable in many cases.This emphasizes a need for risk management for operators in the Arctic  models are the main tool in such forecasting. Work undertaken at the Norwegian Meteorological Institute (MET Norway) as a part of ACCESS has assessed the forecasting capabilities in the Atlantic sector of the Arctic by investigating the forecast performance of the global ECMWF (European Centre for Medium Range Weather Forecasting) NWP and the regional HIRLAM (High Resolution Limited Area Model) model previously run at MET Norway. Pressure at mean sea level (MSLP) is a meteorological quantity not easily felt, unlike wind or temperature, but still the pressure field is closely connected to the weather systems. For instance, away from topography, the wind is closely connected to the pressure gradient through the so-called geostrophic relation. Pressure is much less influenced by local topographic and surface conditions than wind or temperature, and thus comparing pressure verification statistics at different locations is very well suited for getting a picture of how the NWP models are able to capture the main large-scale weather system variations.Figure A candidate for explaining the decline in the quality toward the North would be the corresponding decrease in the observation density of the conventional meteorological observing network. The NWP forecasts depend on determining the initial state of the atmosphere using observations in the so-called data assimilation. For surface observation data, there is a general gap in pressure observations over parts of the sea ice domain and parts of the ocean domain as there is only limited coverage from drifting buoys. There is almost no coverage of near-surface wind observations over sea ice. The wind coverage over ocean is good due to satellite scatterometers providing surface wind observations  observations in the area are too scarce to have a significant effect on forecasts;satellite remote sensing data for vertical profiling of the atmosphere (sounding data) play an important role in improving forecasts quality. This clearly highlights that satellite information will be important also in the future evolution of the Arctic observing system and that enhancing the extraction of information from such satellites could be a key area.During the period of the ACCESS project, a novel state-of-the-art convection resolving regional NWP system covering a high-latitude area, the so-called AROME-Arctic model, was implemented at MET Norway. This is a version of the HARMONIE-AROMEAccounting for the surface contribution to the signal over sea ice: Sounding channels with significant contribution to the measured signal from Arctic Ocean sea ice surfaces are usually rejected in the data assimilation. If we could account for the contribution from the surface by drawing information on emissivity and emitting temperature in new ways, it will enable us using more channels providing temperature and moisture information in the lower troposphere  instruments for NWP in the Arctic are as follows:Finding scenarios for new satellite missions or programs is expensive and subject to well-established long-term processes and thorough investigations in the international satellite agencies. One can hope that further evolution of the satellite observing system will help fill the observation gap in the Arctic, but this can only happen in the long term WIGOS . Here weWhen looking for options for such extensions, it is attractive to look for alternatives with the lowest cost relative to the impact on forecast. There is extensive literature on the impact of observations or observation types on NWP forecast quality; however, few studies consider also the cost of each observation to rank observation types according to impact per cost. Nevertheless, Eyre and Reid  made theThe three-dimensional nature of the atmosphere and previous experience certainly indicate that profile information (sounding) is important. A relatively cheap way to obtain more conventional sounding data is to perform more frequent radiosonde launches at already existing launch sites (at least twice per day or four times per day). There are a few existing radiosondes stations with staff and necessary infrastructure at some remote Arctic islands. Since basic infrastructure for these launch sites are already in place, increasing the number of launches per day at these sites could be a good solution to provide cost-efficient extended data coverage. Our investigation showed that guaranteeing at least two launches per day of radiosonde at already existing sites provides benefit for the updated initial state  and forecasts of geopotential and humidity, while four times launch per day provides a further significant beneficial impact on analysis and forecast of all verified parameters.Based on the indications from Eyre and Reid , we shouAs far as Arctic navigation is concerned, sea ice extent, sea ice concentration, and sea ice thickness really matter. From a climatic point of view, sea ice volume combining sea ice extent, concentration, and thickness is the key element. Based on one of the most pertinent IPCC Earth system models for predicting Arctic sea ice extent, concentration, and thickness and referring to the best available Pan-Arctic Ice-Ocean Modeling and Assimilation System (PIOMAS) and newly estimated sea ice volume deduced from Freezing Degree Days (FDD), we evaluated potential model predictive capacity for supporting marine transportation across the Arctic Ocean for the next three decades. Both PIOMAS and FDD confirm a drastic Arctic sea ice volume loss of about 75% at the end of the summer season (September) when comparing sea ice volume for recent years with those obtained 35 years ago. This results from a 50% reduction of both sea ice extent and sea ice thickness over the entire Arctic Ocean and over a 35-year time period. Both PIOMAS and FDD sea ice volume-based estimations confirm that the drastic Arctic sea ice volume reduction (and consequently sea ice extent and sea ice thickness) is mainly due to a decrease in winter ice growth which does not compensate for ice loss in summer ice melt anymore. CMIP5 climate simulations confirm the trend for sea ice reduction during most of the years extending over the next 30 years facilitating marine transportation across the Arctic Ocean. Earth system models still have deficits though. The long-term trend of sea ice volume indicated in the assimilated PIOMAS model is stronger than that found in the Earth system models used in the current project. It is unclear how much of the PIOMAS trend is due to climate change and how much is due to internal variability which Earth system models would not necessarily show in the same time period. Observational data show that sea ice variability at the interannual and intradecadal time scale is quite significant. The same variability is not yet properly simulated by Earth system models. This even holds for the seasonal cycle. Earth system models need to improve sea ice production during winter and sea ice melting during summer, also impacting sea ice thickness. Finally, higher resolution is clearly needed when it comes to support planning safe marine transportation across the Arctic Ocean along the Northeast Passage (NEP) and/or the Northwest Passage (NWP), e.g., when taking into account critical narrow passages for ship navigation. Weather forecasts for operational use are also suffering from undersampled radiosondes coverage that is falling drastically at high latitudes. For supporting Arctic operations with better weather forecasting capabilities in the near future, it is recommended to improve both regional and seasonal coverage of drifting buoys by a factor of 3 and an increase of operation/launch of radiosondes to 4 times per day.Supplementary material 1 (PDF 1009 kb)Below is the link to the electronic supplementary material.


In this report, we identify Abl-mediated phosphorylation of a highly conserved Tyr residue in the P + 1 loop of protein kinase D2 (PKD2) during oxidative stress. Remarkably, we observed that the three human PKD isoforms display very different degrees of P + 1 loop Tyr phosphorylation and we identify one of the molecular determinants for this divergence. This is paralleled by a different activation mechanism of PKD1 and PKD2 during oxidative stress. Tyr phosphorylation in the P + 1 loop of PKD2 increases turnover for Syntide-2, while substrate specificity and the role of PKD2 in NF-κB signaling remain unaffected. Importantly, Tyr to Phe substitution renders the kinase inactive, jeopardizing its use as a non-phosphorylatable mutant. Since large-scale proteomics studies identified P + 1 loop Tyr phosphorylation in more than 70 Ser/Thr kinases in multiple conditions, our results do not only demonstrate differential regulation/function of PKD isoforms under oxidative stress, but also have implications for kinase regulation in general.Protein kinases are essential molecules in life and their crucial function requires tight regulation. Many kinases are regulated via phosphorylation within their activation loop. This loop is embedded in the activation segment, which additionally contains the Mg Proper control of these processes requires tight regulation, and deregulation of kinase activity causes a variety of diseases2.Protein kinases are essential as receivers, transmitters and executors of a wide variety of cellular stimuli. Their activation results in a plethora of biological responses such as cellular movement, proliferation, and differentiation4. The activation segment can be subdivided into the Mg2+ binding loop, the activation loop and the P + 1 loop5. In many kinases, the activation loop is phosphorylated, inducing a conformational change of the activation segment, thereby establishing the active conformation4. The P + 1 loop was originally named for its involvement in contacts with the P + 1 amino acid residue in Protein Kinase A (PKA) substrates , but actually makes extended contacts with the substrate. Remarkably, while phosphorylation events in the activation loop are well-documented, kinases can also be phosphorylated in their P + 1 loops42. This phenomenon has become increasingly clear as a consequence of large scale proteomics studies that give unprecedented insight in post-translational modifications (PTMs) in a variety of proteins. However, in most cases the functional consequences of these P + 1 loop phosphorylation events remain undefined.The activity of many kinases is regulated via the conformation of their activation segment, which is defined as the region between the conserved DFG and APE motifs43. The activity of PKD is regulated through auto-inhibition by the C1 and PH domains. Classically, PKDs are DAG responders that in many cases signal downstream of PKC pathways44. They bind at DAG-containing membranes through their C1 domains, where they co-localize with PKC isoforms. PKC subsequently phosphorylates the activation loop Ser-738/742 residues (hPKD1 numbering), resulting in alleviation of auto-inhibition of the PH domain and activation of PKD45.The protein kinase D (PKD) family belongs to the CAMK group of the kinome and consists of three highly homologous members  in humans. They have a modular structure, consisting of two diacylglycerol (DAG) binding C1 domains that connect via an acidic stretch to a PH domain, followed by the kinase catalytic domain46. Tyr-463 phosphorylation induces a conformation permissive for subsequent Src-mediated phosphorylation of Tyr-95 in the N-terminus of PKD147. Phospho-Tyr-95 serves as a docking site for the C2 domain of PKCδ, which phosphorylates PKD1 at its activation loop Ser-738/742 residues, an event that has been shown to be essential for PKD1 activation under oxidative stress49. One of the downstream effectors of stress activated PKD1 is the NF-κB pathway. PKD1 activates the transcriptional activity of NF-κB target genes via the IKK complex; however a direct target of PKD1 in this pathway remains elusive50. Activation of NF-κB via mitochondrial ROS results in expression of MnSOD, thereby detoxifying the cell from damaging ROS51. On the other hand, PKD activated under oxidative stress conditions also increases JNK activity downstream of DAPK in a PKC- and pSer-738/742 independent manner, promoting cell death53.PKDs are also responsive to oxidative stress conditions. Here, PKD1 activation is dependent on the hierarchical phosphorylation of several tyrosine residues. First, Tyr-463 in the PH domain is phosphorylated by Abl47. Furthermore, PKD2 is phosphorylated at tyrosine residues by the BCR-Abl fusion protein in BCR-Abl+ CML cell lines54. BCR-Abl phosphorylates PKD2 at Tyr-438 in its PH-domain .Studies on the activation mechanisms of PKD enzymes by tyrosine phosphorylation have been largely limited to PKD1. However, under oxidative stress conditions, PKD2 is phosphorylated at Tyr-87In this study, we reveal phosphorylation of a key tyrosine residue in the P + 1 loop of PKD2 under oxidative stress conditions. Despite its very high conservation, we observe remarkable isoform-specific phosphorylation, revealing unprecedented insight into differential regulation of the PKD family that impacts on their activity towards substrates.www.phosphosite.org), selecting those found in at least 5 independent studies, see Supplementary Table 2O2 indicated that indeed, Tyr-717 is a target site on PKD2 under oxidative stress conditions  or activator (DPH) of Abl, and then stimulated them with H2O2. Phosphorylation of Tyr-717 was markedly decreased in presence of STI-571, while incubation with DPH, resulting in stronger Abl activation, enhanced Tyr-717 phosphorylation even further  and Ser-706/710 phosphorylation (the last step in the PKD1 activation model) using phosphomimetic and non-phosphorylatable mutants of these sites.Interestingly, we observed that Tyr-717 phosphorylation was partly dependent on Tyr-438 phosphorylation, with some increase in the case of a phosphomimetic substitution, but no decrease in Tyr-717 phosphorylation in a Y438F mutant Fig. .2O2, we observed a potent (about 4-fold) increase in Tyr-717 phosphorylation compared to WT. Conversely, a Ser-706/710-Ala mutant showed complete loss of Tyr-717 phosphorylation  and the PKC activator PDB. Treatment with BimI and Gö6983 resulted in an expected decrease of Ser-706/710 phosphorylation Fig. , but als47, we did not observe a dependency of Tyr phosphorylation on the activation loop Ser phosphorylation state in PKD1, since both Ser-738/742-Glu and Ala mutants had similar levels of overall Tyr phosphorylation  or STI-751 (Abl). We could indeed show that this interaction is not affected by tyrosine kinase inhibition Fig. . To furtted Fig. . This inWe next wondered which  motifs could favor interaction of PKD2 with PKCδ by deleting different regions in the regulatory domain of PKD2 Fig. . We obsein cellulo . Since PKD1 is known to be phosphorylated by Abl in the PH domain, but is not phosphorylated in the P + 1 loop, we reasoned that a local molecular difference between PKD1 and PKD2 might contribute to their differential phosphorylation in the activation segment. Interestingly, while the kinase domains of hPKD1 and hPKD2 are 95% identical, the transition region between subdomain VIII (which ends with the APE motif) and subdomain IX is different between the two isoforms. Indeed, PKD1 contains a positively charged 764RNK766 motif, while PKD2 contains a non-charged, partially hydrophobic 724LNQ726 motif in this region  is entirely conserved in all three PKD isoforms, the antibody we raised against the phospho-Tyr-717 site of PKD2 is fully cross-reactive to the other isoforms. Strikingly, we observed that the degree of Tyr phosphorylation in the YxAPE motif in response to oxidative stress is significantly different between the isoforms. Indeed, PKD2 is most abundantly phosphorylated, with 70% less phosphorylation in PKD3 and 90% less in PKD1 when compared to PKD2 Fig. . To eluculo Fig. . It is kion Fig. . The reg766 Fig. . Conversely Fig. . Similarely Fig. . These dThe tyrosine residue identified as a phospho-acceptor site in this study resides in the P + 1 loop of the activation segment of the kinase Fig. . Phospho2O2-stimulated (Tyr-phosphorylated) PKD2. However, we observed no obvious consistent differences in amino-acid preference between the two conditions  and HTo probe for potential effects of Tyr-717 phosphorylation on activity, we investigated the kinetics of the different PKD isoforms and mutants towards the model substrate Syntide-2 (Syn-2).m values for PKD1 and PKD2, but interestingly, kcat values for PKD2 were about 1.5 fold higher than for PKD1  indicating that Tyr-717 phosphorylation is responsible for the increase in kcat for PKD2 . Indeed, this substitution results in drastically increased Kted Fig. . This incat values for PKD2 phosphorylated at Tyr-717 by about 1.5 fold compared to a non-Tyr phosphorylated species .To directly assess how Tyr-717 phosphorylation affects PKD2 activity, we purified PKD2 from unstimulated cells co-expressing Abl. It should be noted that in conditions of high-level expression, (unstimulated) Abl can phosphorylate PKD2 independent of activation loop Ser phosphorylation, although still preferring Ser-phosphorylated species as indicated by a PKD2 S706/710E mutant Fig. . This isies Fig. . In absoSince PKDs are known to signal to NF-κB in oxidative stress, we wondered whether the phosphorylation of PKD2 on its regulatory sites, including Tyr-717, might influence the signaling output. Therefore, we tested WT PKD2 and several mutants for their ability to signal to NF-κB via a luciferase reporter assay Fig. . WT PKD2In this work, we show that PKD2 can be phosphorylated in oxidative stress conditions in the P + 1 loop of the kinase activation segment at a highly conserved Tyr residue just N-terminal of the APE motif.60 and in EGF signaling for PKCε, PKCζ and PKB62. Also, the residue was implicated in activation of PKB downstream of RET/PTC22. IKKβ has also been reported as a kinase regulated by this site in TNF signaling to NF-κB64. Recently, ERK has been shown to be phosphorylated in vitro at this residue by MEK165. Chk2 is also phosphorylated at this residue after irradiation12. All these studies make use of Tyr to Phe substitutions as non-phosphorylatable mutants, and one must be cautious to not over-interpret functional results with these mutants. This is especially evident in our NF-κB assays, where we observed a strong increase in signaling with the Y717F mutant, but not with the LNQ.RNK exchange mutant, which causes a disruption of Tyr-717 phosphorylation without directly affecting the phosphoacceptor. These misrepresentative results stem from the fact that a Tyr to Phe substitution results in drastic effects on kinase activity, with about a 7-fold increase in Km values for Syn-2 phosphorylation, occurring even in PDB stimulated conditions where WT PKD2 is not phosphorylated on Tyr-717. A similar increase in Km has been shown for an analogous Y204F substitution in PKA, which has not yet been identified to be phosphorylated at this residue66. This indicates that for Tyr containing kinases the hydroxyl group is of critical importance for their activity. It would be interesting to determine why Phe containing enzymes do not have this requirement.This Tyr residue shows very high conservation among Ser/Thr kinases, with Tyr occurring in most (73%) cases, followed by Phe (15%) and Trp (11%). Despite the high conservation of the Tyr residue in the P + 1 loop, there are only a few reports of phosphorylation in the YxAPE motif in other kinases. For example, phosphorylation is also reported for PKCδ in oxidative stress conditions67.Interestingly, while a Y717F substitution in PKD2 resulted in impaired kinetics, activation loop Ser-706/710 phosphorylation is even higher than in WT PKD2. This is possibly due to alterations in the conformation of the activation segment, facilitating phosphorylation of the Ser residues by PKCδ. These observations emphasize the fact that activation loop phosphorylation cannot always be used as a readout for kinase activity. This was also shown in a previous study using the allosteric PKD inhibitor CID-755673, where PKD was shown to display elevated activation loop Ser phosphorylation upon inhibitionwww.phosphosite.org and Supplementary Table Strikingly, the phosphosite database reports phosphorylation at the YxAPE motif in more than 70 kinases , suggesting that this phenomenon might be more widespread than was previously appreciated  is only 48% identical between the 3 isoforms.The differential regulation of PKD isoforms in oxidative stress extends beyond Tyr phosphorylation in the P + 1 loop. Our experiments also revealed that the activation mechanism of PKD isoforms is entirely different. While PKD1 has been shown to be dependent on Tyr-463 and Tyr-95 phosphorylation for activation loop Ser-738/742 phosphorylation, we do not see this dependency in PKD2. In fact, we see the opposite: activation loop Ser-706/710 phosphorylation primes for subsequent Tyr phosphorylation. PKD2 can also be phosphorylated at Tyr-438 in the PH domain, (corresponding to Tyr-463 in PKD1), for example in BCR-ABL70. Furthermore, PKD2, and not PKD1 specifically regulates MMP9 secretion71. It was also shown that in cardiomyocytes α1-ARs selectively activate PKD1 while PAR-1 and PDGFRs selectively activate PKD272. PKD2 was specifically activated during murine myoblast differentiation73. While differential activation is well documented, it is unclear what molecular mechanisms underlie this isoform-specific regulation. Our current study provides biochemical evidence for isoform-specific regulation in oxidative stress conditions through differential phosphorylation of Tyr in the P + 1 loop.The differential biochemical regulation of PKD isoforms in oxidative stress exposes a potential mechanism for functional divergence between the isoforms. Such divergence has been reported in restricted cases. For example, PKD1 and PKD2, but not PKD3 phosphorylate PI4KIIIβ54. Since Tyr-717 phosphorylation results in increased PKD2 activity, it consequently does not potentiate signaling to NF-κB. The effects we observed are in marked contrast with PKD1 signaling to NF-κB, where PKD activity is crucial for signaling output48. The final signaling output is likely determined by the differential regulation and different protein levels of PKD1 and PKD2, which is an intriguing question for further investigation. However, while Tyr-717 phosphorylation does not affect NF-κB signaling, it might differentially affect other pathways in oxidative stress.Since oxidative stress induces signaling from PKDs to NF-κB, we wondered if the ROS-induced Tyr-717 phosphorylation in PKD2 might play a role in this pathway. Intriguingly, we found that, while WT PKD2 signals to NF-κB, signaling is highly potentiated with Y717F and S706/710A mutants . By using the PKD2 LNQ.RNK exchange mutant we found that the observed effects are not due to loss of Tyr-717 phosphorylation, but rather to an abolishment of PKD2 activity. We therefore conclude that PKD2 activity is counterproductive for its signaling output to NF-κB signaling, which is consistent with results obtained by the Seufferlein lab, where it was also shown that NF-κB signaling by PKD2 is potentiated by a kinase dead mutantIn conclusion, in this paper we show differential phosphorylation of PKD isoforms at a highly conserved Tyr residue in the activation segment P + 1 loop, which modulates kinase activity towards peptide substrate. These results might have broader implications for our understanding of kinase regulation in general since the residue is highly conserved among Ser/Thr kinases and is found to be phosphorylated in more than 70 cases in proteomic studies.HEK293 and HeLa cell lines were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) Fetal Bovine serum , 2 mM glutaMAX , 100 U/ml Penicillin and 100 µg/ml Streptomycin (ThermoFisher scientific). Anti-GST, Anti-FLAG M2 antibody, HA antibody and agarose resins were purchased from Sigma . Glutathione sepharose 4B beads were from GE healthcare . GFP trap beads were from Chromotek . Anti-phosphotyrosine antibody (4G10) was from Millipore , PKD anti-pSer-744/748 antibody, anti-PKCδ antibody, secondary HRP-linked goat anti-Rabbit and Horse anti-Mouse antibodies were from Cell Signaling Technologies . Phorbol 12,13-dibutyrate (PDB), ATP, Neurotensin, Bradykinin, Lysophosphatidic acid, STI-751, PP2, DPH and Hydrogen peroxide 30% (v/v) were from Sigma . Gö 6983 was from Selleckchem . Polyethyleneimine (PEI) was from Polysciences Inc. .75. Plasmids encoding FLAG-PKD2.Y438F, Y438D, S706/710E and S706/710 A were also provided by the Seufferlein lab and have been described elsewhere54. Plasmids encoding HA-PKD1 WT, Y463D, Y463F, S738/742E and S738/742A were gifts from the Toker lab, and obtained via Addgene50. NF-κB-luciferase (NF-κB-LUC) and pCS2-(n) β-Galactosidase reporters have been described elsewhere50. For siRNA experiments we used the TriFECTa DsiRNA kit .Plasmids encoding for FLAG-PKD2 WT (pdcDNA-FLAG-PRKD2) and GST-PKD2 (pDEST27-PRKD2) were generated via an LR recombination reaction  of pDONR223-PRKD2, genetically modified to contain a stopcodon . Mutagenesis of pDONR223-PRKD1/2 was carried out by site directed mutagenesis via the Quikchange kit  for the following mutants: Tyr717 to Phe , Tyr87 to Phe , PKD1 RNK-LNQ substitution , PKD2 LNQ-RNK substitution . Expression clones were generated via the LR reaction as for PKD2-WT. PdcDNA-FLAG-ABL1 and pDEST27-ABL1 were generated using the LR reaction using pDONR233-ABL1 as a template. Plasmids encoding GFP-PKD2 WT, ΔCRD, ΔC1a, ΔC1b, ΔPH, Δ315–396 and Δ510–550 were a gift from Thomas Seufferlein and have been described elsewhereTransfections of HEK293 cells were carried out with polyethyleneimine (PEI) at a 1:3 (m/m) plasmid/PEI ratio. For siRNA-mediated knockdown of Abl, cells were plated and transfected the next day with 10nM of control or antisense nucleotide using lipofectamine 2000 . Twenty-four hours later, a second transfection was carried out with pcDNA-FLAG-PRKD2 using PEI at a 1:3 (m/m) plasmid/PEI ratio. Cells were treated and lysed 48 h post-transfection.https://admin.kuleuven.be/raden/en/animal-ethics-committee) (project P146/2010). Antibodies were precipitated from the rabbit serum with 50% ammonium sulphate and dissolved in PBS . Phospho-specific antibodies were purified sequentially on a non-phospho-peptide column and a phospho-peptide column. These were prepared by binding peptides to SulfoLink resin (Pierce) according to the protocol of the manufacturer . Antibodies were eluted using 100 mM Glycine pH 3.0 and immediately neutralized with 1 M Tris-HCl, pH 8. The specificity of the antibody was determined in an ELISA using BSA coupled phospho and non-phospho peptide and via immunoblotting.For the production of the site-specific phospho-antibody (PSSA) directed against phospho-Tyr-717, rabbits were immunized with KLH coupled peptide (CPApYLAPEV) using a standard immunization protocol. All animal procedures were done in accordance with KU Leuven - University of Leuven guidelines and regulations. Animal experiments were approved by the animal ethical committee (ECD) of the KU Leuven - University of Leuven (2O2 or left unstimulated. GST-PKD2 was eluted from glutathione-sepharose beads and subjected to TCA/aceton precipitation and trypsin digestion (10 µg modified trypsin (Promega) in 200 mM AmBic, 5% CH3CN, 0.1% RapiGest). Digested proteins were subjected to desalting on C18 Micro Spin Columns (Harvard Apparatus) and equal fractions were loaded on a hybrid quadrupole-orbitrap nano LC-MS system  using a data dependent analysis method. Number of ‘queries matched’ for the PKD2 protein was compared between conditions, and equal amounts of protein were subjected to phospho-enrichment on 50 µl IMAC bead suspension . The eluates were again desalted by C18 Micro Spin Columns and loaded on a QExactive LC-MS system using a targeted analysis method (tMS2). Data analysis was executed by using the MASCOT (Matrix Science) search engine together with the Proteome Discoverer 1.4 PhosphoRS 3.0 workflow. Data mining was done with XCalibur 3.0.63 Qual Browser and MZmine software. The phosphorylation site assignment was manually verified.To detect Tyr-717 phosphorylation after oxidative stress via mass-spectrometry, we performed a pull-down of GST-tagged PKD2 from HEK293 cells stimulated with HBefore lysis, cells were treated with the indicated compounds (see figures) for 20 min or left untreated. Lysis was done in 50 mM Tris, pH 7.4, 150 mM NaCl, 15 mM EDTA, 1% NP-40 supplemented with phosphatase inhibitors , and protease inhibitors . Cell lysates were incubated with affinity beads for 2 hours at 4 °C while rotating , GFP nanobody coated beads , anti-FLAG M2 beads , anti-HA beads ). Alternatively, for immunoprecipitation of endogenous PKD2, lysates were incubated with 2 µg anti-PKD2 antibody  for 2 h, after which protein A beads were added for another hour. Next, the beads were washed twice with NENT500  and once with NENT150 (cf. NENT500 but containing 150 mM NaCl). Alternatively, in co-IP experiments, three washes with NENT150 were performed. Elution was done in 50 mM Tris.HCl pH 7.4, 50 mM NaCl, 25% glycerol using competing peptide (FLAG/HA pulldowns), 20 mM glutathione (GST-pulldowns) or boiling in SDS sample buffer for 5 min at 95 °C.In vitro phosphorylation of PKD2 by Abl, purified FLAG-PKD2 (K580A mutant) was incubated with 50 ng of purified Abl  and allowed to incubate for 30 min at 30 °C. Reactions were stopped by addition of SDS sample buffer. Subsequently, samples were boiled at 95 °C for 5 min prior to loading of an SDS-polyacrylamide gel.For 2, 50 ng PKD and 100 µM ATP added with 2 µCi [γ-32P]ATP . Reactions were started with Syntide-2 (Syn-2) peptide at different concentrations (15–480 µM). After 10′ (in the linear range) the reaction was stopped by spotting 30 µl on a Whatman P81 filter paper. The filter papers were washed 3 times in 0.5% phosphoric acid, followed by one wash in 100% acetone. Subsequently, the papers were air-dried and counted using the Tri-Carb 2810 TR scintillation counter (Perkin-Elmer). Non-linear regression analysis and determination of kinetic parameters was performed using Graphpad (PRISM).For kinetic analysis of FLAG-PKD and its mutants, enzymes were purified from HEK293 cells as described above, but the NENT500 wash was substituted by a stringent NENT750  wash. Protein purity (100%) and concentrations were determined on an SDS polyacrylamide gel using a BSA standard. To determine Michaëlis-Menten kinetics the following reaction mixture was prepared: 50 mM Tris, pH 7.4, 10 mM MgCl2O2 for 18 hours. At the endpoint, cells were washed twice with ice-cold PBS, scraped in 250 µl Passive Lysis Buffer (Promega) and centrifuged . Assays for luciferase and β-gal activities were performed using standard assays (as described in ref. HeLa cells per well of a 6 well plate were transiently co-transfected with 3 µg NF-κB-luciferase reporter (NF-κB-LUC), 1 µg pCS2-(n) β-Galactosidase reporter and the 1 µg cDNA of interest, using Superfect (Qiagen). 4 hours after transfection, cells were stimulated with 500 µM H76. Briefly, 180 peptide mixtures (50 μM) with the general sequence Y-A-x-x-x-x-x-S/T-x-x-x-x-A-G-K-K(biotin), where “x” indicates a mixture of the 17 proteogenic amino acids  and S/T is an even mixture of Ser and Thr, were arrayed in 1536 well plates in 2.5 μL of 50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1% Tween 20. In each well, one “x” position was fixed as one of the 20 amino acids. In addition, two wells contained peptides in which the central S/T position was fixed as either Ser or Thr. Reactions were initiated by adding FLAG-PKD2 that had been isolated from cells either activated with PDB (not phosphorylated at Tyr-717) or H2O2 (to induce Tyr-717 phosphorylation) together with ATP . Plates were incubated for 2 h at 30 C, and then 200 nl aliquots were spotted onto streptavidin coated membrane (Promega SAM2 biotin capture membrane). The membrane was washed extensively as described and exposed to a phosphor storage screen. Radiolabel incorporation was quantified by phosphor imaging. Data were normalized so that the average value at each peptide position was equal to 1. Data from 3 independent experiments were averaged, and log2 transformed data were converted to a heat map in Microsoft Excel.Peptide array analysis to determine PKD2 phosphorylation site specificity was carried out essentially as describedSupplementary information for differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P + 1 loop


Axon guidance involves the spatiotemporal interplay between guidance cues and membrane-bound cell-surface receptors, present on the growth cone of the axon. Netrin-1 is a prototypical guidance cue that binds to deleted in colorectal cancer (DCC), and it has been proposed that the guidance cue Draxin modulates this interaction. Here, we present structural snapshots of Draxin/DCC and Draxin/Netrin-1 complexes, revealing a triangular relationship that affects Netrin-mediated haptotaxis and fasciculation. Draxin interacts with DCC through the N-terminal four immunoglobulin domains, and Netrin-1 through the EGF-3 domain, in the same region where DCC binds. Netrin-1 and DCC bind to adjacent sites on Draxin, which appears to capture Netrin-1 and tether it to the DCC receptor. We propose the conformational flexibility of the single-pass membrane receptor DCC is used to promote fasciculation and regulate axon guidance through concerted Netrin-1/Draxin binding. •Crystal structure of cysteine knot domain of Draxin in complex with DCC•Crystal structure of Netrin-1 in complex with a Draxin fragment•Netrin-1 contains a competing binding site for DCC and Draxin on the EGF-3 domain•Draxin tethers Netrin-1 and DCC together to promote fasciculation Liu et al. report through structural investigations how Draxin associates both with Netrin-1 and its cognate receptor DCC to mediate axon guidance and fasciculation. The wiring of commissural neurons in the developing spinal cord is central to the development of bilateral symmetry. Commissural neurons extend axons dorsoventrally in the spinal cord that eventually cross over the midline to establish this symmetry . During The guidance cue Draxin  was characterized as a repulsive cue that prevents axons from misprojecting before midline crossing . Draxin cis interactions involving the same axon, and trans interactions involving other axons or cellular substrates, to facilitate axonal adhesion and fasciculation.To date, it is not clear how Netrin-1 and Draxin coordinate to affect axon fasciculation and guidance. Cell binding assays indicated Draxin binds DCC at the N-terminal, membrane-distal region containing four immunoglobulin (Ig) domains . These dIg1-Ig4), since Draxin binding was found to involve the N-terminal four domains of DCC  abolishes binding of rDraxin to DCC, whereas an Arg282Ala mutant seems to have little effect  and the Netrin-1/DCC interface (609 Å2). Similarly, the surface complementarity values between the Draxin/Netrin-1 interface (0.67) and the Netrin-1/DCC interface (0.66) are comparable. Since there does not seem to occur any refolding of the hDraxin-22 peptide upon binding to Netrin-1, we speculate that solvation-related energy effects are the main determinant in the relatively strong binding of Draxin to Netrin-1 .Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Rob Meijers (Ig1-Ig4 construct previously described (KM655686) was amplified and subcloned into modified AVEXIS bait vector (Addgene plasmid #36148) with C-terminal removal of CD4 and replaced by a purification tag (TEV-StrepII-linker-StrepII-linker-9xHis) for use in SAXS. Human Netrin-1 covering residues 39 to 457 was subcloned into pXLG as previously described  through transient transfection in roller bottles. Cells were cultured in Dulbecco’s modified eagle medium , containing nonessential amino acids, 2 mM L-glutamine and 0.2% fetal-bovine serum  at 37°C in a 5% CO2 atmosphere in a Wheaton incubator. The expressed protein was secreted into the culture medium, which was harvested four to six days post transfection. The medium was filtered through filter paper (Whatman). Expression was confirmed by western blot analysis using a penta-His primary antibody  and a HRP-linked secondary antibody . Protease Inhibitor Cocktail tablets  were added to the medium (2 tablets per liter) immediately after harvesting.The DNA fragment of full-length rDraxin was amplified from rat embryonic cDNA library, and subcloned into the pXLG vector kindly provided by David Hacker and Florian Wurm . The rat rDCC escribed  was subcescribed . For rDCIg1-Ig4 containing media were dialyzed in 20 mMTris-HCl, pH7.5, 200mMNaCl. These proteins were affinity purified by Ni column (QIAGEN Ni-NTA agarose), followed by size exclusion chromatography using a HiLoad 16/60 Superdex 200 column from GE Healthcare. Harvested media containing recombinant hNetrin-1 was then incubated at 4°C with 1.5 ml Ni-Sepharose Excel resin  per liter medium, overnight with slow stirring. hNetrin-1 was eluted in 1ml fractions with 20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole at pH 7.4. The total elution volume collected was 5ml from 2 l of conditioned media. Next, the eluent from the affinity chromatography was subjected to size exclusion chromatography using a HiLoad 16/60 Superdex 75 prep grade column equilibrated in 50 mM MES buffer pH 6.0, containing 250 mM NaCl and 1 mM DTT, with a flow rate of 1 mL per minute. The hNetrin-1 peak from the size exclusion step was collected in 0.5 mL fractions in a deep well block. After inspection through SDS-PAGE gel, fractions containing hNetrin-1 with > 90% purity were combined and concentrated using an Amicon Ultra-4 Centrifugal Filter  to 1mg/ml (∼20 uM).Both rDraxin and rDCC Ig1-Ig4 were combined in 1.2:1 molar ratio, and incubated at 4°C for 2 hours before loading onto the size exclusion column. The complex peak fractions were collected and concentrated to ∼12mg/ml for crystallization. Crystals appeared readily at a few conditions, the best of which is in the No.8 condition of PEG/Ion Screen (HR2-126 Hampton). It was optimized to 0.2 M KCl, 3% PEG 3350 to get well-diffracting crystals. Diffraction data were collected at SSRF  beamline BL17U and processed with HKL2000 (HKL Research) (Ig1-Ig4 structure (PDB: 3LAF) as the search model. The structure was built in Coot (Purified rDraxin and rDCCesearch) . The stresearch)  using th in Coot  and refi in Coot , with 5% in Coot . The ste in Coot , indicat in Coot .A 22-mer peptide (sequence: GEVMPTLDMALFDWTDYEDLKP) corresponding to the residue spanning 222-243 of human Draxin (UniProtKB entry: Q8NBI3 DRAXI_HUMAN) was ordered from Genscript. The purity of the synthesized peptide was > 75%. A peptide stock of 1mM was prepared by dissolving the peptide in water, first by lowering the pH to 5.5 and then gradually bringing it back to neutrality.4URT served as a search model. Each EGF domain was placed separately by MOLREP , 1.6M ammonium sulfate, 15% (v/v) Ethylene glycol prior to flash-cooling to 100K. X-ray diffraction data were collected on the P14 beamline of EMBL Hamburg situated at the PETRA3 synchrotron. The beamline was equipped with a Pilatus 6M detector and an MD3 EMBL diffractometer. Two X-ray datasets collected on a single crystal of the hNetrin-1/hDraxin-22 complex were merged using MOSFLM  and scaly MOLREP  after thy MOLREP . Electroy MOLREP  and refiy MOLREP , with 5%y MOLREP . An omity MOLREP  . Based on comparison of successive frames, no detectable radiation damage was observed. Data from the detector were normalized to the transmitted beam intensity, averaged, placed on absolute scale relative to water and the scattering of buffer solutions subtracted. All data manipulations were performed using the ATSAS software package  the intensity is represented as I(s) = I(0)exp(-(sRg)2/3)). These parameters were also estimated from the full scattering curves using the indirect Fourier transform method implemented in the program GNOM (p(r) and the maximum particle dimensions Dmax. Molecular masses (MMs) of solutes were estimated from SAXS data by comparing the extrapolated forward scattering with that of a reference solution of bovine serum albumin, the hydrated-particle/Porod volume Vp, where molecular mass is estimated as 0.588 times  and from the excluded solvent volume (Vex) obtained from ab initio modeling in the program DAMMIF (http://www.sasview.org/), respectively. The radius of gyration for folded and unfolded hDraxin was calculated from the Flory relation, Rg = R0Nv  and radius of gyration, ram GNOM , along wm DAMMIF . Ensemblm DAMMIF  using thg = R0Nv  for an aA construct of codon optimized human Netrin-1 (VI+V)  was added to the culture medium. After 2 hours’ incubation, the medium was removed and cells were washed 5 times by PBS and fixed for immunostaining with FLAG  antibody.∗∗∗) p < 0.001; (∗∗) p < 0.01; (∗) p < 0.05.Statistics were done using GraphPad Prism software. Student’s t test was performed for the cell binding and AVEXIS assays.  and 6FKQ (hNetrin-1/hDraxin-22 complex). The accession number for the SAXS analysis of hDraxin reported in this paper is deposited in the SASBDB database: SASDBZ6.


Furthermore, in vitro assays using a hippocampal neural stem/progenitor cell (HNSPC) line indicated that draxin inhibited apoptosis in differentiating HNSPCs, which express DCC. Taken together, we postulate that draxin plays a pivotal role in postnatal DG neurogenesis as a dependence receptor ligand for DCC to maintain and promote survival of neuroblasts.Hippocampal neurogenesis in the dentate gyrus (DG) is controlled by diffusible molecules that modulate neurogenic processes, including cell proliferation, differentiation and survival. To elucidate the mechanisms underlying hippocampal neurogenesis, we investigated the function of draxin, originally identified as a neural chemorepellent, in the regulation of neuronal survival in the DG. Draxin was expressed in Tbr2 (+) late progenitors and NeuroD1 (+) neuroblasts in the dentate granule cell lineage, whereas expression of its receptor DCC  was mainly detectable in neuroblasts. Our phenotypic analysis revealed that  Accumulating evidence has recently revealed that hippocampal neurogenesis plays a pivotal role in many physiological brain functions, especially those associated with learning and memory. Moreover, altered or impaired neurogenesis is associated with neurological disorders such as Alzheimer’s disease, schizophrenia and depression6. Intriguingly, a reduction in hippocampal volume and number of newborn dentate granule cells (DGCs) is observed in patients suffering these diseases, as well as in the relevant animal models. Conversely, enhanced neurogenesis in the hippocampus is seen in other neurological diseases such as the epilepsy, ischemia and traumatic injury8. These opposing observations imply that the disruption of different molecular pathways is likely to regulate hippocampal neurogenesis in each neuropathological condition. To date, elucidation of the mechanisms underlying hippocampal neurogenesis has identified a variety of diffusible factors able to modulate key hippocampal neurogenic processes, including cell proliferation, differentiation and survival10. In this study, we further expand our understanding of hippocampal neurogenesis through the identification of the axon guidance cue, Draxin, as an important regulator of neuronal precursor survival.In the hippocampal dentate gyrus (DG), granule cell production begins in the embryo and continues throughout life20. Furthermore, our study revealed that draxin is important not only for axon navigation but also for hippocampal development. Draxin loss leads to enhanced apoptosis at embryonic day 18 (E18), impaired DG development, fewer dentate granule cells and reduced DG size in juveniles20. However, little is known regarding the underlying mechanism responsible for such DG phenotypes in draxin knockout (KO) mice. Previous studies reported that draxin interacts with netrin receptors physically, although only DCC  and neogenin were proven to mediate the inhibitory effect of draxin21. These transmembrane molecules are also known as dependence receptors, which trigger neuronal apoptosis and promote survival in the absence and presence of their ligands, respectively27. Thus, one possible explanation for the DG phenotype in draxin KO mice described above is deregulation of these dependence receptors due to lack of draxin. In the present study, we elucidated the cellular and molecular mechanisms underlying draxin-regulated hippocampal neurogenesis by investigating the role of draxin in dependence receptor-induced apoptosis.The neural chemorepellent draxin, which we previously isolated using a signal sequence trap, is indispensable for proper navigation of growing axons and migrating neurons in developing embryosdraxin KO mice is obvious at early postnatal stages and thereafter, but not E17.5 , progenitors (type-2a/b), neuroblasts (type-3), immature and mature granule cells28  neuroblasts  caspase-3 confirmed that apoptosis was not observed in draxin KO mice at E17.5 but is present at early postnatal stages P0, P2 and P21  cells are still detectable in the SGZ of P21 Fig. . FurtherP21 Fig. . In contP21 Fig. .draxin KO mice, to investigate the impact of draxin deficiency on the granule cell population. As expected, the number of NeuroD1(+) neuroblasts exhibiting excessive apoptosis was significantly decreased in the KO SGZ  derived from the juvenile/adult rat (see M&M). When the rat-derived HNSPCs were cultured in medium containing the differentiation inducers, retinoic acid (RA) and forskolin (FK), for 4 days, expression of nestin was rapidly downregulated, while that of an early neuronal marker Tuj1 was gradually upregulated in almost all the cells . Furthermore, knockdown of neogenin in addition to DCC did not lead to further attenuation of draxin-induced neuronal survival compared to the single-knockdown of DCC when draxin was present . On the other hand, DCC knockdown in the absence of draxin resulted in the opposite effect on neuronal survival. DCC knockdown significantly increased the viability of differentiating HNSPCs . In contrast to DCC, knockdown of neogenin never affected cell survival , but suppressed it in the presence of the ligand . In contrast, neogenin overexpression had no effect on the viability in the presence or absence of draxin  in DCC resulted in decreased apoptosis in differentiating HNSPCs in vitro, suggesting that DCC-induced apoptosis in the absence of ligands is also mediated through a caspase-dependent mechanism. It has been proposed that inhibition of apoptosis is dependent on conformational change which masks the caspase cleavage site on DCC upon Netrin-1 binding25. Therefore, binding of draxin to DCC might also result in a conformational change, leading to apoptotic inhibition due to the inaccessibility of the caspase target in the DCC intracellular region. Based on the in vitro data obtained in the present study, we believe that the draxin-mediated attenuation of the DCC-induced HNSPC death is mainly due to inhibition of a caspase-dependent apoptosis in the presence of its ligand. This hypothesis is supported by our observation that cell death was drastically prevented in HNSPCs expressing a caspase-resistant DCC mutant compared to the wild-type in the absence of draxin  or DCX (+) neuroblasts, and that draxin and decreased levels of DCC are expressed in Tbr2 (+) late progenitors. Furthermore, expression of draxin and DCC is below the detection limit in neural stem cells/early progenitors and mature granule cells. Since robust apoptosis was observed only in neuroblasts in draxin KO mice, DCC expression would explain why neuroblasts but not the cells representative of other differentiation stages in the granule cell lineage undergo apoptosis in the SGZ of draxin KO mice. Based on these findings, we propose that draxin secreted from late progenitors and neuroblasts inhibits apoptosis of DCC-expressing neuroblasts in the DG via autocrine and possibly paracrine signaling.Immunohistochemical and 20. Excessive apoptosis of cells in the granule cell lineage would generally result in a reduced number of mature dentate granule cells that represent a considerable portion of the DG, leading to DG malformation. This is mechanistically supported by studies of mice lacking genes such as Sox2, Tbr2, Prox1 and neurogenin2, which are known to be essential for hippocampal neurogenesis. In each case, the neuronal population was drastically decreased due to excessive apoptosis, leading to reduced size of the DG42. The malformation of the DG in draxin KO mice may also be attributable in part, to the deregulation of neurogenic processes including neuronal proliferation and differentiation. Previous studies indicated that canonical Wnts play pivotal roles in regulating neuronal proliferation and differentiation in the developing DG46, and that Neucrin, a zebrafish homologue of draxin, attenuates the canonical Wnt/β-catenin signaling by competing with Wnts for their receptor component, lipoprotein receptor-related protein 6 (LRP6)48. Thus, deletion of the draxin gene may lead to defective neuronal proliferation and/or differentiation of SGZ cells due to deregulation of the canonical Wnt signals. Supporting this, the present study indicated that draxin deficiency resulted in reduction of not only neuroblasts, but also early/late progenitors, which do not express DCC or exhibit excessive apoptosis. Future studies would focus on the impact of loss of the draxin gene on Wnt-driven proliferation/differentiation of SGZ cells to further elucidate the functions of draxin in hippocampal neurogenesis. In summary, we have identified the cellular and molecular mechanisms underlying draxin-mediated regulation of neuronal survival in the postnatal SGZ, and found that draxin acts as a dependence ligand for DCC to prevent the proapoptotic effect of DCC in neuroblasts  was performed using the digoxygenin (DIG)-labeled cRNA probes corresponding to 3384–3982 bases of the mouse DCC (NCBI Reference Sequence: NM_007831.3) on hippocampal slices (200 µm) obtained from juvenile mice, respectively, following the procedure reported before49. To perform double staining with immunohistochemistry, hippocampal slices were cut into 10 µm sections using a cryostat (Leica) after developing color for ISH, and then sections were immunostained following the procedure described in the Supplementary Text.2 incubator at 37 °C. Cells were sub-cultured upon reaching 80% confluency using Accutase (Innovative Cell Technologies).Hippocampal neural stem and progenitor cells (HNSPCs) derived from the juvenile/adult rat  were cultured in a mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s Nutrient Mixture F-12  containing B-27 supplement (Invitrogen), human basic fibroblast growth factor  and human epidermal growth factor , in a COth amino acid residue of DCC, a PCR-based site-direct mutagenesis was performed on pMX-CAG-rat DCC-HA using KOD Plus Mutagenesis Kit (Toyobo) according to the instruction. The sequence of the primers used for the mutagenesis was shown below. 5′-AACCGAGGTTTCGGAGCAGGAAGAA-3′ and 5′-CACAGACAGTGTTGGGAATGGTACTGG-3′.The neogenin expression plasmid was constructed by ligating the FLAG tagged-DNA fragment containing the entire mouse neogenin (NM_008684: 314 - 4789) coding region amplified from cDNA prepared from P0 whole mouse brains using KOD DNA polymerase (Toyobo) into Hind III and Not I sites of the pMX-CAG vector (Cell Biolabs). The sequence of the constructed plasmids was confirmed by sequencing. The rat DCC expression plasmid was constructed by inserting the Sal I-Not I HA-tagged rat DCC fragment derived from pcDNA-rat DCC-HA  into Xho I and Not I sites of the pMX-CAG vector . To substitute aspartic acid for asparagine at the 1290Knock-down of gene expression was achieved by introduction of siRNA duplexes (Sigma-Aldrich) designed for the draxin receptors, DCC and neogenin, into rat HNSPCs using the Lipofectamine RNAiMax Reagent (Invitrogen) according to the instructions. The sequences of siRNA duplexes used in this study were: 5′-CAGUGAACGGCUCCCAUAATT-3′ and 5′-UUAUGGGAGCCGUUCACUGTT-3′ for rat neogenin; and 5′-CUAUGUAUUACUUUCGAAUTT-3′ and 5′-AUUCGAAAGUAAUACAUAGTT-3′ for rat DCC. Overexpression was achieved by introduction of the expression plasmids described above into rat HNSPCs using the NanoJuice Transfection Kit (Merck-Millipore) according to the instruction.rd day of culturing, medium was replaced with DMEM/F-12 medium containing various concentrations of the recombinant proteins for human alkaline phosphatase (AP) and AP-fused draxin (draxin-AP) without B-27 supplement and differentiation inducers, and then cells were cultured for one more day. After fixation with 4% PFA in PBS, the recombinant protein-treated cells were immunostained with antibodies to an apoptosis marker, active caspase-3, following the procedure described in the Supplementary Text. Production of the AP and draxin-AP recombinant proteins were described before11. In some experiments, cells were transfected with siRNA duplexes or expression plasmids on the 2nd day of culturing with RA/FK-containing medium, and then cultured for one more day in the presence of 50 nM of the recombinant proteins for draxin-AP and AP, to determine whether DCC and neogenin are implicated in transducing draxin signals.Rat HNSPCs were cultured in DMEM/F-12/B-27 medium containing the differentiation-inducing compounds, retinoic acid  and forskolin , to direct neuronal differentiation. On the 3Quantitative data were analyzed using Student’s t-test for a paired sample group or Tukey-Kramer test following ANOVA for more than three sample groups, with JMP Pro 12 software (SAS Institute Inc.).Supplementary information