Impaired insulin signaling has been thought of as important step in both Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Posttranslational modifications (PTMs) regulate functions and interaction of insulin with insulin receptors substrates (IRSs) and activate insulin signaling downstream pathways via autophosphorylation on several tyrosine (TYR) residues on IRSs. Two important insulin receptor substrates 1 and 2 are widely expressed in human, and alternative phosphorylation on their serine (Ser) and threonine (Thr) residues has been known to block the Tyr phosphorylation of IRSs, thus inhibiting insulin signaling and promoting insulin resistance. Like phosphorylation, O-glycosylation modification is important PTM and inhibits phosphorylation on same or neighboring Ser/Thr residues, often called Yin Yang sites. Both IRS-1 and IRS-2 have been shown to be O-glycosylated; however exact sites are not determined yet. In this study, by using neuronal network based prediction methods, we found more than 50 Ser/Thr residues that have potential to be O-glycosylated and may act as possible sites as well. Moreover, alternative phosphorylation and O-glycosylation on IRS-1 Ser-312, 984, 1037, and 1101 may act as possible therapeutic targets to minimize the risk of AD and T2DM. Hyperphosphorylated forms of tau act as major constituents of neurofibrillary tangles (NFTs), while Aβ is major constituent of plaques [β-cells destruction, respectively. Impaired insulin signaling resulting in neurodegeneration and cognitive damages is the main systematic link between these two diseases. Many studies found that DM almost doubles the risk of AD. It was also found that diabetes is not only the risk factor of AD but it also accelerates the onset of AD. Recent studies also elaborate the involvement of impaired insulin signaling in tau hyperphosphorylation. AD and DM also share several enzymes  and growth factor receptors  [Alzheimer's disease (AD) is a neurodegenerative disease associated with a progressive loss in memory and other mental processes, resulting in dementia. In the AD brain, some changes occur in tertiary structures of microtubule-associated protein tau and  plaques . By 2050 plaques . In DM,  plaques . Hoyer iceptors) –9.β-cells and glucose homeostasis are main functions of IRS-2 [Insulin receptor substrate proteins (IRSs) act as interaction platforms that are phosphorylated on multiple tyrosine residues and attract different signaling proteins to spread the stimulus. Two isoforms of IRSs, that is, IRS-1 and IRS-2, are most abundant in mammals and have many tyrosine phosphorylation sites. In general, IRS-1 is important for growth, while proper function of pancreatic of IRS-2 , 11. Knoof IRS-2 . Irreconof IRS-2 , 14. Howof IRS-2 . Loss ofof IRS-2 . Apart fof IRS-2 .In biological systems, posttranslational modifications (PTMs) determine protein activity, localization, and their interaction with other proteins . Multiplβ-GlcNAc. Interplay between these two PTMs on the same residues also known as yin yang sites has been reported in several nuclear and cytoplasmic proteins [β-GlcNAc, and yin yang sites on IRS-1 and IRS-2 proteins, involved in metabolic functions in the brain by using various bioinformatics tools. Impaired phosphorylation and O-glycosylation on IRS 1 and 2 lead to defective insulin signaling that resulted in Alzheimer- and diabetes-like complications. The sites where this type of crosstalk occurs are useful targets for designing new drugs for resulting diseases.Analogous to phosphorylation, O-glycosylation is also one of the important PTMs during which N-acetyl-glucosamine (O-GlcNAc) molecule is introduced on Thr or Ser residue by O-GlcNAc transferase (OGT). Phosphorylation on Thr or Ser residue can be inhibited by the addition of O-proteins . These Pproteins . Variousproteins –34. Althhttp://www.uniprot.org/uniprot/?query=irs&sort=score) with entry name IRS1_HUMAN and accession number P35568 and entry name IRS2_HUMAN with accession number Q9Y4H2, respectively. Blast of these two sequences was made by Blast/Uniprot. A total of 37 hits for IRS-1 and 24 hits for IRS-2 were obtained with E-value of zero or less. Sequences based on uncharacterized, putative, hypothetical, predicted, and unnamed proteins were neglected. For the multiple alignments of IRS-1 proteins, eight sequences were selected from 37 retrieved sequences. The accession numbers of these selected sequences are given in http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used for the multiple alignments of all the sequences of IRS-1 and IRS-2 to get the conservation status of serine, threonine, and tyrosine residues.The FASTA sequences of human IRS-1 and IRS-2 were taken from the Uniprot sequence database (https://modbase.compbio.ucsf.edu/scgi/modweb.cgi) [http://zhanglab.ccmb.med.umich.edu/I-TASSER/) [http://swissmodel.expasy.org/) [As there were no complete 3D structures of human IRS 1 and IRS 2 available in protein data bank, ab initio models were designed by using software MODELLER (ModWeb: web.cgi) , I-TASSETASSER/) , and Swisy.org/) .http://molprobity.biochem.duke.edu/) server [http://fatcat.burnham.org/fatcat-cgi/cgi/fatcat.pl?-func=pairwise) was used to perform pairwise alignment of protein 3D structure. Chimera (http://www.cgl.ucsf.edu/chimera/) [http://jmol.sourceforge.net/) software were used to view and analyze 3D structure.On the basis of Ramachandran plots, best ab initio models were selected. Ramachandran plots were taken from MolProbity  server . FATCAT himera/)  and Jmolβ-GlcNAc attachment sites in eukaryotic nuclear and intracellular protein sequences. These bioinformatics tools have been used efficiently in many studies [We used three distinct algorithms: support vector machine (SVM), neural network (ANNs), and hierarchal searches  to reduc studies , 41, 42.http://www.cbs.dtu.dk/services/NetPhos/) [http://scansite.mit.edu/motifscan_id.phtml) [Neural network-based programs NetPhos 2.0 (etPhos/)  and Scand.phtml)  were usehttp://scansite.mit.edu/motifscan_id.phtml) [http://cbs.dtu.dk/services/NetPhosK/) [http://kinasephos2.mbc.nctu.edu.tw/) [Prediction of kinase specific phosphorylation sites in human IRS 1 and IRS 2 was made by Scansite (d.phtml) , NetPhostPhosK/) , and Kinedu.tw/) . Kinase http://phospho.elm.eu.org) [http://www.uniprot.org/uniprot/) databases were used to obtain the data regarding experimentally verified phosphorylation sites on human IRS 1 and IRS 2.Phospho.ELM (.eu.org)  and Uniphttp://www.cbs.dtu.dk/services/YinOYang/) database [ O-β-GlcNAc modification potential sites. The potential phosphorylation sites can also be predicted by YinOYang 1.2 program. Hence, this program predicts the yin yang sites with highly uneven threshold that is adjusted in accordance with amino acid surface accessibility. This method also helps to predict the potential yin yang sites. Modification potential and conservation status of Thr and Ser residues were used to determine the false negative (FN) yin yang sites.YinOYang 1.2 (database  was usedhttp://www.cbs.dtu.dk/services/NetSurfP/) [http://scansite.mit.edu/motifscan_id.phtml) [The surface accessibility of predicted Ser, Thr, and Tyr residues for posttranslational modifications was assessed by using NetSurfP (tSurfP/) . Scansitd.phtml)  was alsoTo determine the evolutionary conservation of Ser/Thr/Tyr residues, IRS-1 was aligned with orthologous sequences of other species including green monkey, pig, chicken, Chinese hamster, mouse, rat, and western clawed frog . Out of IRS-2 was aligned with only known orthologous sequences of species rat and mouse . Like IRz-score. The energy of these structures was minimized using YASARA software. To determine whether these structures truly represent or are nearer to possible future true structures, their geometry had been accessed by drawing Ramachandran plots that represent most reliable and suitable source to check predicted 3D structure's geometry. The Ramachandran plot analysis is a method to determine the phi and psi torsion angles between amino acids and compare this information to experimentally verified data as well as standard geometrical properties of protein 3D structures. Five models each were predicted by I-TASSER for constructing Ramachandran plots assessing both IRS-1 and IRS-2. Figures 3D structure of protein plays important role in determining the exact and precise functions of any protein. However, the whole 3D structures of IRS-1 and IRS-2 proteins have not been determined yet. To assess this difficulty we predict the 3D structure of these proteins by homology and ab initio modeling. We used many servers to predict the whole structure of both proteins and I-TASSER was the only server that predicts the complete structure of these proteins. I-TASSER predicted five models for each 3D structure with minimum β deviations for these predicted models were not predicted by server.On the basis of Ramachandran plot analysis we proposed predicted model #2  as possiβ deviations > 0.25A. Only 5.69% of residues exist within the disallowed region of the plot. The 3D structures of IRS-1 and IRS-2 proteins predicted by I-TASSER were submitted to online database PMDB—Protein Model Database (http://bioinformatics.cineca.it/PMDB/). PMDB accession numbers are given in front of each respective model in Figures Ramachandran plot analysis was also performed for predicted structures for IRS-2 protein as shown in P value 9.01 E-01; Raw score = 251.71). The structure alignment has 546 equivalent positions with an RMSD of 11.03, with five twists. Both structures were found to have identity 4.02% and similarity 8.71%. This information leads to the conclusion that both IRS-1 and IRS-2 proteins regulate insulin singling in different ways.IRS-1 and IRS-2 predicted 3D structures had been compared with each other by FATCAT and visualized by using Chimera as shown in Phospho.ELM is the website that contains data about experimentally verified phosphorylated residues and kinases involved in proteins to ease research work. Human IRS-1 has more than 40 Ser, Thr, and Tyr residues that were found to be phosphorylated experimentally. These include Ser-24, 268, 270, 272, 274, 307, 312, 323, 341, 345, 348, 374, 527, 531, 574, 616, 629, 636, 639, 794, 892, 1078, 1101, 1145, 1222, and 1223 Thr-530, and 1111, Tyr-46, 612, 662, and 896. Uniprot database also showed phosphorylation potential on Ser-3, 99, 329, 1223, and Tyr-465, 989, and 1229 on basis of similarity. Different kinases and groups of kinases like CK2, PLK1, IKK, PKC, MAPK3, SIK, AMPK, INSR, and RSK1 were involved in phosphorylation of IRS-1 on same or different residues.For human IRS-2 protein phosphorylation had been verified on Ser-304, 306, 312, 334, 346, 365, 384, 388, 391, 518, 523, 550, 560, 577, 594, 608, 620, 679, 714, 730, 731, 735, 736, 770, 772, 828, 894, 915, 973, 1100, 1103, 1109, 1148, 1149, 1162, 1174, 1176, 1203, and 1283, Thr-350, 363, 520, 527, 713, 777, 779, 1151, 1159, and 1202, and Tyr-75, 184, 653, 675, 823, and 919 residues. While IKK kinase group was involved in phosphorylation, Uniprot database showed phosphorylation on four more residues including above, that is, Thr-527, 579, 580 and Tyr-1253.http://dx.doi.org/10.1155/2014/324753.To predict phosphorylated residues in human IRS-1, NetPhos was used. Similar to IRS-1, web server NetPhos 2.0 also made prediction of phosphorylated residues in human IRS-2. Various kinases involved in phosphorylation of human IRS-1. NetPhosK 1.0, Scansite, and KinasePhos 2.0 were used to assess possible kinases on IRS-1. The results obtained from these three servers had shown the involvement of various kinases in phosphorylation of human IRS-1. GSK3, PKC, and PKA showed maximum involvement in phosphorylation while other kinases like PKG, p38MAPK, cdk5, CKII, cdc2, ATM, and CKI also showed potential for phosphorylation of many Ser and Thr residues. INSR, syk, SRC, and insulin receptor kinase are the most predicted kinases for tyrosine phosphorylation as shown in Table 1S.β-subunit of IR is known to phosphorylate diverse substrates, including IRS-2 [Similar to IRS-1, NetPhosK 1.0, Scansite, and KinasePhos 2.0 predicted many kinases involved in phosphorylation of human IRS-2. GSK3, PKC, PKA, 14-3-3 Mode 1, PKG, p38MAPK, cdk5, CKII, cdc2, ATM, and CKI are the most predicted kinases involved in phosphorylation of many serine and threonine residues in IRS-2. INSR, syk, SRC, and insulin receptor kinase are the most predicted kinases for tyrosine phosphorylation as shown in Table 2S. Intrinsic tyrosine kinase in the ng IRS-2 .β-GlcNAc modifications  yin yang sites. These residues include Ser-383, 412, 1037 and Thr-387.YinOYang prediction server showed that the human IRS-1 has 57 potential sites for O-ications . It had fication –54, thusfication . These sfication . Out of -β-GlcNAc modification and 64 Ser/Thr residues were predicted to be O-glycosylated  via tyrosine autophosphorylation of IR. Activation of IR subsequently phosphorylates its substrates (IRSs). Insulin receptor substrates 1 and 2 are specific substrates for IR/IGFR and widely expressed in mammalian tissues. Both IRS 1 and 2 contain phosphotyrosine-binding (PTB) domains. Tyrosine phosphorylation of IRS proteins is well studied and more than 20 potential tyrosine phosphorylation sites are mapped on IRSs. During insulin signaling, IRS proteins phosphorylate and activate AKT/PKB/MAPK pathways that regulate insulin mediated metabolic actions and control cell growth and differentiation , 20.β, mTOR, PKC, and JNK [Despite Tyr phosphorylation, IRSs also phosphorylate on Ser/Thr residues and phosphorylation on these residues may effect downstream insulin signaling. More than 30 Ser/Thr residues on IRS-1 and 50 on IRS-2 have been mapped using different analytical techniques. However, function of all mapped Ser/Thr residues on IRSs is still undetermined. Our results showed that both IRS-1 and IRS-2 have high potential to be phosphorylated on Ser and Thr residues and more than 100 Ser/Thr residues have been predicted including previously mapped residues on IRS 1 and 2 on both N- and C-terminals including PTB domain Figures . These p and JNK , 25, 56. and JNK . S6K act and JNK . Ser-312 and JNK , 23, 25. and JNK , 58, 59.β-GlcNAc modification is known to be influential and analogous to phosphorylation. Some researchers indicated increased phosphorylation and reduced O-GlcNAc levels in the AD and diabetes patients [β-GlcNAc modification on IRS-1 (Figures β-GlcNAc modification in human IRS-1 has been reported at Ser-984 or 985, 1011, and possibly at multiple residues within 1025–1045 [β-GlcNAc modification. Ser-1037 is conserved in human IRS-1 and identification of this site will be helpful in exploring the biological implication of the O-β-GlcNAc modification [The O-patients –62. Thes Figures  and 4. O025–1045 . Based ofication . Althougfication . In this-β-GlcNAc can mimic or inhibit phosphorylation on the neighboring or same residues and if this interplay happened on same Ser/Thr residues these sites are called yin yang residues. These yin yang residues can be used as therapeutic targets to overcome different diseases [It is established fact that Odiseases , 52. Prodiseases , 41. Howdiseases . Furtherdiseases . All theHere we propose that alternative O-glycosylation on Ser-312 at N-terminal and Ser-984, Ser-1037, and Ser-1101 at C-terminal of IRS-1 is an important modification that becomes reduced in diabetes and AD due to impaired glucose metabolism. During normal circumstances, O-glycosylation can block phosphorylation on these residues and can act as possible therapeutic sites to reduce risk of diabetes and AD.Table S1 provides detailed analysis about experimental and predicted phosphorylation, kinases involved and surface accessibility for Ser/ Thr/ Tyr residues of human IRS-1.


Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains  of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. Bullous pemphigoid (BP) is an autoimmune skin disease resulting in antibody-mediated separation of the epidermis from the dermis. The initial phase of lesion development is characterized by urticarial plaques and eosinophilic infiltration of the upper dermis. As the lesions progress, formation of tense, fluid-filled vesicles corresponds histologically to loss of epidermal adhesion at the basement membrane zone (BMZ). In addition, there is perilesional infiltration of lymphocytes, mast cells and neutrophils, and often, elevated levels of circulating IgE in vitro and in vivoThe severity of BP is correlated with levels of autoantibodies targeting the hemidesmosomal protein BP180, also known as type XVII collagen There is increasing evidence that eosinophils are powerful mediators of immunity through the production of a variety of cytokines, toxic granule proteins, and lipid mediators 2) or trimeric (αγ2) forms, and is expressed on a variety of cells, including mast cells, basophils and eosinophils a∼1010 M−1) of FcεRI for IgE, antibody is always bound to receptors expressed on the cell surface In humans, the high-affinity IgE receptor, FcεRI, is comprised of an α-chain, which is responsible for IgE binding, and β- and γ-chains, which mediate signal transduction Clinical studies indicate a critical role for IgE autoantibodies in BP; however, the exact nature of its contribution is not well understood. As eosinophils are increasingly recognized as mediators of autoimmunity, establishing relationships between eosinophilia and other markers of disease severity may provide additional insight into the mechanisms of the disease process in BP. In this study, we characterized a large cohort of untreated BP patients and examined associations between circulating IgE, eosinophil numbers, IgG and IgE autoantibody levels and disease severity. Furthermore, FcεRI expression was evaluated by mRNA expression, flow cytometry and two immunofluorescent (IF) staining methods to demonstrate that tissue specific expression of this receptor may impact eosinophil function in BP.Serum samples were collected from 48 untreated patients  diagnosed with BP based on clinical , histologic , and direct IF (linear IgG and/or C3 deposition at the basement membrane zone) criteria. Patients were enrolled at the time of diagnosis and had received no prior treatment for BP. Since these samples were collected over several years, disease severity was assessed using the previously reported BP Index De-identified archival blocks of lesional skin biopsies from BP patients were obtained from the University of Iowa Department of Pathology. Skin, discarded at the time of cutaneous surgery, was collected from age- and gender-matched controls with no known autoimmune disease. This study was approved by the Institutional Review Board (IRB #200701758) at the University of Iowa and was performed in adherence to the Declaration of Helsinki Guidelines. Written informed consent was obtained prior to inclusion in the study. The Institutional Review Board waived the need for informed consent to obtain 1) de-identified archival blocks of lesional skin biopsies from BP patients; and 2) skin discarded at the time of surgery.−/CD49d+/CD16− eosinophils. Cytospins were prepared using freshly purified cells (4×104) on glass microscope slides with a Shandon 2 cytospin. Eosinophil purity was also confirmed using Wright’s stain (Fisher Scientific).Eosinophils were enriched from peripheral blood of BP or control subjects using density gradient centrifugation and immunoselection. Briefly, blood was diluted 1∶2 in PBS, subjected to density gradient centrifugation  and granulocytes were obtained after hypotonic lysis of the RBCs. Eosinophils were purified by immunomagnetic negative selection according to kit instructions (Miltenyi Biotec). For flow cytometry, cells were stained on ice with mixtures of CD16 (Alexa-Flour 488 or APC), CD49d-PE, CD203c-PerCP Cy5.5  and FcεRIα-APC (R & D Systems), or FcεRIβ (Abnova) followed by goat anti mouse IgG Alexa Fluor 488 (Molecular Probes) or anti human IgE-FITC (Bethyl Laboratories). CD203c staining was used eliminate basophils. This purification routinely results in >93% purity of CD203cRNA was extracted using an RNeasy Plus mini kit and followed by a genomic DNA Eliminator spin column (Qiagen). The RNA was collected using RNase- free water and quantified using NanoDrop. The RNA was DNase treated using the TURBO DNA-free ™ Kit as directed . One step RT-PCR was performed as follows: RT at 55°C for 30 minutes and inactivation at 94°C for 2 minutes; 35 cycles of PCR utilized 94°C denaturation, 15 sec, 57°C annealing, 30 sec, and 68°C extension for 1 minute. Final extension was at 68°C for 7 minutes. PCR products were run on a 1.5% agarose gel. PCR Primers: Beta Actin F5′-GGA CTT CGA GCA AGA GAT GG-3′ and Beta Actin R5′-AGC ACT GTG TTG GCG TAC AG-3′; IgE Alpha F5′-TGT GGC AGC TGG ACT ATG AG-3′ and IgE Alpha R5′-GAA ATG TGA CCT GCT GCT GA-3′; IgE Beta F5′-TAT TGA AGT CGG CCT CAT CC-3′ and IgE Beta R5′-TCC CCA GAA TGG ATA ACC TG-3′; IgE Gamma F5′-GGA GAG CCT CAG CTC TGC TA-3′ and IgE Gamma R5′- CAG GCA TAT GTG ATG CCA AC-3′.For IF, cytospins or biopsy cryosections (5 µM) were fixed in 50% methanol: acetone for 5 min, washed in tris-buffered saline  and stained in TBS/5% BSA. For MBP staining, cells were permeabilized with 5% Triton x100 in TBS. Primary antibodies included those specific for the following: MBP , FcεRIα , human IgE  or appropriate isotype controls. Species specific secondary antibodies conjugated to Alexa-546 or Alexa-488 (Molecular Probes) were used. Nuclei were counter-stained with diamidino-2-phenylindole (DAPI). Staining was visualized using a Nikon photomicroscope equipped with epifluorescence. Images were pseudo-colorized and occasionally adjusted for overall brightness using NIH Image J .In situ PLA, OLINK Bioscience). Briefly, slides were fixed in 4% paraformaldehyde, washed, and incubated with primary antibodies specific for human IgE , FcεRIα  and/or FcεRIβ . The proximity of bound antibodies was evaluated using species specific probes. If probes bind to sample in close proximity (<40 nm) to each other, ligation and amplification of the signal occurs. Images were captured using a Zeiss 710 confocal microscope at the University of Iowa Central Microscopy Research Facility and processed with NIH Image J .The proximity ligation assay (PLA) Commercially available ELISA kits were used to evaluate the following: BP180 and BP230 IgG, EDN . BP180-specific IgE was quantified using a previously described protocol The degranulation assay was adapted from studies evaluating mast cell degranulation Experiments were conducted with the number of individual patient samples indicated as N and results were expressed as mean ± SD. Assays utilizing human cells or tissues were conducted with duplicate or triplicate samples from the same patient being averaged and represented as n = 1. Statistical analysis was performed using GraphPad Prism software, version 5.0 . A non-parametric unpaired T-test (Mann-Whitney U-test) or ANOVA  was used to determine statistical significance between groups. Spearman’s rank correlation coefficient (r) was used to determine the statistical dependence between variables. The P-value of ≤0.05 was considered to be statistically significant.Patients with a confirmed diagnosis of BP who had not received any prior immunosuppressive treatment were enrolled in the study. Disease activity was assessed using either a BP Index both BP180 specific IgG and IgE were detected 75% (36/48) of BP samples, which is similar to our previous findings 3±1110, range 10–5830) were also above the normal range (40–390/mm3).Sera were collected from untreated BP patients , patients evaluated for other autoimmune skin diseases , or age- and gender-matched controls  and evaluated for BP180 IgG, BP230 IgG, total IgE and BP180 IgE by ELISA  D–G. BP To better understand the role of IgE and eosinophils in BP, we evaluated these factors in relation to the BP180 and BP230 IgG levels and disease activity. A correlation matrix was performed so that relationships between all variables could be explored. The strength of these associations is expressed as Spearman’s correlation coefficient (r) with the likelihood this relationship is due to chance decreasing as the p value gets smaller . As expeWhen analysis was focused on sera from patients (n = 16) with levels of total IgE≥400 IU/ml, some additional relationships were revealed (Table II). BP180 IgG levels correlated strongly with disease severity, but also with BP230 IgG, total circulating IgE and BP180 IgE. The concentration of total IgE was additionally correlated with disease severity and BP230 IgG levels. Although total IgE and BP180 IgE increased together in most BP samples (r = 0.438), this was not statistically significant. Interestingly, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG in this cohort. Overall, these analyses provide additional evidence for a relationship between IgE autoantibodies and peripheral eosinophilia in BP.3 and circulating IgE ranging from 34–3855 IU/ml. Two control cell lines were utilized: RBL-SX-38, a rat basophilic line which expresses all three chains of the human FcεRI . Peripheral blood granulocytes were enriched via density centrifugation prior to staining. FcεRI was not detected on eosinophils from controls subjects, regardless of circulating IgE concentration   . EosinopIgE≥400) , but notpatients , or contEosinophils were also examined for surface expression of CD23 and bound IgE. As expected, CD23 was detected on eosinophils from 10/10 BP patients and controls . Likewis+ eosinophils (red) from the circulation (n = 12) and lesional skin (n = 9) of BP patients (P = 0.008) higher frequency of circulating FcεRIα+/MBP+ cells in 12 BP patients, compared to 11 controls. As expected in the BP skin + cells were observed in both the blister cavity (indicated by the bar) and the dermis of biopsied skin . Immunofluorescent staining was utilized to evaluate expression of the FcεRI α-chain (green) on MBPpatients . Normal patients . Staininied skin . A subse2) or trimeric (αγ2), is expressed. To address this, we employed an in situ PLA to assess interaction of high affinity receptor chains and provide signal amplification. Antibodies specific for IgE and the α- and β-chains of FcεRI were used to label slides of enriched peripheral eosinophils or matched biopsy tissue from the same patient. The γ-chain of the FcεRI was not examined as it is present in both receptor forms Thus far, our RT-PCR, flow cytometry and IF staining suggest that eosinophils from BP patients express the FcεRI; however, the level of surface staining does not provide enough resolution to determine which receptor form, tetrameric  to one another, indicating interaction. Eosinophils were identified by their characteristic bi-lobed nucleus . Antigen specific degranulation is indicated as a percentage of total (non-immunologic) release triggered by ionomycin (100 µg/ml) for each sample. Antigen-specific EDN release was observed only in a subset (3/5) of samples from active BP patients, while no BP180-specific degranulation was observed in eosinophils from patients in remission (n = 8) or controls (n = 13). Additional studies will elucidate the mechanisms of eosinophil degranulation and its relevance to BP.The presence of BP180-specific IgE, coupled with eosinophil FcεRI expression, provides a potential mechanism for antigen specific responses in BP. Eosinophils primarily respond to FcεRI engagement through release of preformed mediators via piecemeal degranulation BP patients often exhibit increased serum IgE levels accompanied by peripheral or lesional eosinophilia. Previously, we and others proposed a relationship between circulating total IgE The current studies reveal a significant correlation between eosinophil count and disease severity when a large number of untreated BP patients were evaluated. However, the strongest associations were observed in the subset of patients with circulating IgE≥400 IU/L where i) total IgE correlated strongly with disease severity; and ii) peripheral eosinophil counts correlated with both total and BP180 specific IgE. These studies provide further clinical evidence for a link between IgE and eosinophil activation in BP.2) FcεRI, which is largely accepted as the receptor form expressed by eosinophils 2) receptor form under certain conditions, mRNA expression does not necessarily reflect expression on the cell surface. Furthermore, the complete lack of receptor mRNA in some samples, despite elevated IgE and/or eosinophilia, further underscores the complex nature of eosinophil FcεRI receptor expression. In humans, IL-4 is required for the production of the FcεRI α-chain Eosinophil expression of FcεRI is reported in diseases associated with elevated circulating IgE and eosinophilia −/CD16−/CD49d+ eosinophils. Surface FcεRI α-chain is representative of an intact receptor since the α-chain is not translocated to the surface in the absence of the γ-chain Typically, eosinophil surface expression of FcεRI is quite low when compared to basophils 2) receptor form in some patients.CD23 is known to be expressed on the surface of eosinophils and basophils where it plays a role in cellular activation and degranulation. In our hands, CD23 expression was detected on peripheral eosinophils from BP patients and controls as well as basophils from BP patients. Furthermore, interaction of CD23 with IgE was detected on the surface of a variety of cells in lesional biopsies and normal skin using the PLA (data not shown). Thus, evaluation of surface bound IgE likely represents total IgE receptor expression, rather than FcεRI or CD23 alone. Since the clinical manifestations of BP are exhibited in the skin, eosinophil FcεRI expression was evaluated in lesional biopsies using two approaches. In the first, eosinophils were identified by the presence of eosinophil major basic protein (MBP), which requires permeabilization of the samples. FcεRI staining prior to permeabilization resulted in loss of signal, therefore colocalization of FcεRI α-chain protein with MBP reflects both surface bound and intracellular receptor stores 2) FcεRI on the surface of eosinophils in the skin; however, others 2) receptor in BP is associated with IgE levels, eosinophilia, disease activity, or some other factor. It is well established that the antigenic crosslinking of the tetrameric FcεRI on basophils results in degranulation To our knowledge, this is the first report of the tetrameric (αβγ2) FcεRI may contribute to eosinophil degranulation in BP lesions.In summary, these studies utilized clinical samples to explore the relationships between IgE autoantibodies and eosinophilia in BP. In patients with IgE≥400 IU/ml, peripheral eosinophil count correlated strongly with IgE autoantibodies directed against BP180. Expression of FcεRI by circulating and lesional eosinophils from BP patients provides a novel mechanism of action for IgE in BP. Furthermore, these studies suggest that expression of the tetrameric  on z-series image stacks using high resolution confocal microscopy. Interaction of FcεRIα/IgE on tissue eosinophils from BP patient results in a red fluorescent signal. The image stacks have been smoothed and pseudo-colored in NIH ImageJ. Scale bar = 50 uM.(AVI)Click here for additional data file.Movie S2Representative interaction of FcεRIα and FcεRIβ on the surface of eosinophils in BP lesions. Interaction of specific molecules was evaluated using the proximity ligation assay on non-permeabilized skin cryosections from BP patients. Eosinophils were identified by their unique bi-lobed nuclear morphology  on z-series image stacks using high resolution confocal microscopy. Interaction of FcεRIα/FcεRIβ on tissue eosinophils from BP patient results in a red fluorescent signal. Circled area contains several eosinophils. The image stacks have been smoothed, annotated and pseudo-colored in NIH ImageJ. Scale bar = 50 uM.(AVI)Click here for additional data file.


We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known splN mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit  The Notch signaling pathway is highly conserved among metazoan organisms and plays a pivotal role in cell fate determination throughout development Over the years a remarkably detailed picture has emerged on the conserved components and mechanisms controlling ligand binding, Notch receptor processing, the nuclear functions of its intracellular domain (NICD), and factors mediating expression of the HES repressors. Despite this progress, our understanding of the mechanisms by which the large number of HES repressors mediate the diverse functions of Notch still remains incomplete. Because of their conserved (bHLH) structure, it has been thought that the HES proteins are functionally redundant and that they act as dosage-dependent effectors of Notch signaling. Arguments against both views (see below) have emerged from studies in Drosophila, specifically during the patterning/selection of the ‘founding’ R8 photoreceptors in the compound eye and the bristle sensory organ precursors (SOPs).atonal (ato) in the case of the R8 photoreceptor achaete scute Complex (ASC) in the case of the bristle SOP The development of the R8 or SOP is dependent upon bHLH proneural activators Enhancer of split Complex, E(spl)CE(spl)C elicits the birth of supernumerary R8s/SOPs Suppressor of Hairless (Su(H)) E(spl)D) elicits dominant loss of R8s and the eye A body of genetic studies have sought to analyze the roles of the seven HES repressors of Drosophila encoded by the This paradox was resolved for E(spl)-M8 whose ability to bind and antagonize Ato requires phosphorylation by protein kinase CK2 Notch (splN) backgrounds microtubule star (mts), the unique catalytic subunit of Drosophila PP2A, elicits twinned R8s/SOPs, defects that closely mimic loss of Notch or CK2. The possibility thus arises that PP2A antagonizes Notch signaling. However, the participating PP2A regulatory subunit remained to be identified, and it was unknown if this phosphatase impacted E(spl)-M8 activity in vivo.The aforementioned studies, in turn, raise the question on control of E(spl)-M8 by dephosphorylation. A candidate enzyme is the phosphatase PP2A, whose role emerged in assays for impaired signaling in wild type and mutant widerborst (wdb), twins (tws), B52 and B56. The studies described here focus on wdbNotch and E(spl), we provide evidence that Notch signaling is potentiated by loss of wdb and antagonized by its overexpression, and that wdb mitigates the activity of ectopically expressed M8 protein. The multiple lines of evidence in the bristle, eye and wing identify wdb as a component of Notch signaling and suggest that repression by E(spl) proteins is controlled by phosphorylation and dephosphorylation.Drosophila PP2A, like the mammalian enzyme, is composed of a catalytic (C) and a scaffolding (A) subunit that associate with a regulatory (R) subunit 109-68Gal4 (B6479), scaGal4E(spl)Gal4 (B8225). The following mutants were obtained from the Bloomington Stock Center; KG02977wdb (B12977), 55e11, frt19AN (B28813), splN  and E(spl)D (B2447). EP3559wdb (see ref UAS-m8 and UAS-m8S159D flies have been previously described by us UAS-RNAi lines to the PP2A regulatory subunits were obtained from the Drosophila Genetic Resource Center and include PP2A–EY22564B’ (Flybase #22569) and 02414tws (Flybase #108872).Flies were raised at 24°C on standard Yeast-Glucose medium. All crosses were performed at 24°C, unless indicated otherwise. The Gal4 drivers were generously provided by other investigators or obtained from the Bloomington Stock Center (denoted by the prefix B). The Gal4 drivers are  see ref  flies weFly heads were removed from newly eclosed flies. Heads were passed through a graded alcohol series for 24 hours each (25%–50%–75%-absolute), passed through Hexamethyldisalizane, and mounted on EM stubs using carbon tape (Ted Pella). Fly heads were dried for 24 hours, sputter coated with gold, and examined with a Hitachi scanning electron microscope at an accelerating voltage of 20 kV. Images were acquired as TIFF files that were collated using Adobe Illustrator. For quantitative analysis of eye size (facet number), crosses were established in duplicates, and ≥15 adult flies for each genotype were photographed using a Leica MZ16 stereomicroscope equipped with a Leica DFC-480 digital camera. Facet numbers were manually counted from TIFF images of the eyes and subjected to statistical analysis. For bristle phenotypes, newly eclosed adults were photographed using a Leica MZ16 stereomicroscope or by scanning EM. For quantitative analysis of the bristle phenotypes, multiple crosses were established (triplicates), and adults were scored for bristle defects. Wing margin phenotypes were documented using a Leica MZ16 stereomicroscope. Eye size (facet counts) and bristle and wing defects were statistically analyzed using the Student’s t-test, with the exception of Imaginal discs were isolated from late third instar larvae, fixed in freshly prepared 4% paraformaldehyde in 1x phosphate buffered saline (PBS) for 45 minutes at 4°C, and washed three times with PBS containing 0.3% Triton X-100 (PBS-TX). The discs were incubated for 12 hours at 4°C in PBS-TX containing 5% normal goat serum and primary antibody, washed three times with PBS containing 0.3% Triton X-100 (PBS-TX) and immunostained for 2–3 hours in secondary antibody. Following this, eye discs were washed three times with PBS containing 0.3% Triton X-100 (PBS-TX) and mounted in Vectashield. The following antibodies were used in this study; guinea pig anti-Sens  at a dilution of 1∶500 and mouse anti-ELAV  at a dilution of 1∶500. The mouse anti-ELAV antibody (9F8A9) developed by Gerald Rubin was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Secondary antibodies (Molecular Probes) were goat-anti mouse-IgG coupled to Alexa Fluor 594 (1∶1000) and goat anti-guinea pig-IgG coupled to Alexa Flour 488 (1∶1000).An Olympus FluoView (FV1000) was used for imaging. Images were generated from scans acquired every 1 µm along the apicobasal axis of the discs. Scanning was limited along the Z-axis to acquire full spectral output of the fluorophores, and no layers were removed from confocal stacks. Confocal images were compressed as a Z-stack, exported as TIFF files and collated in Adobe Illustrator.mts, in Notch signaling wdb because it had been identified in a GoF screen for genes that affect Drosophila bristle development EP3559wdb (UAS-wdb) by scaGal4 elicits ectopic and split macrochaetes (MCs) on the notum , we used the enhancer trap 109-68Gal4sca, a driver of weaker strength . 109-68Gal4/+ flies display a notably lower incidence of ectopic MCs , but importantly do not exhibit any split MCs (EP3559)wdb Gal4. We find that expression of wdb elicits notched wings with a high penetrance (EP3559/+wdb flies or in E(spl)Gal4/+ flies Gal4wdb would modify the classical wing margin defect of the Notch allele 55e11N. Consistent with a wdb-GoF role in reducing Notch pathway activity, reduction of wdb dosage  yields a significant rescue of the wing margin defect of 55e11N heterozygotes , similar to the inactivity of ectopic Mts, reported by us Loss of CK2 compromises lateral inhibition and elicits ectopic (twinned) R8s and rough eyes wdb plays a role in R8 selection, we employed splN, a widely studied mutation that mainly affects the eye. Specifically, splN increases receptor sensitivity to the ligand Delta, thereby allowing inappropriate Notch activity in R8 precursors leading to loss of R8s ato expression and progression of eye development, further R8 specification becomes impaired. This reduces both, the number of ommatidia (facets) and their hexagonal phasing, which manifest as reduced and rough eyes, respectively  or E(spl)CCK2-RNAi or Mts-GoF To evaluate if ectively . ConsistKG02977wdb modifies the retinal defects of splN. Indeed, when homozygous, KG02977wdb exacerbates the reduced and rough eye of splN males  was exacerbated in splN/Y; KG02977wdb/KG02977wdb flies  rescues the reduced eye of splN . Importantly, no modulation was seen in relevant control genotypes , but found that neither modulated splN (data not shown). As an alternative, we expressed UAS-RNAi lines that target tws and the B’ regulatory subunits with 109-68Gal4 but found that they did not modulate splN (data not shown). Similarly, the recessive lethal allele XE2258mts did not exacerbate the reduced eye of splN (data not shown), indicating that a 50% reduction in tws or mts activity is not limiting for PP2A activity. Therefore, our studies do not formally exclude the contributions of the regulatory subunits tws or B’.We also tested a recessive lethal allele of KG02977wdb on splN involve the R8 cells, the founding photoreceptors splN eye discs  mutant background. We employed the dominant m8 mutation E(spl)D, which elicits a reduced eye, but only in splN flies D/+N males display a severely reduced eye field D/+N females CWe next evaluated the role of lies see . Given twdb with 109-68Gal4 rescues the reduced eye of spl/+; E(spl)D/+N females, appears to restore facet phasing D/+N eye discs display gaps between differentiated R8s, regions also devoid of secondary (ELAV+) photoreceptors D/+N females D.We find that overexpression of  compare , and sig compare . These eenotypes . We nextenotypes , Nspl/+;eceptors . Moreove females , an outc females . Overexpruitment . Thus inwdb overexpression would attenuate the activity of ectopic M8, as this would provide in vivo evidence that PP2A affects E(spl) activity, rather than expression of endogenous E(spl) proteins by perturbed Notch signaling. This was deemed important, because the modulation observed in our studies (wdb-GoF/LoF. We have previously shown that expression of UAS-M8 (wild type) with scaGal4 elicits strong loss of the IOBs, but does not affect R8 birth, eye size or facet phasing (UAS-wdb significantly restores IOBs (UAS-LacZ (data not shown), excluding competition for limiting amounts of Gal4 protein. Expression of wdb-alone elicits only occasional loss or duplicated IOBs  effects on components of the Notch receptor pathway.We next tested if  studies –4 could  phasing . Indeed,res IOBs . This efIOBs see . As thesscaGal4 elicits a severely reduced eye , whereas that with 109-68Gal4 is more muted  photoreceptors D/+N flies D/+N females  mutants, and upon ectopic expression of E(spl)-M8 or its CK2-site variant M8-S159D. Our studies suggest that PTM of HES repressors constitutes a sophisticated yet poorly studied component of Notch signaling.Control of protein activities by phosphorylation is a widespread regulatory mechanism, and reflects the need for rapid switching between two activity states. These modifications are often fine-tuned by the coordinated activities of phosphatases. One such enzyme is PP2A, whose role in Notch signaling was unclear. The studies described here identify wdb elicits bristle and wing margin defects that mimic loss of Notch (Notch and E(spl) mutations -M8, one of the bHLH effectors of this pathway.Specifically, we demonstrate that ectopic of Notch , rescuesutations , 3 and 4utations  and thatutations . The effn N55e11  and Nspl11  activity. If any of these were the case, ectopic wdb would attenuate expression of endogenous E(spl) proteins or the mutant (M8) protein encoded by the E(spl)D allele -M8 activity.It has been previously shown that decreased dosage of E(spl) or CK2 rescues the eye/R8 defects of D allele , either D allele  and the D allele  were in SP motif , raising the prospect that M8, like HES6, requires multi-site phosphorylation. In light of these findings, PP2A may target the MAPK site or modifications at the other Ser residues , which requires multi-site phosphorylation of fly/human Period /HES/HER proteins display length and sequence heterogeneity of the CtD. Computational analysis of fly E(spl) proteins reveals that the P-domain and its flanking sequences (aside from WRPW) are intrinsically disordered (ID), as is also the case for the HES/HER proteins , a window of time over which mutations would have accumulated were these inconsequential for repressor activity.Firstly, how widespread is the regulation of E(spl)/HES proteins by PTM? The P-domain is highly conserved amongst M8, M7 and M5 , and ourato-enhancers ato-enhancer ASC enhancer, whose mutations lead to impaired repression. Given the roles of HES proteins in myogenesis, oogenesis, somitogenesis, it is conceivable that either one or a combination of mechanisms regulate repression, or that in vivo targets of HES proteins are isoform-specific. Consistent with the latter possibility, the direct physical interaction of M8 with Ato has not been observed with E(spl)-Mγ and-Mδ Second, why are some E(spl)/HES proteins devoid of a P-domain? We speculate that this may reflect different modes by which E(spl)/HES proteins repress cell fate. Perhaps, the best example is the M8-Ato interaction. It was initially thought that E(spl) proteins bind N-box sequences to mediate repression doubletimeshaggysplN mutants Third, can the principles gleaned from other biological contexts apply to our studies on CK2, PP2A and Notch? The regulation of the central clock protein Period (PER) provides an attractive model. Detailed studies have revealed that PER activity/levels are regulated by CK2 and PP2A On its own, the shared regulation of M8 and HES6 makes for a strong evolutionary argument. Although the role of PP2A in HES6 regulation remains unknown, we note that HES6 regulates transitions in retinal cell fate specification and during development of the cerebral cortex, where progenitors give rise to the ordered specification of neurons (first), astrocytes (second) and oligodendrocytes (third) Future studies to identify the site on M8 that is a target for PP2A, and whether HES6 is similarly regulated by dephosphorylation will be required to more fully reveal the mechanism(s) by which phosphorylation and dephosphorylation control repression. Thus, the observation of M8 control by PTM fortuitously opens a new window into regulation of Notch signaling, and raises the prospect that this mechanism is differentially employed in tissue patterning.


Dermatophagoides pteronyssinus)-specific IgE and IgE secretion B cells. The mRNA expression of FcεR1α was significantly increased after Der p2 stimulation in PBMCs with SNPs of the FcεR1α promoter region. Despite the increased Cε mRNA expression in PBMCs and histamine release from PMNs and the up-regulated mRNA expression of interleukin (IL)-6 and IL-8 secretions after Der p2 stimulation, there was no statistically significant difference between SNPs of the FcεR1α promoter region and the wild type. SNPs of FcεR1α promoter region were associated with IgE expression, IgE producing B cells, and increased Der p2-induced FcεR1α mRNA expression. These SNPs may be used as a disease marker for IgE-mediated allergic inflammation caused by Dermatophagoides pteronyssinus.The high affinity immunoglobulin E (IgE) receptor-FcεR1 is mainly expressed on the surface of effector cells. Cross-linking of IgE Abs bound to FcεR1 by multi-valent antigens can induce the activation of these cells and the secretion of inflammatory mediators. Since FcεR1 plays a central role in the induction and maintenance of allergic responses, this study aimed to investigate the association of FcεR1 with the allergic phenotype of Cε expression and cytokine and histamine release from peripheral leukocytes. Peripheral leukocytes from 67 allergic and 50 non-allergic subjects were used for genotyping analysis. Peripheral mononuclear cells (PBMCs) were used for Cε expression and ELISpot analysis, while polymorphonuclear cells (PMNs) were used for histamine release. The association between genotype polymorphism of the FcεR1α promoter region (rs2427827 and rs2251746) and allergic features of Cε expression and histamine were analyzed, and their effects on leukocytes function were compared with wild type. The genotype polymorphisms of FcεR1α promoter region with CT and TT in rs2427827 and TC in rs2251746 were significantly higher in allergic patients than in non-allergic controls. Patients with single nucleotide polymorphism (SNP) of FcεR1α promoter region had high levels of total IgE, mite-specific Der p 2 (Group 2 allergen of  During the IgE-mediated response, IgE binds with a high-affinity IgE receptor consisting of a tetramer of a ligand-binding α chain FcεR1α), a signal-augmenting β, and a signal-transducing γ chain dimer. The receptor is abundantly expressed on the surface of mast cells and basophils, and is thought to be involved in allergic inflammation of the asthmatic airway. Because high total serum IgE levels are closely correlated with the clinical expression and severity of asthma and allergy, IgE is thought to play a key role in the pathogenesis of allergic diseases α, a sign. The reg samples .FCER1A) using 1000 Genomes Project datasets and identified its concomitant contribution to IgE regulation [The FcεR1, also known as high-affinity IgE receptor, is the high affinity receptor for the Fc region of IgE and is a tetrameric receptor complex consisting of one α (FcεR1α-antibody binding site), one β , and two disulfide bridge connected γ chains . The binding itself of monomeric IgE to FcεR1 can promote the survival of mast cells without cross-linking of the receptor ,8, suggeH) gene to be expressed from Cμ(IgM) to other CH genes, resulting in switch of the Ig isotype from IgM to either IgG, IgA or IgE. Stimulation of B-cells with anti-CD40 plus IL-4 induced CSR from Cμ to Cε (IgE), which contributes to the pathogenesis of allergic diseases. This study investigated the association of genetic polymorphisms in the FcεR1α promoter region with the clinical features of allergy, and analyzed its genetic effect on the mRNA expression of FcεR1α and Cε.Although no interaction between the single nucleotide polymorphism (SNP) in the FcεR1α and environmental exposures including endotoxin was observed, the SNP in the FcεR1α exhibits strong effects on total serum IgE levels . It suggThe association of genetic polymorphisms of the FcεR1α promoter region with clinical features of allergy was analyzed in patients with allergy. Two different genotypes of FcεR1α promoter regions, rs2427827 (−344) and rs2251746 (−95), were analyzed . The genComparisons of the genotype and frequency of FcεR1α polymorphism in the promoter region between allergic patients and healthy controls revealed that the genotype of the FcεR1α promoter region with CT and TT in rs2427827 (−344) and TC in rs2251746 (−95). The genotypes of the rs2427827 (−344) CT/TT and rs2251746 (−95) TC were significantly higher in allergic patients . The freThe PBMCs derived from allergic subjects with genotypes rs2251746  and rs2427827  of the FcεR1α promoter region were collected to investigate the effects of Der p2 on FcεR1α mRNA expression. The PBMCs were cultured with or without Der p2 stimulation for 48 h. The cell pellets were collected to extract RNA for mRNA expression. There were significantly higher levels of FcεR1α mRNA expression in the PBMCs after Der p2 stimulation in patients with genotypes rs2251746  and rs2427827  of the FcεR1α promoter region .The PBMCs derived from allergic patients were cultured and analyzed with or without Der p2 and LPS stimulation. The cell pellets were collected for Cε mRNA expression and supernatant were collected for cytokine analysis. There were significantly higher levels of Cε mRNA expression in PBMCs of patients with SNPs of the FcεR1α promoter region compared to those derived from the wild type. However, there was no significantly increased Cε mRNA expression in PBMCs derived from either group after Der p2 stimulation .The PMNs with different FcεR1α promoter SNPs were used for the histamine release assay, and the results showed there were no statistically significant differences between the two different FcεR1α promoter genes A,B. NoneThe PBMCs derived from subjects with genotypes rs2251746  and rs2427827  of the FcεR1α promoter region were collected for cytokine secretion analysis. There was no significant difference between the two groups except for IL-6 and IL-8 levels before and after Der p2 stimulation. There were increased secretions of IL6 and IL8 in the PBMCs derived from patients with SNPs of the FcεR1α promoter region after Der p2 stimulation. Both IL-6 and IL-8 also increased in subjects with the wild type of FcεR1α promoter region . There wELISpot assay was used to analyze the IgE producing B cells . The resDermatophagoides pteronyssinus.The associations between SNPs (rs2427827 and rs2251746) in the FcεR1α promoter region and allergic features of Cε expression, histamine release were analyzed, and their effects on leukocyte function compared among the different SNPs were performed in this study. The results showed the two SNPs in the FcεR1α promoter region with CT/TT in rs2427827 and TC in rs2251746 were significantly higher in allergic patients than in non-allergic controls. Patients with the two SNPs (CT/TT in rs2427827 and TC in rs2251746) in the FcεR1α promoter region had high levels of total IgE, mite-specific Der p2-specific IgE and IgE secretion B cells. The mRNA expression of FcεR1α was significantly increased after Der p2 stimulation in PBMCs with the two SNPs. Thus, the results suggest that the SNPs (CT/TT in rs2427827 and TC in rs2251746) in the FcεR1α promoter region are associated with IgE expression, IgE producing B cells, and increase Der p2-induced FcεR1α mRNA expression. These SNPs may be used as a disease marker for IgE-mediated allergic inflammation caused by FCERIA on human chromosome 1q23 [FCERIA polymorphisms not only drive IgE levels but also influence IgE in association with asthma, suggesting that disease-specific mechanisms in IgE regulation exist [et al. [Genetic determinants were suspected to influence IgE regulation and IgE levels considerably, and recently genome-wide association studies (GWAS) allowed the identification of the linkage and candidate gene for the total serum IgE levels such as ome 1q23 . A largeome 1q23 . The FCEon exist . The polon exist . The ass [et al. .2 heterotetramer, but on the other cells only two subunits forming αγ2 heterotrimer. The structure and function of the FcεR1A gene, encoding for α-subunit of FcεRI (FcεRIα), plays an important role in the pathogenesis of allergic diseases. It has been reported that the FcεR1α receptor gene play important roles in IgE synthesis and IgE-mediated response [FcεR1A variants and total serum IgE levels in allergic subjects. Similar findings of significantly higher levels of Cε mRNA expression in PBMCs derived from subjects of genotypes with −95C and −344T alleles of the FcεR1α promoter region compared to those derived from genotypes with −95T and −344C alleles have been observed. These results suggest that the two SNPs of the FcεR1α promoter region not only affect FcεR1α expression but also Cε expression.The high affinity receptor for IgE, FcεRI, is expressed on several different types of cells such as basophils/mast cells, epidermal Langerhans cells, dermal dendritic cells, monocytes, and eosinophils. Among these cells, the receptor on basophils/mast cells is composed of three different subunits that form αβγresponse . The basThe functional investigation of the FcεR1α promoter showed the −66T/C polymorphism affecting the transcription activity GATA-1-binding affinity, which is one of the determinants for allergic diseases . The −34FCER1A), 5q31 , 12q13 (STAT6), 6p21.3 (HLA-DRB1) and 16p12 , suggesting that genetic variants in these loci may influence IgE regulation [FCERIA have functional associations, which were reported and replicated in several research groups. These associations do not confer susceptibility to allergic diseases, but rather modulate serum concentrations of IgE. FCERIA is a good candidate in allergic diseases, and appears to participate in system regulation of IgE levels [The IgE mediated immune responses are best known for their involvement in allergies, and genetic determinants are suspected to influence IgE regulation and IgE levels considerably. Recently GWAS have provided the power to identify genetic determinants for serum IgE levels such as 1q23  and each participant provided written informed consent before enrollment. Sixty-seven patients with airway allergy and 50 non-allergic subjects who consulted at the Allergy Clinic of Taichung-Veterans General Hospital were recruited. There were 36 males and 31 females in these allergic patients. The age ranged from 8 to 48 years old, with the mean and standard deviation as 27 ± 15.6. Patients with airway allergy were defined as having a history of recurrent nasal stuffiness, sneezing, and/or asthma. Serum total IgE and mite specific IgE were measured by using the UniCAP system  as background value. Cytokine and histamine release from leukocytes derived from different genetic polymorphisms were also examined.n = 52) and healthy controls (n = 50) were compared. Two SNPs rs2427827 (−344) and rs2251746 (−95) in the promoter region were identified by direct sequencing of the PCR products of genomic DNA.Blood samples (5 mL) were collected with EDTA syringes and DNA from the buffy coat was purified using a Genomic DNA Mini kit . A set of primers was designed for the amplification 684 base pair PCR product based on FceR1α promoter region. The forward and reverse primer sequences were 5'-AAGAAAAGCGTTGGTAGCTCTGGTG-3' and 5'-ATCTTCTTCATGGACTCCTGGTGC-3', respectively. The genotype and frequency of the FcεR1α promoter region with CT and TT in rs2427827 (−344) and TC in rs2251746 (−95) between allergic patients (®-Allele 2) was conducted using 96-well plates in an ABI Prism 7900 Real-Time PCR system (Applied Biosystems). Duplicate samples and negative controls were included to ensure genotyping accuracy.The FcεR1β and FcεR1γ genotypes were determined using a TaqMan real-time polymerase chain reaction (PCR)  in a StepOnePlus™ Real-Time PCR System . The Assay IDs of rs1441586 and rs11587213 were C_1842226_10 and C_27848237_10, respectively. Negative controls were included in each PCR to avoid contamination. Detection of the probe signal with two ABI fluorescent dyes . The cells were stimulated with or without Der p2 , or LPS  for 3 days in RPMI-1640 medium containing 10% heat inactivated FBS and 1% streptomycin/penicillin in a humidified 5% CO2 atmosphere. After the PBMCs cultured with or without Der p2 stimulation, the cell pellets were collected to extract RNA for mRNA expression. The cell culture supernatant were collected and evaluated for cytokine concentrations using commercially available Luminex MAP® kits according to the manufacturers’ directions. Correlations between data sets were evaluated using Pearson’s correlation coefficient (r).Human PBMCs were prepared by density centrifugation Ficoll-paque2 incubator. ELISpot assays were performed utilizing Mabtech™ ELISpot plus Human IgE kits . The number of spot-forming cells in each well was automatically counted with a Cellular Technology Limited (CTL)-ImmunoSpot Analyzer Human PBMCs were prepared by density centrifugation Ficoll-paque™ PLUS. B cells were then purified by negative selection with a B cell isolation kits. Non-B cells were indirectly magnetically labeled by using a cocktail of biotin-conjugated antibodies against CD2, CD14, CD16, CD36, CD43, CD235a (Glycophorin A), and Anti-Biotin MicroBeads . Isolation of highly pure B cells is achieved by depletion of magnetically labeled cells. Purified B cells were cultured with Der p2 (10 μg/mL) in the presence of IL-4 (30 ng/mL), IL-21 (25 ng/mL) and anti-CD40 (1 μg/mL) for 5 days in the COThe PBMCs derived from allergic patients were cultured with or without Der p2 or LPS for 3 days, and the cell pellets were then collected to extract RNA for Cε mRNA expression and supernatant for cytokine analysis. Total RNA from cells was extracted using the RNeasy mini kit . The first standard cDNA was synthesized by the RevertAid M-MuLV reverse transcriptase  according to the manufacturer’s protocol. The cDNA then served as a template in a PCR using the G-Storm PCR machine . The IgE synthesis from B-cells carries out from the IgM to isotype IgG, which mRNA expression from Cμ gene to Cε gene. Detection of excision circular transcripts from direct Cμ gene to Cε switching events was performed using a forward primer in Iε (5'-CGTCTTCCCCTTGACCCGCTGCTG-3') together with a reverse primer in Cμ (5'-CACGTCCATGACCTGCCCGTCCTC-3'). The amplification cycles were as follows: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. The PCR products were then subjected to electrophoresis on a 2% agarose gel after 30 cycles and the electrophoresis products were visualized by ethidium bromide staining. The mRNA of GAPDH was used to control sample integrity and loading.i.e., anti-histamines and systemic corticosteroids) for at least 24 h before blood sampling.Sera from 30 patients with airway allergy or non-allergic controls were collected for basophil histamine release assay. The patients were instructed to avoid the use of systemic anti-allergenic drugs (6 cells/mL using trypan blue assessment. Passive sensitization of KU812 cells with sera from patients or controls (1/5 volume of subject sera) was performed for 4 h at 37 °C.Histamine release assay was analyzed after incubation with Der p2 for 30 min. The basophil cell line KU812 was used as control. The polymorphonuclear cells (PMNs) were isolated from the blood of allergic patients by centrifugation of the leukocytes on Ficoll-paque™ PLUS. KU812 cells  were re-suspended in medium RPMI-1640 by adjusting to 1 × 10O-phthalaldehyde  for 7 min. The reaction was stopped by adding H2SO4 (0.04 M). The histamine released into the supernatant was measured by a fluorescence spectrophotometer. The percentage of histamine release was calculated using the following formula:After incubation with sera, the sensitized KU812 cells or PMNs from allergic patients were then incubated with 10 μg/mL of Der p2 for 30 min at 37 °C and with 1 μg/mL of A23187  as total release histamine. The supernatant was collected and reacted with p < 0.05.All statistical analyses were performed using SPSS software . The SPSS Sample Power 2.0 was used for power calculation analysis. Values were presented as mean ± standard error of mean (SEM). All analyses were performed by analysis of variance (ANOVA) with non-parametric testing, followed by Spearman’s coefficient and Kruskal-Wallis Test. Statistical significance was set at Dermatophagoides pteronyssinus.SNPs of the FcεR1α promoter region (rs2427827 and rs2251746) are associated with the phenotype of IgE-mediated allergic diseases, including total IgE, Der p 2-specific IgE and IgE producing B cells. The SNPs of the FcεR1α promoter regions rs2427827 and rs2251746 are associated with Der p2-induced FcεR1α mRNA expression and high levels of Cε expression. These findings suggest that SNPs may play an important role in the allergic inflammation caused by


This article reviews the historical context, indications, techniques, and complications of four posterior fixation techniques to stabilize the subaxial cervical spine. Specifically, posterior wiring, laminar screw fixation, lateral mass fixation, and pedicle screw fixation are among the common methods of operative fixation of the subaxial cervical spine. While wiring and laminar screw fixation are now rarely used, both lateral mass and pedicle screw fixation are technically challenging and present the risk of significant complications if performed incorrectly. With a sound understanding of anatomy and rigorous preoperative evaluation of bony structures, both lateral mass and pedicle screw fixation provide a safe and reliable method for subaxial cervical spine fixation. Posterior fixation of the subaxial spine is routinely performed for cervical spinal instability from any etiology, such as trauma, infection, primary or metastatic malignancy, or decompressive laminectomy. It is often done to augment subaxial spine stabilization in conjunction with multilevel anterior decompression fusion procedures. Multiple posterior approaches for surgical fixation of the subaxial spine have been developed and continually modified over the years with a better understanding of subaxial spine anatomy and biomechanics, concurrent with improved spine implant technology. This article reviews four common fixation techniques, which comprise posterior cervical wiring, laminar screw fixation, lateral mass screw fixation, and pedicle screw fixation. With each technique, we detail its historical context, indications, contraindications, techniques, and recommendations for complication avoidance.IntroductionPosterior cervical wiring historically has played a major role in stabilizing the cervical spine. Hadra first introduced spinous process wiring in Pott’s disease in 1891 -3. RogerIndicationsPosterior cervical wiring requires that the posterior bony ring is preserved . Sublaminar wiring has been shown to be cost-effective with low neurological risks in patients with cervical spine trauma -10. BecaContraindicationsAny traumatic or pathologic process that compromises the integrity of the posterior bony elements  is a contraindication for posterior cervical wiring. In addition, wiring only offers resistance against flexion, so additional fixation technique(s) must be employed to provide extension, rotation, and lateral bending stability. Tension band wiring is at risk for failing in osteoporotic bone. In general, stand-alone posterior fixation is relatively contraindicated when there is instability of the anterior or middle column.  TechniquesThere are many posterior wire stabilization techniques that have been developed and described in the literature. We will review the original Rogers’ interspinous wiring technique and one of its modifications, Abdu's triple-wiring technique, which has been shown to impart greater biomechanical stability .In the Rogers’ interspinous technique, a burr hole is drilled transversely at the base of the upper spinous process and the lower spinous process. A stainless steel or titanium wire or cable is passed through the burr holes in a figure eight pattern. Finally, the wire is tightened using a tensioner Figure .With Abdu’s triple-wiring, the wire is first secured under tension as described by the Rogers’ technique. Subsequently, the second wire is passed through the upper burr hole and looped around the upper spinous process. Similarly, the third wire is passed through the lower burr hole and looped around the lower spinous process. These latter two wires are then passed through corresponding holes drilled in two autologous bone graft struts, each placed lateral to the spinous processes. These wires are then tightened under tension Figure  13-14].-14.13-14ComplicationsThe main complications from posterior cervical wiring are wire pullout and injury to the spinal cord or exiting spinal nerves from the misdirection of the wire. Fortunately, these complications are rare . Over-tiComplication AvoidanceAs with any surgery, comprehensive preoperative planning is necessary to study the anatomy and evaluate for anatomical variations in order to plan the operation accordingly. Patients with anterior or middle column instability will need additional anterior fusion and instrumentation. Sublaminar wiring should be avoided in patients with a narrowed spinal canal or neural foramen. Avoid kinking during wire passage to prevent creating a stress riser where wire breakage can occur. The tension should be sequentially tightened with the tensioner as to avoid loosening as well as over-tightening. When drilling at the base of the spinous process, avoid drilling too deeply, and angle the drill transversely  to avoid violating the epidural space.   IntroductionTraditionally, translaminar screw fixation is better described and more frequently performed in the atlantoaxial and thoracolumbar spine -17. LamiIndicationsLaminar screws may be used in cases of deficient lateral masses or after failed attempts to place a lateral mass screw. They appear to have better biomechanics at C7 when compared to the C3-C6 levels. As with any posterior fixation technique, translaminar screw fixation requires intact posterior elements, specifically intact laminae, at the levels to be instrumented. ContraindicationsAny traumatic or pathologic process that leads to insufficiency of the lamina is a contraindication to laminar screw fixation . As with any posterior fixation technique, stand-alone translaminar screw fixation is insufficient to restore stability in cases involving the anterior and/or middle columns. TechniquesWe will review the technique for C7 screw placement as described by Hong, et al. and Jang, et al. in 2008 and 2010 Figure  20-22]..22].Preoperative axial CT imaging is obtained to measure the translaminar screw length as well as trajectory angle and path. The screw length is estimated as the length from the contralateral spinolaminar junction to the ipsilateral lamina/lateral mass junction . An entrComplicationsThe complications of laminar screw fixation include laminar cortical breach and violation of the facet joint. Breach of the medial cortex can lead to spinal cord injury and penetration of the thecal sac. Screw loosening and hardware failure are other reported complications -22.Complication AvoidanceAlthough injury to the vertebral artery is theoretically lower with intralaminar screw placement, a long drill or screw can potentially injure the lateral structures, including the vertebral artery. Preoperative CT is warranted to measure the size of the lamina and estimate the length of the screw to ensure there is adequate bone stock and laminar diameter for good screw purchase. Intraoperative fluoroscopic imaging can assist in identifying the ideal trajectory for screw placement to prevent penetration of the cortical walls.IntroductionLateral mass screw fixation is widely considered the mainstay technique to achieve posterior fixation of the subaxial spine. Roy-Camille first introduced posterior cervical spine fixation with lateral mass screws in 1964 . This fiIndicationsLateral mass screw fixation provides strong posterior fixation in patients with instability of the subaxial spine. Lateral mass screws have been implemented with success in cervical spine trauma, infections , neoplasms, degeneration, and failed anterior fusions. Lateral mass screw construct offers comparable or superior stability compared to pedicle screw fixation or laminar screw/sublaminar wiring and is a useful surgical option in patients whose pedicles or laminae are deficient , 30.    ContraindicationsSince lateral screw fixation requires an intact lateral mass, any traumatic or pathologic process that compromises the integrity of the lateral mass is a contraindication for this type of fixation. In cases of severe instability, lateral mass screws may not offer sufficient stabilization. As such, posterior fixation may have to be supported with anterior stabilization, thus subjecting the patient to an additional procedure . In certTechniquesWe will describe two commonly used techniques for lateral mass screw placement, the Roy-Camille’s technique and the Magerl’s technique, which differ in regards to the entry point and screw trajectory Figure .The starting point for the Roy-Camille’s technique is at the midpoint of the lateral mass. The lateral mass is rectangular-shaped when viewed from behind (posteriorly). A vertical line can be drawn connecting the facet joints at the midline, and a horizontal line can be drawn dividing the lateral mass into equal upper and lower halves. The intersection of these lines is the ideal entry point for the Roy-Camille’s technique. An entry hole is created using a 2 mm drill bit, angling perpendicular to the posterior lateral mass wall and 10 degrees lateral to the sagittal plane , 28, 34.With the Magerl’s technique, begin by identifying the center of the lateral mass, which is the entry point for the Roy-Camille’s technique described above. The Magerl’s starting point is 1 mm medial and 1 mm cephalad in relation to the Roy-Camille’s starting point. The trajectory is angled at 45-60 degrees anterosuperiorly  and 25 degrees lateral to the sagittal plane , 34. TheComplicationsNeurovascular structures are at risk when inserting lateral mass screws. Misplacement of the lateral mass screws can cause injury to the spinal cord, vertebral artery (extremely rare), spinal nerves, and facet joints. Since the trajectory is directed away from the spinal cord, lateral mass screw insertion, in theory, has a lower risk of injuring the spinal cord. Neurologic injury can also be caused by insertion of long screws leading to a violation of the ventral cortex of the lateral mass. Aiming the screw anteromedially rather than anterolaterally can lead to penetration of the transverse foramen and thus put the vertebral artery at risk. Other common complications include screw loosening and pull out . AlthougComplication AvoidancePreoperative anterolateral (AP) and lateral plain radiographs should be obtained to identify the level(s) of interest and to understand the anatomy. Computed tomography (CT) imaging is recommended for preoperative planning. Intraoperative fluoroscopy is utilized to identify the ideal starting point and guide the trajectory during screw placement. If a patient becomes symptomatic at follow-up, plain films followed by CT imaging are recommended to determine screw loosening or failure. MRI is often required to determine whether an over-penetrated lateral mass screw has damaged the exiting spinal nerve. The vertebral artery lies within the transverse foramen, medial and anterior to the lateral mass. Injury of the vertebral artery is avoided by directing the screw laterally, in an "up and out" direction. Although bicortical screw purchase offers greater pullout strength compared to unicortical screw purchase, it inherently carries a greater risk of over-penetration and thus injury to nerve roots and vertebral artery .IntroductionPedicle screws are the standard of care in the thoracic and lumbar spine but are not routinely used in the cervical spine because they are technically difficult to place. This is due to small pedicle diameter and high medial angulation. Pedicles get smaller caudal to C2, reaching a nadir around C3-C4 , and 75%IndicationsLateral mass screws are increasingly being used for posterior cervical fixation in the United States. Although they may serve well in the majority of cases, they have their limitations. Given their low pullout strength in cases of severe instability, they may not offer sufficient stabilization . As suchSeveral biomechanical studies have demonstrated that pedicle screws offer superior fixation when compared to lateral mass screws -50. TheiTechniquesAbumi, et al. first described a technique for pedicle screw placement in 1994 . The staA high-speed burr is used to decorticate the starting point to expose the pedicle canal. A small pedicle probe is then inserted into the canal with the help of a lateral image intensifier. The pedicle is tapped under fluoroscopic guidance, and finally, an appropriately sized screw is inserted Figure .While placing pedicle screws, it is important to consider the location of the pedicle in three-dimensions. The medial to lateral pedicle angulation is variable and often determined from preoperative imaging. In general, this angle is lowest at C2 and increases caudally. Abumi, et al stated that most of their screws ranged from 25 to 45 degrees from the transverse process in the horizontal plane . Figure The cranial-caudal angulation of pedicles is superior to the vertebral endplate at C2-C3, parallel to the end plate at C3-C4, and inferior to the endplate at C5-C6 .Since Abumi’s initial description in 1994, several different techniques have been described. They vary in how the starting point is obtained, and options include using surface landmarks, performing a laminoforaminotomy to probe the pedicle borders, and using computer navigation. The relative merits of these techniques are discussed later in the chapter.ComplicationsGiven the small pedicle diameter, high medial to lateral angulation, and thin lateral cortex, this is a technically demanding procedure. Complications can be broadly categorized into injury to the vertebral artery, spinal cord, or exiting nerves. A lateral pedicle perforation would lead to violation of the transverse foramen with potential vertebral artery injury Figure .A medial perforation would violate the spinal canal and risk dural tear or spinal cord injury Figure .A superior or inferior breach would violate the neural foramen and could cause nerve injury.Five studies in the literature have analyzed complications from pedicle screw fixation in the subaxial cervical spine. The rates of screw perforation ranged from 6.7% to 30%, and most cases breached the lateral wall , 59-62. Despite the relatively high rate of pedicle perforation with screw placement, the incidence of neurovascular injury is relatively low. Out of the 350 patients across the five studies, only two patients had a vertebral artery injury, five patients had a nerve root injury, and zero patients had a spinal cord injury , 59-62. This high incidence of pedicle perforation, yet the low incidence of neurovascular injury, can be explained on an anatomic basis. On average, the vertebral artery occupies only 35% of its foramen. Furthermore, the distance from the vertebral artery to the lateral pedicle wall increases from C2 to C7 . The criComplication Avoidance: Learning CurveSubaxial cervical pedicle screw placement is not a commonly used technique and is technically demanding. As such, there is a learning curve and published results demonstrate improved outcomes with surgeon experience. In one study, screw misplacement was 13% for the first 20 screws and decreased to 4% for the subsequent screws . In anotComplication Avoidance: Computer NavigationAs described earlier, the starting point can be obtained based on a number of methods. As expected, the literature shows improved accuracy with more detailed delineation of pedicle anatomy. One study reported a 65% pedicle breach with using surface landmarks alone, 39.5% with laminoforaminotomy, and 10.5% with computer navigation . AnotherComplication Avoidance: ImagingThe main complications with pedicle screw placement - pedicle breach, vertebral artery injury, and nerve injury - can be minimized with appropriate preoperative imaging. Pedicle diameters are small in the subaxial cervical spine and tend to vary across the population. In general, pedicle screws are unsafe when pedicle diameters are less than 4 mm. Therefore, preoperative CT scan should be obtained to make sure pedicles are large enough to be instrumented. Vertebral artery occupancy in the transverse foramen can be obtained with preoperative CTA. There have been described cases where CTA reveals a  tortuous artery with a high occupancy or one that erodes into the pedicle wall. In such cases, pedicle screws should be avoided. In general, a sound understanding of the pedicle anatomy and its associated neurovascular structures for individual patients will help reduce complications.Surgical techniques for posterior stabilization of the subaxial spine include wiring, laminar screw fixation, lateral mass screw fixation, and pedicle screw fixation. Posterior cervical wiring is rarely performed today as a stand-alone procedure because it only offers resistance against flexion. Translaminar screw fixation is more frequently performed in the atlantoaxial and thoracolumbar spine, although it may be an option for subaxial spine fixation in selected cases when there are deficient lateral masses. Care must be taken to avoid laminar cortical breach and violation of the facet joint. Pedicle screws are the standard of care in the thoracic and lumbar spine but are technically challenging to place in the subaxial spine due to small pedicle diameter and high medial angulation in this region. Lateral mass screw fixation is the mainstay technique for posterior subaxial stabilization and offers exceptionally high fusion rates. Comprehensive preoperative planning is critical to evaluate for anatomical variations, and a sound knowledge of the anatomical relationships between screw trajectory and surrounding structures is key in avoiding and minimizing intraoperative and postoperative complications.


Hypsibius dujardini. We first demonstrated that inhibition of transcription or translation drastically impaired anhydrobiotic survival, suggesting that de novo gene expression is required for successful transition to anhydrobiosis in this tardigrade. We then screened 81 chemicals and identified 5 chemicals that significantly impaired anhydrobiotic survival after severe desiccation, in contrast to little or no effect on survival after high humidity exposure only. In particular, cantharidic acid, a selective inhibitor of protein phosphatase (PP) 1 and PP2A, exhibited the most profound inhibitory effects. Another PP1/PP2A inhibitor, okadaic acid, also significantly and specifically impaired anhydrobiotic survival, suggesting that PP1/PP2A activity plays an important role for anhydrobiosis in this species. This is, to our knowledge, the first report of the required activities of signaling molecules for desiccation tolerance in tardigrades. The identified inhibitory chemicals could provide novel clues to elucidate the regulatory mechanisms underlying anhydrobiosis in tardigrades.Upon desiccation, some tardigrades enter an ametabolic dehydrated state called anhydrobiosis and can survive a desiccated environment in this state. For successful transition to anhydrobiosis, some anhydrobiotic tardigrades require pre-incubation under high humidity conditions, a process called preconditioning, prior to exposure to severe desiccation. Although tardigrades are thought to prepare for transition to anhydrobiosis during preconditioning, the molecular mechanisms governing such processes remain unknown. In this study, we used chemical genetic approaches to elucidate the regulatory mechanisms of anhydrobiosis in the anhydrobiotic tardigrade,  For terrestrial organisms, desiccation is one of the most commonly encountered environmental stresses. To avoid deleterious water loss, most animals escape from a desiccated environment using their mobility, and retain their body water by the proper intake of water and by preventing surface water evaporation ,2. In coTardigrades are tiny animals comprising the phylum Tardigrada, in which more than 1000 species have been reported . All tarPolypedilum vanderplanki, and the anhydrobiotic nematode, Aphelenchus avenae, accumulate huge amounts of a non-reducing disaccharide, trehalose, reaching almost 10% to 18% of the dry weight during the preconditioning period ) after 48 h preconditioning, and surprisingly, brain-deprived larvae also exhibit similar tolerance, suggesting that the central nervous system plays no role in sensing desiccation in this animal [Caenorhabditis elegans can tolerate desiccation at 23% RH or above after preconditioning at 98% RH for 4 days [Ramazzottius varieornatus, produces huge amounts of unique heat soluble proteins [Hypsibius dujardini is an anhydrobiotic tardigrade which requires longer preconditioning in a high humidity condition to acquire tolerance against severe desiccation [H. dujardini is easy to maintain in the laboratory, and the strain is established [http://www.ncbi.nlm.nih.gov/nucest/?term=hypsibius+dujardini). Therefore, this species is suitable for molecular dissection of the regulatory mechanisms of anhydrobiosis in tardigrades.Although preparation for anhydrobiosis would start with sensing surrounding water evaporation, the mechanisms of sensing desiccation are likely species dependent. Larvae of s animal . In contr 4 days , and ther 4 days . Therefor 4 days , and an proteins ,20, suggiccation . This imde novo gene expression is required for entering anhydrobiosis in H. dujardini. In addition, we identified 6 chemicals that significantly and specifically impaired the anhydrobiotic survival of this tardigrade. Two inhibitors against protein phosphatase (PP) 1 and PP2A exhibited the most profound inhibitory effects, suggesting an important role of PP1/PP2A activity in anhydrobiosis in this species. This is, to our knowledge, the first report of required activities of signaling molecules for desiccation tolerance in tardigrades, and the inhibitory chemicals identified in this study could be powerful tools for further elucidation of the molecular regulatory mechanisms of tardigrade anhydrobiosis.Here, we used a chemical genetic approach and suggested that H. dujardini was purchased from Sciento (UK) and maintained at 18°C. Tardigrades were reared on 1.2% agar plates overlaid with volvic water containing Chlorococcum sp.  as food. Water and food were replaced once or twice a week.The Z151 strain of α-Amanitin, cycloheximide, J-8, and cantharidic acid were purchased from Enzo Life Sciences (USA). Triptolide was purchased from MedChem Express (USA). 3,4-Methylenedioxy-β-nitrostyrene (MNS), 2-aminoethyl diphenylborinate (2-APB), and okadaic acid were purchased from Santa Cruz Biotechnologies (USA). The 81 chemicals used for the screening were provided by the Drug Discovery Initiative, The University of Tokyo (Japan) and are listed in H. dujardini were placed on each filter in the experiments other than screening. For preconditioning, the dishes were immediately transferred in a sealed plastic box moistened at a defined RH and incubated for 0 to 4 days. RH was controlled by equilibrium with 60% glycerol (62% RH) or saturated solutions of potassium nitrate (95% RH), potassium chloride (85% RH), or magnesium chloride (33.5% RH) [All procedures were essentially performed at the rearing temperature (18°C). For desiccation, a nylon net filter  was placed on Whatman 3MM filter paper  in a plastic dish (35 mm diameter), and tardigrades were dropped onto the net filter with 125 μl of stMQ water. Approximately 20 3.5% RH) ,25. AfteThe stock solutions were diluted to an appropriate concentration with stMQ water with an adjustment of the DMSO concentration to 1% just prior to chemical treatment. Tardigrades were transferred with 3 μl stMQ water into 50 μl chemical solution of the defined concentration and thus, tardigrades were exposed to slightly diluted concentration of the chemicals (94% of the defined concentration). Tardigrades were incubated in chemical solution for 5 h, except incubation in okadaic acid was for 10 h. Tardigrades in 53 μl solution were then dropped onto a nylon filter placed on the filter paper, and lightly washed with an additional 72 μl stMQ water  and subjected to desiccation tolerance assays.H. dujardini were treated with each chemical solution as described above and subjected to a desiccation series of 95% RH for 2 days, 10% RH for 2 days, and 95% RH for 1 day. Three or four dishes of 10 to 30 tardigrades/dish were assayed for each chemical. The detailed conditions for each group are listed in We screened 81 chemicals that were provided to us by Drug Discovery Initiative, The University of Tokyo (Japan) as a numbered list of compounds. We were blinded to the identification of the compounds. We were not able to analyze all 81 chemicals simultaneously, due to limitation in animal supply and too laborious task. Thus, the 81 chemicals were divided into 20 groups and each group was separately assayed with control treatment (1% DMSO). Ten to 30 t-test or Dunnett’s test were performed against the DMSO control in the same group.Statistical tests were performed using Statcel 3 . Specific inhibitory effects on anhydrobiotic survival were examined using the Tukey-Kramer test, comparing four conditions for each chemical. To detect significant inhibition in the chemical screening, the Student’s H. dujardini with an uncertain origin and reported that this species requires pre-incubation at 85% RH for 150 to 200 min to tolerate exposure to a low RH environment (25–31% RH) [H. dujardini was established in 1987 [H. dujardini in the previous work, however, is unclear. To address this, we examined the desiccation tolerance of the Z151 strain in a low humidity environment with varying lengths of preconditioning periods . Preconditioning at 95% RH for 1 day was more reliably sufficient to confer tolerance to exposure to 10% RH for this strain.In a previous study, Wright (1989) examined the tolerability of the tardigrade identified as –31% RH) . The Z15 in 1987  and rece in 1987  and an e periods . Without periods . A 1-day periods . We furt periods . In cont periods , though H. dujardini also induced the expression of protection molecules necessary for transition to anhydrobiosis during the preconditioning period. To examine this possibility, we pre-treated the tardigrades with the transcription inhibitor α-amanitin [H. dujardini requires de novo transcription and translation for successful transition to anhydrobiosis. The tardigrade likely produces gene products necessary for survival in a low humidity environment during preconditioning periods.Many anhydrobiotic animals induce the expression of putative protection molecules upon desiccation, such as late embryogenesis abundant proteins ,22. Accoamanitin  for 5 h amanitin , which hH. dujardini.Pre-incubation for 5 h with chemicals was sufficient for both inhibitors against transcription and translation to interfere with anhydrobiotic survival. Thus, we conclude that this experimental scheme is useful for evaluating the effects of various chemicals on anhydrobiotic survival in de novo gene expression for anhydrobiotic survival prompted us to postulate the presence of signal transduction pathways connecting desiccation stimuli to responsive expression of genes related to desiccation tolerance. To elucidate such signaling pathways, we conducted chemical screening to identify chemicals that inhibit anhydrobiotic survival in H. dujardini. We selected 81 chemicals as candidates from available inhibitory chemicals whose target molecules were known, based on knowledge of the stress-responsive pathways in other organisms, general signaling molecules, and the gene repertoire encoded in the tardigrade genome  instead of exposure to low humidity (10% RH). At the appropriate concentrations, all five chemicals exhibited significant inhibitory effects specifically on survival after exposure to low humidity, with little effect on survival after exposure to only high humidity . These fH. dujardini.Among the five identified chemicals, cantharidic acid and triptolide exhibited the most profound inhibitory effects on anhydrobiotic survival . CantharH. dujardini  is clearly dephosphorylated during the preconditioning period [H. dujardini and dauer larvae of C. elegans.In addition to PP2A, PP1 activity may also be involved in anhydrobiosis. In dauer larvae of g period , althougg period . Thus, dIn addition to phosphatase inhibitors, MNS, a protein tyrosine kinase inhibitor, also significantly inhibited anhydrobiotic survival . MNS is Panagrolaimus superbus, pre-treatment with MAPK inhibitors against JNK  and p38  partially inhibits anhydrobiotic survival only when nematodes were exposed to 10% RH without preconditioning [Mitogen-activated protein kinase (MAPK) pathways are representative phosphorylation signaling pathways involved in various environmental stress responses ,58. Thesitioning . In the itioning . CorrespTriptolide was another chemical with strong inhibitory effects . This coH. dujardini, in part because their inhibitory effects were relatively weak . Low humidity, low humidity exposure; High humidity, high humidity exposure.Specific inhibitory effects on anhydrobiotic survival were reproduced for all five identified chemicals. The optimal concentration varied slightly for J-8 and 2-APB see , possibl(TIF)Click here for additional data file.S2 FigP<0.05; **, P<0.01). Low humidity, low humidity exposure; High humidity, high humidity exposure.The effects of identified chemicals on tun formation were examined. The effects on tun formation are generally much smaller than those on anhydrobiotic survival, and the significance of the effects varied among experiments, suggesting that these chemicals have subtle inhibitory effects on tun formation, if any. Tun formation rates are shown for experiments corresponding to (TIF)Click here for additional data file.S1 Tablet-test (Groups 16–20). Pink highlighting indicates five chemicals that exhibited significant inhibitory effects (P<0.01). With regard to the other chemicals that did not exhibit significant inhibitory effects, we cannot exclude the possibility that concentrations used in the screening were not appropriate and/or had insufficient effects on the tardigrade.The effects of 81 chemicals on anhydrobiotic survival were examined. The 81 chemicals were divided into 20 groups and each group was assayed independently with a separate DMSO-treated control for each group. Tardigrades were treated with chemical solution or 1% DMSO solution  for 5 h prior to the desiccation tolerance assay. After chemical treatment, they were preconditioned at 95% RH for 2 days, followed by dehydration at 10% RH for 2 days, and humidification at 95% RH for 1 day. Recovery was examined at 1 h and 24 h after rehydration. In the list, recovery rates are shown as raw data as well as relative values normalized with that of DMSO-treated control (% of DMSO) in the same group . For each group, statistically significant differences compared with the DMSO-treated control were determined using Dunnett’s test (Groups 1–15) or Student’s (XLS)Click here for additional data file.