Background: The mechanism for rapid biosynthesis of platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is unclear.Results: Phosphorylation of lysophosphatidylcholine acyltransferase 2 (LPCAT2) at Ser-34 via PKCα enhanced rapid PAF biosynthesis following PAF or ATP stimulation.Conclusion: The molecular basis for rapid PAF biosynthesis in response to inflammatory stimuli is elucidated.Significance: These data suggest a better understanding of PAF production in early-phase inflammation. 2+-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKCα-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of Vmax with minimal mK change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent proinflammatory lipid mediator that triggers various cellular functions through binding to its G protein-coupled receptor, PAF receptor (PAFR), resulting in pleiotropic biological effects such as platelet activation, airway constriction, hypotension, and systemic anaphylaxis . Under inflammatory conditions, 1-alkyl-PC is hydrolyzed in a Ca2+-dependent manner by the action of cytosolic phospholipase A2 to produce polyunsaturated fatty acid and lyso-PAF, a lysophosphatidylcholine (LPC) derivative with an alkyl ether bond at the sn-1 position. Biosynthetic enzymes of various lipid mediators subsequently utilize polyunsaturated fatty acid and lyso-PAF as substrates. Our previous studies showed that macrophages and neutrophils from cytosolic phospholipase A2α-deficient mice are unable to produce PAF in response to extracellular stimuli ) activity that catalyzes the incorporation of arachidonoyl-CoA into membrane phosphatidylcholine (PC). In fact, 1-alkyl-PC represents approximately half of the phospholipid content in the biomembranes of hematopoietic cells . Thus, LThere are a number of reports showing that lyso-PAFAT activity is enhanced by several distinct mechanisms. We reported previously that, in mouse macrophages, lyso-PAFAT activity was increased within 30 s after PAFR stimulation  and with17–Vmax value of LPCAT2, whereas the mK value was almost unchanged. Our report identifies the enzyme and signaling pathway that mediate rapid activation of PAF production, which is critical in the early stages of inflammatory responses.In this study, we identified phosphorylation at Ser-34 of LPCAT2 via PKCα signaling as the underlying mechanism that led to rapid enhancement of lyso-PAFAT activity following PAF- or ATP-stimulation. Enzymological analyses showed that phosphorylation increased the Salmonella minnesota, ATP, anti-FLAG M2 antibody, and anti-β-actin antibody were from Sigma. Deuterium-labeled d4-16:0 lyso-PAF, d4-16:0 PAF, 16:0 lyso-PAF, and 16:0 methylcarbamyl PAF  and horseradish peroxidase-linked anti-rabbit or anti-mouse IgGs were from GE Healthcare . [1-14C]Arachidonoyl-CoA (2.22 GBq/mmol) was from Moravec Biochemicals . TLC silica gel plates (type 5721) were from Merck . Acetyl-CoA, dimethyl sulfoxide, acrylamide, and Phos tag acrylamide were from Wako . U73122 (PLCβ inhibitor), U73343 , bisindolylmaleimide I , Ro-31-8220, Ro-31-8425, bisindolylmaleimide V , and 12-O-tetradecanoylphorbol-13-acetate (TPA) were from Calbiochem. The siRNAs , and L-052470-01-0005 (LPCAT2)) were from Thermo Scientific . The cell line Nucleofector kit V was from Lonza . Anti-MK2, anti-phospho-Thr-334-MK2, and anti-PKCα antibodies were from Cell Signaling Technology . The protease inhibitor mixture, EDTA-free Complete, was from Roche. The anti-LPCAT2 and anti-phospho-LPCAT2 antibodies and FLAG-LPCAT1, FLAG-LPCAT2, S34A, and S34D plasmids have been described previously  were frad libitum. All animal studies were conducted in accordance with the guidelines for animal research at The University of Tokyo and were approved by The University of Tokyo Ethics Committee for Animal Experiments.C57BL/6J mice were obtained from Clea Japan, Inc. . Mice were maintained in a light-dark cycle with lights on from 08:00–20:00 h at 22 °C. Mice were fed with a standard laboratory diet and water Thioglycolate-induced mouse peritoneal macrophages were isolated as described previously . Cells wm BAPTA/AM, 10 μm U73122, or 10 μm U73343 for 10 min; 1–20 μm of BIM-I, 1–10 μm of Ro-31-8220, 1–10 μm of Ro-31-8425, and 10 μm of BIM-V for 30–60 min; and 100 nm TPA for 24 h. Cells were then stimulated with 200 nm mcPAF or 250 μm ATP for 30 s. After stimulation, cells , 0.3 m sucrose, and 1 mm sodium orthovanadate and collected in buffer containing 20 mm Tris-HCl (pH 7.4), 1 mm sodium orthovanadate, 5 mm 2-mercaptoethanol, and 1× EDTA-free complete protease inhibitor mixture. Subsequently, cells were sonicated twice on ice for 30 s each time and centrifuged at 9000 × g for 10 min at 4 °C to remove cellular debris, intact cells, and mitochondria.Cells were pretreated with the following reagents for the indicated times: 10 μing PAFR , non-traing PAFR ) were wag was separated at 100,000 × g for 1 h at 4 °C. The resultant pellet was resuspended with ice-cold buffer containing 20 mm Tris-HCl (pH 7.4), 1 mm sodium orthovanadate, 5 mm 2-mercaptoethanol, and 1× EDTA-free Complete protease inhibitor mixture. The concentration of each protein was measured by the Bradford method   were seem mcPAF for 30 s.For the knockdown of PKCα, CHO-S-PAFR cells were seeded in 6-cm collagen-coated dishes 24 h before transfection of 180 pmol siRNA using 17 μl of RNAi MAX (Invitrogen). The cells were incubated for 24 h and subsequently cotransfected with 4 μg of FLAG-LPCAT2. After another 24 h of incubation, cells from each 6-cm dish were divided into two 6-cm dishes and incubated for 2 h. The medium was changed to DMEM containing 0.5% BSA. The cells were cultured without serum for 24 h and then stimulated with 200 nm ATP for 30 s.RAW264.7 cells stably expressing FLAG-LPCAT2 were suspended in 100 μl of Nucleofector kit V and mixed with 30 pmol PKCα siRNA or negative control siRNA. The mixture in the cuvette was set onto the Amaxa Nucleofector and electroporated with program D-032. Then, cells were seeded onto 6-cm dishes. Forty-eight hours after transfection, cells were stimulated with 250 μm Phos tag acrylamide and 100 μm manganese ions (Mn2+) were used. The Phos tag forms a complex with two Mn2+ ions and serves as a phosphate-binding molecule  were incubated in buffer containing 100 mm Tris-HCl (pH 7.4), 0.015% Tween 20, 1 μm CaCl2, deuterium-labeled lyso-alkyl-PC (d4-16:0 lyso-PAF), and acetyl-CoA or arachidonoyl-CoA at 37 °C for 5 min. The concentrations of each substrate are described in D–G. Other assays were performed under the following conditions: 10 μg/ml proteins (supernatant after centrifugation at 9000 × g for 10 min) were incubated in buffer containing 100 mm Tris-HCl (pH 7.4), 0.015% Tween 20, 1 μm CaCl2, 5 μm d4-16:0 lyso-PAF and 1 mm acetyl-CoA at 37 °C for 5 min. Lipids were extracted with methanol containing dimyristoyl phosphatidylcholine (14:0/14:0 PC) or 17:0 LPC as an internal standard and subsequently analyzed by LC-MS/MS.The lyso-PAFAT and LPCAT assays were performed using radioisotopes, as described previously , 25, or For endogenous PAF analysis, lipids from cells grown on 35-mm dishes were extracted with 500 μl of methanol containing d4–16:0 PAF as an internal standard and purified with Oasis HLB cartridges  as described previously , 27.TM UPLC BEH C8 column  using an AcquityTM ultra-performance LC system (Waters) as described previously . Nexera UHPLC  was connected to an LCMS-8040 (Shimadzu) mass spectrometer  in positive ion scan mode. Deuterium-labeled 1-alkyl-PC was monitored by selected reaction monitoring transition (m/z 832.6–303.2) in negative ion scan mode. Signal intensities were determined relative to the internal standard or the percentage of the positive control. Mass spectra were processed using Xcalibur 2.0 software (Thermo Scientific) or LabSolutions 5.53 software (Shimadzu).Chromatography was performed on an Acquityeviously . Lipids eviously . Endogentrometer . DeuteriTM real-time PCR system (Applied Biosystems). The following primers were used: Lpcat2, a 64-bp fragment, CCCTGCCAATACAGAAGAGATCA (forward) and GCCGTCCTCATCAACATCAA (reverse); Prkca, a 172-bp fragment, GTGTCCTACCCCAAATCCTTGTC (forward) and GTTGGATCTCCCTGTTCTCCAGT (reverse); and Rplp0, a 136-bp fragment, CTGAGATTCGGGATATGCTGTTG (forward) and AAAGCCTGGAAGAAGGAGGTCTT (reverse).Total RNAs were prepared using the RNeasy mini kit (Qiagen), and first strand cDNA was subsequently synthesized using Superscript III (Invitrogen). The PCRs were performed using Fast SYBR Green Master Mix and the StepOnePlusAll statistical calculations were performed using Prism 5 .in vitro: LPCAT1 and LPCAT2 . Lyso-PAFAT activity was not detected in vector-transfected samples.There are two known enzymes that possess lyso-PAFAT activity d LPCAT2 , 25. In d LPCAT2 . To examresponse . Cells wenhanced A. Lyso-PB).Previously, we observed that phosphorylation of LPCAT2 at a single residue, Ser-34, occurs 30 min after LPS stimulation and leads to an increase of lyso-PAFAT activity . To inveFig. 1m mcPAF for 30 s, and the microsomal proteins  were analyzed by Western blot analysis using antibodies to detect total or phospho-Ser-34 LPCAT2. Phosphorylated LPCAT2 was barely detectable in untreated cells but detected readily following either 30 min of LPS or 30 s of mcPAF stimulation. To examine whether phosphorylation of LPCAT2 following mcPAF stimulation is also MK2-dependent, the samples were probed with antibodies that recognize total or activated MK2. Phosphorylated MK2 was observed following LPS but not mcPAF stimulation (A), indicating that the rapid phosphorylation of LPCAT2 in response to mcPAF is independent of MK2 signaling and occurs via a separate signaling pathway.To determine the phosphorylation site of LPCAT2 following mcPAF stimulation, peritoneal macrophages were stimulated with 100 ng/ml LPS for 30 min or 200 nmulation A, indicam mcPAF for varying times (0–2430 s), and microsomal proteins were analyzed by Western blot analysis using antibodies to detect total or phospho-Ser-34 LPCAT2. The amount of total LPCAT2 was similar in all samples. Phosphorylated LPCAT2 was not detected in vehicle-treated cells, but stimulation with mcPAF rapidly induced phosphorylation of LPCAT2 in as little as 10 s, reached a maximum by 30–90 s, and was decreased by 270 s (B), consistent with our previous study (C).To investigate the rapid time course of LPCAT2 phosphorylation, peritoneal macrophages were stimulated with 200 nby 270 s B, consisus study . Next, wus study C.A). Next, we examined the effect of phosphorylation on the dual activities of LPCAT2 (lyso-PAFAT and LPCAT) by measuring the incorporation of radiolabeled acyl-CoA ([3H]acetyl-CoA or [14C]arachidonoyl-CoA) into PAF or PC, respectively. Both the lyso-PAFAT and LPCAT activities of WT LPCAT2 were enhanced by mcPAF stimulation (B and C). Basal S34A activities were similar to WT but not increased by mcPAF stimulation. Conversely, basal activities of LPCAT2 containing the S34D mutation, which functions as a phosphomimetic, were higher than in the WT, but enhancement by mcPAF stimulation was not observed (B). Because the expression level of each construct was similar (A), our results indicate that both the lyso-PAFAT and LPCAT activities of LPCAT2 are enhanced by Ser-34 phosphorylation in response to mcPAF stimulation.To further examine the role of Ser-34 phosphorylation following mcPAF-stimulation, two FLAG-tagged mutant constructs (S34A and S34D) of LPCAT2 were transiently expressed in CHO-S-PAFR cells. The cells were stimulated with mcPAF for 30 s, and the cellular proteins were analyzed by Phos tag acrylamide gel electrophoresis and Western blot analysis. An upward mobility shift in LPCAT2 indicative of phosphorylation was detected in WT LPCAT2 following mcPAF-stimulation but not in the S34A or S34D mutant proteins A. Next, mulation , B and Cobserved B. Becaus similar A, our reVmax values than control proteins in both lyso-PAFAT and LPCAT activities in vitro (D–G), potentially because of a conformational change in LPCAT2, which is phosphorylated at Ser-34. Changes in the mK values were minimal following mcPAF stimulation.Next, we investigated how Ser-34 phosphorylation affects enzymatic activities of LPCAT2 using microsomal proteins from CHO-S-PAFR cells overexpressing LPCAT2. Proteins from mcPAF-treated cells exhibited higher in vitro , D–G, poA). Therefore, we examined signaling pathway activation after mcPAF stimulation.Although the phosphorylation of LPCAT2 by 30 min of LPS stimulation was dependent on MK2, mcPAF stimulation for 30 s utilized a different pathway A. Theref2+ concentration is increased rapidly via the Gq/11-PLCβ-inositol 1,4,5-trisphosphate pathway . Therefore, we examined the effect of pretreatment with a cytosolic Ca2+ chelator (BAPTA/AM) (A), indicating that this phosphorylation requires PLCβ activation and intracellular Ca2+ elevation.It has been shown that, following PAFR stimulation, intracellular Ca pathway –32. We aAPTA/AM) , 34 as wAPTA/AM)  on LPCATAPTA/AM) A, indicam) for 30 min and then stimulated with mcPAF for 30 s. Microsomal proteins were analyzed by Western blot analysis using antibodies against total or phospho-LPCAT2. mcPAF-induced LPCAT2 phosphorylation was diminished by BIM-I pretreatment in a dose-dependent manner. Pretreatment with 5 μm BIM-I was sufficient to obtain this effect (B).Next, we investigated kinase activation following PAFR signaling. Because previous reports showed that PKC is activated rapidly after PAFR stimulation , 37, thes effect B.C). These compounds selectivity inhibit conventional PKC (cPKC) isozymes  also showed dose-dependent inhibition of LPCAT2 phosphorylation in CHO-S-PAFR cells transiently expressing FLAG-LPCAT2, whereas pretreatment with BIM-V, an inactive analog of BIM-I, had no effect C. These isozymes  over theisozymes . We concm TPA for 24 h. Consistent with previous reports (D). The phosphorylation of LPCAT2 by mcPAF-stimulation was reduced by TPA treatment, accompanied by a decrease in lyso-PAFAT activity in vitro (E).Next, CHO-S-PAFR cells transiently expressing FLAG-LPCAT2 were pretreated with 100 n reports , 42, pro reports D. The phin vitro E.F). We then examined the effect of PKCα siRNA on lyso-PAFAT activity by measuring the incorporation of acetyl-CoA into deuterium-labeled PAF. Knockdown of PKCα resulted in a significant decrease of lyso-PAFAT activation induced by mcPAF (G). From these data, we concluded that PKCα mediated the Ser-34 phosphorylation of LPCAT2 following mcPAFstimulation.To further confirm the involvement of PKCα in phosphorylation of LPCAT2 at Ser-34, PKCα siRNA and FLAG-LPCAT2 were cotransfected into CHO-S-PAFR cells. PKCα protein was reduced by the knockdown, and mcPAF-induced phosphorylation of LPCAT2 was also decreased F. We theby mcPAF G. From tA and B). Endogenous expression of LPCAT2 in RAW264.7 cells was much lower than FLAG-LPCAT2. Phosphorylation of LPCAT2 following ATP stimulation was diminished correspondingly in cells transfected with LPCAT2 siRNA (A). Lyso-PAFAT activity in cellular lysates was measured by incorporation of acetyl-CoA into deuterium-labeled PAF. RAW-LPCAT2 cells had higher basal lyso-PAFAT activity than RAW-VEC cells, and ATP-stimulation increased the activity (C). When LPCAT2 was knocked down, both RAW-VEC and RAW-LPCAT2 showed lower lyso-PAFAT activity (C).In addition to mcPAF, we investigated the ability for other G protein-coupled receptor ligands to mediate phosphorylation of LPCAT2 in RAW264.7 cells. Treatment with ATP led to phosphorylation of endogenous LPCAT2 within 30 s . To confT2 siRNA A. Lyso-Pactivity C. When Lactivity C.B and D), compatible with lyso-PAFAT activity in vitro (C). From these data, we conclude that LPCAT2 is the enzyme that catalyzes the rapid PAF production following ATP stimulation.PAF production from RAW-VEC and RAW-LPCAT2 after 1 min of ATP stimulation was quantified by LC-MS/MS. The amount of PAF was elevated in RAW-LPCAT2 and increased upon ATP stimulation for 1 min. Knockdown of LPCAT2 by siRNA led to decreased LPCAT2 phosphorylation and PAF production , B and Din vitro C. From tm ATP for 30 s. The proteins were analyzed by Western blot analysis (A). Similar to the experiments using CHO-S-PAFR cells . The phosphorylation of LPCAT2 by ATP stimulation was also reduced, accompanied by decrease in lyso-PAFAT activity in vitro (C). These two pharmacological experiments showed that PKCα mediated Ser-34 phosphorylation of LPCAT2.To down-regulate cPKC, RAW-LPCAT2 cells were treated with 100 n protein B. The phin vitro C. These D). Knockdown of PKCα led to reduced phosphorylation of LPCAT2 after ATP stimulation. However, some residual phosphorylation was still detected, possibly because of residual PKCα protein still present in the cell because of incomplete knockdown (D).We targeted PKCα by siRNA to examine the involvement of this isoform. Forty-eight hours after transfection with PKCα siRNA, the cells were treated with ATP for 30 s. PKCα protein was decreased by transfection of PKCα siRNA D. Knockdnockdown D.E). These data demonstrate that PKCα mediates the phosphorylation of LPCAT2 following ATP stimulation. However, a partial redundancy of other kinases cannot be fully excluded.We then examined the lyso-PAFAT activity of LPCAT2 by measuring incorporation of acetyl-CoA into deuterium-labeled PAF. Knockdown of PKCα resulted in significant decrease of lyso-PAFAT activation after ATP stimulation E. These This study establishes that rapid PAF biosynthesis in response to mcPAF or ATP stimulation requires PKCα-dependent Ser-34 phosphorylation of LPCAT2  and 6, w2+ for its activation, whereas neither novel PKC nor atypical PKC exhibit Ca2+ dependence. Our knockdown experiments established the positive involvement of PKCα on Ser-34 phosphorylation. However, a partial redundancy of other isoforms or kinases cannot be fully excluded in this study , suggesting that this role of PAF is required for a very short period, consistent with the rapid degradation of PAF by PAF acetyl hydrolase in the cytosol and plasma . LPCAT2 selectively catalyzes the incorporation of polyunsaturated fatty acids, such as arachiconic acid, into 1-alkyl-PC at the sn-2 position. 1-alkyl-PC containing arachidonic acid is a major membrane phospholipid of inflammatory cells (On the other hand, the LPCAT activity of LPCAT2 was also increased during the same rapid time course C. LPCAT2ry cells . The phyin vivo studies are required to elucidate the roles of phosphorylated LPCAT2 under inflammatory conditions.The data obtained in this study reveal a novel mechanism for PAF production that may function at the onset or early stages of inflammation. Because LPCAT2 may be a key enzyme balancing pro- and anti-inflammatory lipid metabolism, additional studies are necessary to understand its functions at various stages of inflammation, which may elucidate potential targets for medical intervention. For example, selective inhibition of the lyso-PAFAT activity of phosphorylated LPCAT2 might ameliorate excessive inflammation in response to exogenous or endogenous triggers. Thus, further


Shigella flexneri, the causative agent of bacillary dysentery, invades host cells in a vacuole that is subsequently ruptured to allow growth of the pathogen within the host cytoplasm. S. flexneri invasion has been classically described as a macropinocytosis-like process, however the underlying details and the role of macropinosomes in the intracellular bacterial lifestyle have remained elusive. We applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) to study the highly transient events of S. flexneri’s early invasion into host epithelial cells and elucidate some of its fundamental features. First, we demonstrate a clear distinction between two compartments formed during the first step of invasion: the bacterial containing vacuole and surrounding macropinosomes, often considered identical. Next, we report a functional link between macropinosomes and the process of vacuolar rupture, demonstrating that rupture timing is dependent on the availability of macropinosomes as well as the activity of the small GTPase Rab11 recruited directly to macropinosomes. We go on to reveal that the bacterial containing vacuole and macropinosomes come into direct contact at the onset of vacuolar rupture. Finally, we demonstrate that S. flexneri does not subvert pre-existing host endocytic vesicles during the invasion steps leading to vacuolar rupture, and propose that macropinosomes are the major compartment involved in these events. These results provide the basis for a new model of the early steps of S. flexneri epithelial cell invasion, establishing a different view of the enigmatic process of cytoplasmic access by invasive bacterial pathogens.Intracellular pathogens include all viruses, many bacteria and parasites capable of invading and surviving within host cells. Key to survival is the subversion of host cell pathways by the pathogen for the purpose of propagation and evading the immune system. The intracellular bacterium  Shigella flexneri is an intracellular bacterial pathogen and the causative agent of bacillary dysentery. It possesses the ability to invade and propagate within human cells by injecting bacterial effector proteins directly into host cells. Shortly after entry within a vacuole, S. flexneri induces vacuolar rupture and escapes into the host cytosol via an unknown mechanism. Using large volume correlative light electron microscopy (CLEM) and dynamic microscopy we studied discrete and highly transient steps of S. flexneri early invasion in detail. We provide the first 3D high resolution view of the S. flexneri invasion site and of vacuolar rupture itself. We find that vesicles formed at the invasion site due to injected bacterial effectors, termed macropinosomes, are functionally involved in vacuolar rupture and come into direct contact with the bacterial containing vacuole during this process. This unique and surprising pathogenic strategy stands in stark contrast to other invasive pathogens that induce direct lysis of their surrounding vacuole via the action of destabilizing bacterial proteins. Yersinia pseudotuberculosis, Listeria monocytogenes, Salmonella enterica and Shigella flexneri, are able to induce their own entry into nonphagocytic host cells ..YersiniaS. flexneri is a medically important pathogen . Ind. IndS. fal cells . In shoral cells ,9],10],[10]S. f,[10]S. fal cells . This leal cells . Such rual cells . In thisal cells , entry abacteria , Rho-GTPbacteria , and thebacteria ,16]. En. EnS. flflexneri  to choleflexneri  and to bflexneri ,20] sug sugS. flr routes . Thus, iS. flexneri reside within bacteria containing vacuoles (BCVs), followed by BCV rupture and escape into the cytosol within 10 minutes, a step crucial for the intracellular growth of S. flexneri [S. flexneri vacuolar rupture [in vitro to oligomerize and insert into the plasma membrane of target cells, forming cation selective ion channels involved in vacuolar rupture [S. flexneri subverts host cell pathways for BCV rupture [2 phosphatase, was shown to regulate Rab11 recruitment and to be required for efficient vacuolar rupture [S. flexneri relates to bacterial entry and vacuolar rupture has remained unclear.Upon entry, flexneri . Initial rupture . For ins rupture . In cont rupture . A siRNA rupture . How thiS. flexneri. We analyze the architecture of the S. flexneri invasion site in detail and reveal that it contains two distinct compartments, the BCV and macropinosomes. We demonstrate that Rab11 is recruited directly to macropinosomes and its activity is required for efficient vacuolar rupture. Finally, we reveal that the BCV and macropinosomes come into direct contact at the onset of vacuolar rupture. Our results represent a major step forward in understanding the mechanism of S. flexneri escape to the cytosol, and provide the basis for an updated model of S. flexneri invasion into epithelial cells.In this work, we applied dynamic imaging and advanced large volume correlative light electron microscopy (CLEM) in the form of focused ion beam/ scanning electron tomography (C-FIB/SET) to reveal key features of the pathogenic strategy employed by Salmonella enterica in epithelial cells using phase contrast microscopy and fluorescent dextran added to the infection media, acting as a fluid phase marker [S. flexneri invasion site by measuring internalization of fixable dextran conjugated to Alexa Fluor-647 S. flexneS. flexneri. We examined the relative contribution of pre-existing endocytic vesicles to the endomembrane content at the invasion site  to examine the S. flexneri invasion site in detail [S. flexneri invasion: First, discreet and highly transient (in the order of minutes) stages of invasion can be targeted using fluorescent microscopy with stage-specific fluorescent markers prior to tomography acquisition. Thus, precise access to early invasion (before BCV rupture) and to the vacuolar rupture event itself can be gained. Secondly, the large cellular volumes (in the order of 1000 μm3) acquired using this technique allow visualization of entire invasion sites, accounting for multiple bacteria, vesicles and other cellular structures present at the site. The BCV’s and other vesicles’ membranal integrity and their connectivity to other compartments can be examined in 3D from all axes, providing structural information not easily accessible by classic serial sectioning and tomography approaches [S. flexneri invasion site revealed that BCVs exclude detectable fluorescent dextran and are surrounded by dextran containing vesicles in all our experiments, we first set out to examine the architecture of the early invasion site in detail using C-FIB/SET. A detailed description of the correlative workflow for the data set analyzed in S. flexneri invasion site was imaged by confocal microscopy. As a tight actin enrichment around bacteria was previously shown to appear exclusively prior to vacuolar rupture [S. flexneri invasion site from all axes. Membrane ruffles are observed at the cell surface while BCVs and smaller vesicles are seen in the cytosol.Since fine structural details of the n detail  Fig 2)..S. flexnproaches . Finally rupture , a site S. flexneri invasion site reveals the BCV is a tight, uniform compartment, structurally distinct from the surrounding heterogeneous macropinosomes. We conclude that the BCV and macropinosomes present at the early invasion site represent two distinct compartments.We examined the structure of the BCV in early invasion in detail and delineated the typical BCV structural motifs . The datS. flexneri is occupied by macropinosomes formed in situ during invasion without significant recruitment of pre-existing host endocytic vesicles. We applied C-FIB/SET to correlate the total vesicle population at the invasion site with dextran Alexa Fluor-647 labeling (added during infection)  . FIB/SETresented , 95% andresented . In dataeriments .S. flexneri invasion, is associated with membrane ruffles, and can induce membrane ruffling via stimulation of Rac1 and Cdc42 activities. Like IpgD, it has no known function in disrupting membrane integrity or pore formation to our knowledge [S. flexneri uptake and vacuolar rupture [We have recently shown that the bacterial effector IpgD is required for efficient vacuolar rupture . As IpgDnowledge . Thus, onowledge  did not  rupture  we teste rupture . CK-666 S. flexneri invasion in cells transfected with a marker of phosphatidylinositol 3-phosphate (PI3P), 2XFYVE-GFP, used for labeling macropinosomes, and galectin-3-mOrange used for labeling vacuolar rupture. PI3P has been previously reported to be associated with early macropinosome maturation, in particular with the cup closure step [We therefore set out to examine the cell biology underlying the relation between macropinosomes and vacuolar rupture in detail using dynamic microscopy, functional assays and structural analysis. First, we examined the spatial and temporal proximity of macropinosomes to the rupturing BCV. We performed live microscopy of ure step . We founure step   was chosen so that invasion sites already containing internalized dextran but prior to vacuolar rupture  could be captured. Thus the relation between dextran containing macropinosomes and Rab11-GFP recruited to the invasion site could be examined during live imaging of early invasion. A representative experiment is presented. We found that Rab11 is directly and dynamically recruited to macropinosomes prior and during vacuolar rupture, conforming the non-classical association between macropinosomes and Rab11 at the S. flexneri invasion site. As expected, live imaging also revealed that rupture events occur in close proximity to macropinosomes  [S. flexneri invasion to determine vacuolar rupture timing  . We founsion see .Overall our effector mutant screens, live imaging and functional studies reveal that macropinosomes at the invasion site are the target for direct recruitment of Rabs, they border the BCVs at the onset of vacuolar rupture, and their formation and trafficking are required for efficient vacuolar rupture. Together, these results provide for the first time evidence that vacuolar rupture involves two cellular compartments, the BCV and macropinosomes.ΔipgD strain revealed a reduction in the number of vesicles in contact with the BCV, yet macropinosome-BCV contact points morphologically identical to the wild-type were still always observed  and M90T expressing dsRed : the strains do not express afaI and are a part of a pwR100 collection [S. flexneri serotype 5a (M90T-Sm). Growth medium was supplemented with ampicillin (50 μg/ml) for all strains except for ΔipgD supplemented with tetracycline (5 μg/ml). All bacterial strains were cultured on tryptic casein soy broth (TCSB) agar plus 0.01% Congo red with 20mg/mL agar at 37°C.The following 90T AfaI , expresspgD AfaI   and cultured as above except with 10% CO2.  and NRK fibroblasts (Clone CRL- 1570 from ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS) at 37°C, 5% CO2, 0.8 mM MgCl2, 5 mM glucose, 25 mM HEPES, pH 7.3) and incubated with poly-L-lysine for 15 min if they did not express AfaI. For imaging of fixed samples, bacteria were added to cells at MOI 30 . Prior to infection, bacteria were washed with PBS and resuspended in EM buffer  at a density of 7000 cells per well 24 h prior to transfection. The cells were then transfected as described  with RabFixed samples were imaged using a Perkin Elmer UltraView spinning disc confocal microscope, with a 60X/ 1.2NA water objective and a Z step size of 0.3 μm. For time lapse microscopy, cells were imaged with a confocal microscope . Amira and Avizo (FEI) were used for 3D visualization, data correlation, manual segmentation and supporting movies. Internalized bacteria were used as correlating fiducials. Inverted contrast is presented. Overall 15 C-FIB/SEM datasets of S. flexneri invasion sites were acquired, 11 of WT strain and 4 of ΔipgD strain.Sample preparation and acquisition were performed as previously described . In shorhttp://icy.bioimageanalysis.org/). “Spot detector” plugin was applied to segment dextran containing vesicles in 3D confocal stacks. A spherical assumption was used to extract vesicle diameter. 2300 vesicles from three separate experiments were analyzed. WT vs. ΔipgD strain vesicles/bacterium (http://www.cellprofiler.org/). Macropinosomes were counted only when found in actin labeled ruffles that contained at least one bacterium. Quantification is based on six separate experiments including in total 1186 cells, 1877 bacteria and 4330 vesicles. Unpaired t test was used for significance. Vesicle volume distribution . For rupture timing screen three independent experiments per strain were performed, at least 50 invasion events per strain were measured, with 791 events in total. For Rab11 DN experiments three independent experiments per condition were performed, with 946 rupture events analyzed in total . Vacuolar rupture timing was measured as the time interval between the beginning of ruffle formation and the appearance of a Galectin-3 localized signal. Statistical analysis was performed in GraphPad Prism software v6. The difference between WT and mutants, or DN rupture timing was evaluated using one-way ANOVA. p < 0.05 was considered as significant: *p<0.05, **p<0.01, ***p<0.0001, and ****p<0.0001. Time lapse microscopy Click here for additional data file.S1 FigipgD/IpgD shows partial complementation. Unpaired student t test was used for significance.Invasion sites formed after a 30 min infection were analyzed. Number of vesicles per invasion site was calculated using automated software . Δ(TIF)Click here for additional data file.S2 FigC-FIB/SET was applied to correlate dextran Alexa Fluor-647 labeling (purple) added during 30 min infection to all vesicles detected by FIB/SET at the invasion site . Bacteria are labeled by DAPI (cyan) and segmented from FIB/SET data (blue). Two separate datasets are presented, (A) and (B). Quantitative analysis reveals that 95% and 99% (respectively) of vesicles found at the invasion site reside within the fluorescent dextran label. Vesicles outside of dextran label are highlighted in the overlay view (yellow). In data set B, five of the largest vesicles that reside outside of the dextran labeling are found near the surface of the cell, most likely representing late forming macropinosomes formed after removal of dextran in the washing phase. Scale bars are 10μm.(PNG)Click here for additional data file.S3 FigS. flexneri invasion at five second intervals. Z-projections are shown in the figure. After the onset of membrane ruffling (0s), a transient PI3P enrichment was observed (130s) around the BCV with a lifetime of 20-250s (n = 27), followed by macropinosome formation (310s). Vacuolar rupture begins with macropinosomes bordering the BCV (340s) in 33 out of 36 events observed (92%). Macropinosomes persist around the BCV during rupture (440s). Scale bar is 5μm. See also (A) Full results of the two bacterial effector mutant library screens described in (PNG)Click here for additional data file.S4 FigS. flexneri expressing dsRed for 30 minutes in the presence of dextran Alexa Fluor 647 for 30 minutes, followed by washes and fixation. (B) Cell transfected with Rab11S25N-GFP, a GDP locked Rab11 dominant negative, and infected as in (A). Representative images are shown, scale bars are 10 μm. (C) Rab11 WT vs. Rab11S25N-GFP ruffling formation.(A) Cells transfected with Rab5-GFP, Rab7-GFP or Rab11-GFP were infected with (PNG)Click here for additional data file.S1 MovieS. flexneri and labeled with DAPI (cyan) and actin-phalloidin (Magenta) is presented followed by a view of the FIB/SET data from three different axes. Finally a segmentation showing membrane ruffles is presented.3D confocal stack of an invasion site in a cell infected with (MPG)Click here for additional data file.S2 MovieThe detailed structure of the BCV is presented. Presentation of the ultrastructural data is followed by a segmentation of the bacterial cytosol and membrane (blue), LPS layer (white), BCV membrane (yellow), actin cage (magenta) and surrounding vesicles (orange). A cut-out-view in the context of membrane ruffling is presented.(MPG)Click here for additional data file.S3 MovieConfocal time lapse microscopy of wild-type strain infection of cells transfected with the macropinosome marker 2XFYVE-GFP and vacuolar rupture marker galectin-3-mOrange. Images were taken every 30 s, z-projections are presented, scale bar is 5μm. Channels are from left to right- 2XFYVE-GFP dextran, galectin-3-mOrange, composite.(AVI)Click here for additional data file.S4 MovieCells transfected with the PI3P marker 2XFYVE-GFP, and the vacuolar rupture marker galectin-3-mOrange, were imaged with confocal microscopy  during S. flexneri invasion at five second intervals. Images were taken every 30 s. Z-projections are shown in the movie, scale bar is 5 μm. Channels are from left to right- 2XFYVE-GFP dextran, galectin-3-mOrange, composite(AVI)Click here for additional data file.S5 MovieS. flexneri in the presence of dextran Alexa Fluor 647. After 22 minutes dextran was washed out and live imaging was initiated. Channels are from left to right- Rab11-GFP, dextran Alexa Fluor 647, galectin-3-mOrange, composite. Scale bar is 5 μm.Dextran pulse chase live imaging experiment. Cells transfected with Rab11-GFP and galectin-3-mOrange were infected with (AVI)Click here for additional data file.S6 MovieS. flexneri. Channels are from left to right- Rab11-GFP, galectin-3-mOrange, composite. Scale bar is 10 μmRepresentative movie showing live imaging experiment of cells transfected with Rab11-GFP and gal-3mOrange, and infected with (AVI)Click here for additional data file.S7 MovieS. flexneri. Channels are from left to right- Rab11S25N-GFP, galectin-3-mOrange, composite. Scale bar is 10 μm.Representative movie showing live imaging experiment of cells transfected with Rab11S25N-GFP dominant negative (DN) and gal-3mOrange, and infected with (AVI)Click here for additional data file.S8 MovieWild-type strain. 3D confocal view of an invasion site in a cell transfected with galectin-3-mOrange rupture marker (red) and labeled with DAPI (cyan) is presented. The FIB/SET data set is shown followed by segmentation of a bacterial cytosol and LPS , dissociated BCV membrane (yellow) and macropinosomes in direct contact with the BCV membrane (orange). Frame showing dissociated membrane emanating from the macropinosome-BCV contact point is highlighted.(MPG)Click here for additional data file.S9 MovieipgD mutant strain. Movie notation and progression identical to Δ(MPG)Click here for additional data file.


Lateral inhibition is known to occur at all levels of processing of visual information: from retinal ganglion cells, through principal cells in LGN to primary cortical areas . The rolOur work is based on local field potential (LFP) multichannel signal data  recordedIt was hypothesized that the reason for these different cortical activation patterns was due to lateral inhibition raised by salient visual stimulus during bottom-up task . This woWe have constructed a model of neuronal network comprised of single compartment excitatory and inhibitory cells activated according to extended Hodgkin-Huxley dynamics. The network consists of 16 domains representing cortical patches , where tThe modelled neurons receive two kinds of Poisson inputs, which represent the feed-forward input from the thalamus and feed-back activation from higher order cortical regions. The reinforcement of the latter causes widespread increase in beta band power of LFP and represents diffuse, top-down attentional modulating signal. The input from the thalamus arriving to one or more domains represents bottom-up attentional activation of excitatory and inhibitory neurons evoked by salient visual stimulus. The cortical domains activated by this salient input exert additional lateral inhibition on neighboring domains.Our model shows that the spatial heterogeneity of cortical activation resulting in local decrease of correlation in bottom-up task and homogeneity in top-down task, can be mediated by feed-forward lateral inhibitory influences at the cortical level. The higher cross-correlation variance observed experimentally during bottom-up than in top-down attentional paradigm is also observed in the model.This modelling study, confirmed that mechanism of lateral inhibition may be sufficient in explaining spatial differences in organization of beta activities in visual cortical areas observed during bottom-up and top-down attentional paradigms.The network was modelled by means of PyNEST package.


CD23 the low affinity receptor for IgE plays an important role in allergic disease because it facilitates allergen presentation to T cells. CD23 is a 45 kDa trans-membrane protein consisting of an alpha-helical stalk and a head domain, which is mainly expressed on B cells. In order to analyze the complex formation between allergen, allergen-specific IgE and CD23 on the molecular level, we expressed four different forms of CD23 in baculovirus-infected SF9 insect cells. Construct A consists of the extracellular part of the molecule, the second construct (B) differs from construct A by a single amino acid exchange in the stalk region to abolish the N-linked glycosylation of CD23. Constructs C and D were smaller molecules consisting mainly of the head domain of the protein. Furthermore we expressed human monoclonal IgE specific for the major birch pollen allergen Bet v 1 in a hybridoma cell line and the Bet v 1 allergen in Escherichia coli.Using circular dichroism analysis we found that all molecules were expressed as folded proteins and gel filtration revealed that the CD23 constructs were monomeric. In ELISA we demonstrated that each of the four CD23 constructs binds monomeric IgE and allergen-IgE complexes in a similar way. Concerning the different CD23 constructs, we observed weaker binding signals when measuring the smaller CD23 molecules. Interestingly, construct B, which was devoid of the N-linked glycosylation site, displayed enhanced IgE binding compared to the glycosylated protein.In conclusion, our results reveal a comparable interaction between isolated IgE as well as IgE-allergen immune complexes with CD23 and provide defined molecules for the detailed structural characterization of the allergen-IgE-CD23 interaction.Supported by grants 4605, 4613 and in part by 4604 and P23350-B11 of the Austrian Science Fund (FWF).


N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, and has recently been suggested as a target for the treatment of stroke. Here, we examined DAPK1-mediated in vitro phosphorylation of the GluN2B subunit of NMDA receptors. We established a high-throughput screening assay for the protein kinase DAPK1 by using a Caliper microfluidics capillary electrophoresis system  and identified a novel small-molecule imidazo-pyramidazine inhibitor (6) targeting the catalytic domain of DAPK1. The inhibitor was characterized by enzyme kinetic assays and isothermal titration calorimetry (ITC), and a high resolution crystal structure provided detailed insight into the binding mode of the inhibitor.Death-associated protein kinase 1 (DAPK1) has been associated with 2+ ions through NMDA receptors composed of GluN1 and GluN2B subunits by DAPK1-dependent phosphorylation of Ser1303 in the C-terminal region of GluN2B.1292–1304, KKNRNKLRQHSY) reduced stroke in an animal model, an effect that was attributed to inhibition of the DAPK1/GluN2B interaction.bThe human genome comprises more than 500 different kinases, the majority of which have been linked to regions associated with various diseases by chromosomal mapping.It is well-established that excitotoxicity is primarily mediated by NMDA receptors containing GluN2B; NMDA receptors containing GluN2A subunits do not play a prominent role in this process. This important difference between the two subunits, GluN2A and GluN2B, has been shown to be related to differences in their intercellular C-terminal domains (CTDs).72+/calmodulin-dependent protein kinase II (CaMKII) has been suggested to phosphorylate Ser1303 of GluN2B,Escherichia coli, and purified; the reference substrate Peptide38SNVF) and the 13-mer peptide N2B13 , which has ten additional C-terminal amino acids, we observed phosphorylation to a degree similar to that for the reference substrate. Substituting Ser1303 with Ala  prevented phosphorylation, thus suggesting that Ser1303 was indeed phosphorylated. We examined the inhibitory effect of 1, a putative inhibitor of Ser1303 phosphorylation, on peptide 2, but did not observe any effect (data not shown). Next we studied the corresponding 13- and 23-mer GluN2A peptides, N2A13  and N2A23 ; for both, Ser1291 corresponds to Ser1303 in GluN2B. Interestingly, none of these peptides was phosphorylated by DAPK1, thus indicating that DAPK1 indeed selectively phosphorylates Ser1303 in the GluN2B CTD. We also examined these peptides for in vitro CaMKII phosphorylation; we observed an almost reversed substrate specificity compared to DAPK1: GluN2A derived peptides 4 and 5 were phosphorylated at a similar rate and to the same degree as the control peptide, Autocamtide-2TVDAL-NH2), the shorter N2B13 (1) was phosphorylated in a similar manner. In contrast, neither the longer GluN2B peptide, N2B23 (2), nor its Ala mutant, 3, was phosphorylated by CaMKII. Together, these results indicate that DAPK1 might be responsible for the phosphorylation of Ser1303 in GluN2B, thus validating DAPK1 as a target for inhibition of selective GluN2B CTD phosphorylation; CaMKII phosphorylates the corresponding residue (Ser1291) in GluN2A. Furthermore, it was seen that substrate recognition by DAPK1 is dependent on additional amino acids C-terminal to the phosphorylation site compared to CaMKII substrates, and that these additional residues seemingly inhibit recognition by CaMKII.This prompted us to examine related peptides derived from GluN2B and GluN2A  and we cKm for ATP was determined , and screening was carried out with an ATP concentration of approximately 2×Km, to allow ATP-competitive DAPK1 inhibitors to be discovered. The quality of the screening was determined, and the resulting Z-factor of 0.84 was deemed satisfactory. We then screened approximately 4300 compounds from a kinase-targeted in house compound collection in duplicate at 10 μm. The compound library contained compounds presumed to have an increased probability of binding to the conserved ATP-binding pocket of protein kinases. The compound collection included 244 known, potent kinase inhibitors from the InhibitorSelect library (Millipore). From the first screening, we identified 96 compounds exhibiting more than 58 % inhibition of DAPK1; these were selected for determination of IC50 values with the same assay. Of the 96 compounds, 57 were known kinase inhibitors from the CAL library; the remaining 39 were potentially novel inhibitors of DAPK1. The 96 compounds showed IC50 values ranging from 0.003 μm (staurosporine) to 131 μm. Of these, 26 had IC50 values below 1 μm: 22 were from the InhibitorSelect library (Supporting Information), and the most potent of the others was compound 6 (IC50=0.247 μm).Next, we identified small-molecule compounds that inhibit DAPK1 and are thus potential leads for the development of compounds with neuroprotective properties and potential application for the treatment of stroke. For this, we established a high-throughput enzymatic screening assay for DAPK1. First we prepared a fluorescent peptide substrate, with 5-carboxyfluorescein (5FAM) coupled to the N-terminus of peptide38 . This peptide was used in a Caliper microfluidics capillary electrophoresis assay, which separates phosphorylated peptide product from the non-phosphorylated peptides in microcapillaries. The fluorescently labeled peptide enabled quantification in real time and thus determination of reaction velocities. To ensure that the kinase reaction was at steady-state and that initial velocities were measured, the DAPK1 concentration was adjusted to achieve a substrate turnover of no more than 15 % throughout the time course of the reaction. The 6 was tested in primary screening against a number of other kinases ; 60 % inhibition at 10 μm compound was used as the cut-off limit. PIM1, JAK2, PAK7, and SRC were identified, and IC50 values from the subsequent assays were determined as 1.6, 1.8, 5.6, and 0.15 μm, respectively. Notably, only SRC was inhibited to the same extent as DAPK1; CAMKII did not to pass the primary screening. Thus 6 was clearly an interesting compound. Therefore, we verified the inhibitory activity of 6 towards DAPK1 in a different assay: the equilibrium dissociation constant (Kd) of 6 was determined by ITC to be 0.24±0.09 μm .Compound ±0.09 μm , thus ve6 to DAPK1, and we therefore co-crystallized DAPK1 with 6. Our structure knowledge for small-molecule inhibitors of DAPK1 is currently limited to two published X-ray crystal structures (aside from the broad kinase inhibitor staurosporine):P212121 with one molecule in the asymmetric unit  was explained by mimicking the ARD and binding directly to the catalytic domain of DAPK1;1, thus suggesting that the effect could be mediated by interfering with other interactions.6. This displayed some selectivity towards DAPK1 and reasonable potency, and from the X-ray co-crystal structure we identified the molecular details of the interaction of 6 with DAPK1. Thus, 6 exhibits a range of promising properties for further studies, and the structure suggests specific areas for modification for improving both selectivity and potency.In conclusion two different approaches to develop an inhibitor of DAPK1 were explored: a peptide-based approach based on mimicking the ARD and a more classic small molecule approach. The in vivo effect of the cell penetrating version of GluN2B


The Lévy flight foraging hypothesis predicts a transition from scale-free Lévy walk (LW) to scale-specific Brownian motion (BM) as an animal moves from resource-poor towards resource-rich environment. However, the LW-BM continuum implies a premise of memory-less search, which contradicts the cognitive capacity of vertebrates.Cervus elaphus, strengthens previous empirical support of memory-enhanced and scale-free space use in a northern forest ecosystem.We describe methods to test if apparent support for LW-BM transitions may rather be a statistical artifact from movement under varying intensity of site fidelity. A higher frequency of returns to previously visited patches (stronger site fidelity) may erroneously be interpreted as a switch from LW towards BM. Simulations of scale-free, memory-enhanced space use illustrate how the ratio between return events and scale-free exploratory movement translates to varying strength of site fidelity. An expanded analysis of GPS data of 18 female red deer, A statistical mechanical model architecture that describes foraging under environment-dependent variation of site fidelity may allow for higher realism of optimal search models and movement ecology in general, in particular for vertebrates with high cognitive capacity.The online version of this article (doi:10.1186/2051-3933-1-9) contains supplementary material, which is available to authorized users. Foraging theory aims to identify the complex mixture of behaviour and morphology most efficient to gather energy in different environments . IdentifLW contrasts with a more scale-specific and “fine-grained” kind of movement, Brownian motion (BM), which may be expected to arise in environments with more frequent responses to events and conditions within the perceptual range resulting in low degree of directional persistence of the path. One such event could for example be detection and handling of a food item, followed by a new displacement that is independent on the foregoing ones. The medium range steps are in this case rarer than for LW and the longest ones are completely absent. Consequently, the area covered during a given time period is small relative to LW under similar movement speed. A BM, which describes a stochastic process, will result from sampling an actual movement path at sufficiently large time intervals to ensure that successive relocations (fixes) provide inter-fix distances that reflect a sum of independent and intermediate movement events , 10, 11.On the other hand, the LFF hypothesis defines BM in a more direct and mechanistic manner, linked to actual search behaviour. The model explicitly describes both BM and LW as alternative modes of movement (movement rules), while BM is not considered a consequence of fix sampling at coarse temporal scales as described above. In other words, when an animal is foraging in BM mode, it is assumed to perform successive steps in more or less random direction with a Gaussian-distributed step length at fine temporal resolutions. When in LW mode, the successive directions are still random, but the step length distribution is scale-free (power law compliant) Figure A.et al., has only 6.25% linear range compared to the left-hand arena [ρ =  = 10]. Thus, two processes, based on two different frameworks, may explain more constrained space use in resource-rich environments.Under the Markov condition Figure A, a BM cLj) . Stronger BM compliance apparently emerges with decreasing ρ, which is thereby influencing F(L) with an influence which the LW-BM continuum does not account for Figure ρ ≤ 10, from varying tret and keeping tobs constant, are selected to show a temporal scale range where the site fidelity is particularly influential on the results.Studying the fix samples in Figure ρ = 10 and a narrow memory horizon of 10 time increments  over a sub-range of j  plots from site fidelity gradually shifts towards smaller j in a wave-like manner: the distribution shape-shifts as a consequence of sensitivity to tret relative to observer- defined tobs. Depending on choice of tobs one gets an impression of a truncated LW as ρ is approaching 1, and then the impression of a BM (β >≈3) as the ratio is further reduced to ρ < 1. Hence, observing BM compliance from a F(Lj) distribution based on real GPS data may either represent true BM-like movement or – if site fidelity is involved – a ratio between tret and tobs which masks the underlying power law scaling under the given foraging conditions and average movement speed. Site fidelity may have been too strong  to allow for observation of the scale-free movement component of foraging at the chosen tobs for fix collection. However, deviations from power law as exemplified in Figure As a BM β >≈ as the rz <<1 in I(N) = cNz  and scale-free space use . Figure z in a log-log plot of I(N) is reduced in the transition zone between memory-less and infinite memory and then stabilizes despite further decrease of ρ, from keeping tobs constant and reducing tret. In other words, z is thereby shown to be independent of the spatially explicit site fidelity strength, as expressed by c. For the three conditions of infinite memory and ρ< = 1, the plots illustrate that a reduction in ρ leads to a reduction in c (the intercept with the y-axis). For example, the line y = 0.51x + 0.69 (for the condition ρ = 1), gives c = 20.69 = 1.61 grid cells at the a priori chosen grid resolution. This implies that the “characteristic average scale”, expressing strength of site fidelity in this case is 1/1.61 = 0.62 grid cells.Fortunately the confusing patterns in Figure Four results supported the non-Markovian framework over the Markovian framework from the red deer data.Lj) (see Methods) of the pooled set of step lengths by changing tobs by factor of 10 was 5.7  = X0.79 = 6.16], based on the estimated β = 2.26. This diffusive rate is 80% larger than the expectation for LW-like composite BM; with β > =3 [rms(X) ≈ X1/(3–1) = X0.5 = 3.16]. This result confirms super-diffusion as expected from scale-free movement, which may be caused both by MRW observed from ρ ≫ 1 and by LW.First, the parallel shift of F by a factor of 10 verified that red deer were capable of moving in a truly scale-free manner rather than composite BM  will show a transition towards power law pattern under reduced sampling interval tobs, before showing the over-sampling effect due to entering the scale range for a “smooth” and correlated random walk-like pattern if tobs is reduced too strongly (tobs <<t). In other words, BM from a Markov process lacks the intermediate scale range of power law-distributed steps when path resolution is increased by reducing tobs.A MRW path might also show transition towards a pattern that may resemble a correlated random walk-like path (with Gaussian distribution of step lengths) at very high-frequency fix sampling imagined at temporal scales <<t. However, while a true BM leads to transition towards correlated random walk and a Gaussian distribution directly during such “over-sampling” of a path, an under-sampled MRW . CruciaAdditional details on model simulations and red deer movementhttp://www.movementecologyjournal.com/imedia/9839694510401541/supp1.pdf. (PDF 1 MB)Additional file 1:


This phenotype is sufficiently rescued by the transgenic expression of draxin in neocortical neurons. Genetic interaction data suggest that draxin acts through Deleted in colorectal cancer (DCC) and Neogenin (Neo1), to regulate thalamocortical projections in vivo. Draxin promotes the outgrowth of thalamic axons in vitro and this effect is abolished in thalamic neurons from Dcc and Neo1 double mutants. These results suggest that draxin from neocortical neurons controls thalamocortical projections into the neocortex, and that this effect is mediated through the DCC and Neo1 receptors.The thalamocortical tract carries sensory information to the neocortex. It has long been recognized that the neocortical pioneer axons of subplate neurons are essential for thalamocortical development. Herein we report that an axon guidance cue, draxin, is expressed in early-born neocortical neurons, including subplate neurons, and is necessary for thalamocortical development. In  During neural development thalamocortical axons follow corticofugal projections into the neocortex. Here, using a combination of knock down and rescue experiments, the authors show that Draxin expression in neocortical cells promotes thalamic axon projections from the internal capsule. In the mammalian brain, reciprocal connections between the neocortex and thalamus formed by the corticothalamic and thalamocortical axons are critical for the relay and processing of sensory information. During development, corticothalamic and thalamocortical axons concurrently grow into the subcortical telencephalon, where they meet to form the internal capsule and continue to extend in opposite directions to reach their targets238draxin is expressed in the neocortical neurons of the developing braindraxin is involved in establishing the reciprocal interactions of corticofugal and thalamocortical axons. We found that draxin−/− mice had severe defects in corticofugal and thalamocortical projections. These corticofugal and thalamocortical phenotypes were rescued by the transgenic expression of draxin in neocortical neurons. We showed that draxin genetically interacted with Deleted in colorectal cancer (DCC) and Neogenin (Neo1). Notably, draxin+/−;Dcc−/−, draxin+/−;Neo1Gt/Gt or Neo1Gt/Gt;Dcc−/− mutants displayed severe corticofugal and thalamocortical projection defects, which resembled those in the draxin−/− mice. We observed that the pathfinding errors of thalamocortical axons occurred before those of corticofugal axons in draxin−/− mice, suggesting that draxin plays a direct role in guiding thalamocortical projections. Furthermore, we showed that draxin promotes the axonal growth of thalamic neurons in vitro, which was absent in thalamic neurons from Neo1Gt/Gt;Dcc−/− embryos. These results suggest that draxin from neocortical neurons controls the guidance of thalamocortical projections and this effect of draxin is mediated through the DCC and Neo1 receptors.We previously reported that a chemorepulsive axon guidance protein, draxin, which shares no significant homology with known guidance cues, is necessary for the development of spinal cord and forebrain commissuresdraxin−/− mice, we analysed serial paraffin-embedded coronal sections of wild-type and draxin−/− brains using immunostaining against neurofilament (NF). Corticofugal and thalamocortical axons pass through the internal capsule by forming fasciculated axon bundles in wild-type mice  and the 5-HT transporter  to label thalamocortical axon terminals in the neocortex  staining on the brains of draxin+/− mice, in which the second exon containing the ATG start codon was replaced with the β-gal genedraxin was strongly expressed in the neocortex  mice was achieved by crossing Z/draxin homo;draxin−/− mice with Emx1Cre/+;draxin−/− mice, which show specific Cre expression in the dorsal telencephalon, but not in the ventral telencephalon and the thalamus -tagged draxin protein (draxin-AP)L-lysine (PLL) and L1, because L1 promotes significantly higher neurite outgrowth than PLL or laminin/PLL. This enabled us to evaluate the effects of draxin more easily  of 620 pM , Dcc−/−, Neo1Gt/Gt and Neo1Gt/Gt;Dcc−/− embryos in the absence of draxin-AP proteins. However, in the presence of draxin-AP proteins, we observed 27% and 30% reductions in the neurite outgrowth from thalamic neurons in Neo1Gt/Gt and Dcc−/− embryos, respectively, compared with that in littermate controls  stimulate the neurite outgrowth of neocortical and thalamic neurons through the DCC and Neo1 receptors. We previously reported that 10 nM draxin inhibits neurite outgrowth from the neurons of the dorsal spinal cordOur draxin−/− brains, draxin+/− males were crossed with draxin+/− or draxin−/− females, which were maintained in a mixed C57BL/6-CBA backgrounddraxin−/− mice were essentially identical in the F1 to F6 generations, which were backcrossed with C57BL/6N or ICR mice. DccNeo1 (MMRRC:030400-MU)Unc5a (MMRRC:030749-MU)Unc5b (MMRRC:030410-MU)Dscam (RBRC:05290)Mice were treated according to protocols approved by the Committee on Animal Research at the University of Kumamoto. To obtain MMRRC:0300-MU4041,draxin expression construct, the floxed vector pCAG-loxP-βgeo-loxP, based on the pCALL constructdraxin complementary DNA, together with an IRES2-EGFP (Clontech) sequence, were cloned downstream of the loxP-βgeo-loxP in this vector. Immunocytochemistry and western blot analysis using rabbit anti-draxin antibody draxin construct induced Cre-dependent expression of draxin . Routine genotyping was performed using PCR with the primers 5′-CAAAGGAATGCAAGGTCTGT-3′ and 5′-CATCCTTCAGCCCCTTGTTG-3′, which amplified 260 bp. We selected one transgenic line that exhibited significant β-geo expression levels in the brain. Emx1-Cre knock-in mice (RBRC01345) have been described previously27To generate the pZ/f draxin . A correIn situ hybridization and immunohistochemistry were performed as previously described−1 X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2 and PBS) for 10–16 h at 37 °C and were then counterstained with 1% eosin.N,N-dimethylformamide  was injected into the neocortex of the anaesthetized pups at postnatal day 0 (P0). The pups were perfused at P1 and the brains were postfixed by overnight immersion in 4% paraformaldehyde (PFA). To label thalamocortical axons, 4% PFA-fixed brains at P1 were bisected into hemispheres. Small crystals of DiI were placed into the medial face of the dorsal thalamus with an insect pin. For two-colour fluorescent tracing, DiI and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (Invitrogen) were injected in the neocortex and dorsal thalamus, respectively, of 4% PFA-fixed brains at E17.5. The brains were then incubated in PBS for 2–4 weeks at 37 °C. The labelled brains were embedded in 3% agarose and 100-μm-thick sections were cut on a Vibratome in the coronal plane. Sections were counterstained with Hoechst 33342 (Invitrogen).To label corticofugal axons, a 10% solution of DiI (Invitrogen) in –5 days, the conditioned media was harvested and then concentrated with the Amicon Ultra-15 filter device (Millipore). In the conditioned media, draxin-AP was detected using western blotting with an anti-draxin antibody 12Control-AP (pAPtag-5 vector) and draxin-AP constructs were transfected into HEK293T cells (Riken BRC)4 cells per dish in four-well dishes on coverslips coated with PLL, PLL and laminin, or PLL and L1-Fc chimeric protein–30% of the neurons had very short or no neurites independently of the culture conditions. To exclude these neurons from the quantitative analysis, 21 of the 30 neurons (70%) with the longest neurites were analysed using the BZ-II analysis system. Statistical analyses were conducted with the number of independent experiments and the graphs summarizing the results show average values from independent experiments.Thalamic and neocortical neurons from E14.5 mouse embryos were dissociated using papain . We used the entire dorsal thalamus to prepare the dissociated culture of thalamic neurons, because anterior and posterior dorsal thalamic axons responded similarly to draxin. Dissociated neurons were plated at a density of 3 × 10draxin gene, we used the Flp-In System (Invitrogen), which allows expression of a gene of interest in mammalian cells at a specific genomic location by FLP recombinase-mediated integration. Draxin expression in draxin-293 cells was confirmed by immunohistochemistry and western blotting using the draxin antibody 5 cells per dish in four-well dishes. Twenty-four hours later, dissociated thalamic neurons from wild-type mice at E14.5 were plated at a density of 3 × 104 cells per dish onto the draxin-293 or control-293 cells. Cultures were grown for 40 h and then neurons were stained with a Tuj1 antibody. To establish a neocortical feeder layer, dorsal cortices of draxin−/−, wild-type or Ctx-draxin mice at E14.5 were dissociated with papain and plated at a density of 3 × 106 cells per dish in four-well dishes. Two hours later, 3 × 104 cells of dissociated thalamic neurons from CAG-EGFP mice at E14.5 were plated onto the neocortical feeder layerTo create stable HEK293T cells expressing the Dcc- and/or Neo1-deficient neurons to respond to draxin, thalamic and neocortical neurons were dissected from these mutant embryos at E14.5 after genotyping by PCR and cultured for 40 h with draxin-AP-conditioned medium. Dcc−/− or Neo1Gt/Gt embryos were generated by crossing heterozygous parents. Double mutant embryos were generated from intercrosses of Dcc+/−; Neo1Gt/Gt mice. Neurite outgrowth was analysed as described above.To test the ability of Neo1+/+ and Neo1Gt/Gt embryos at E14.5 was dissected and lysed using lysis buffer  supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride (Sigma), 10 μg ml−1 aprotinin (Sigma) and 10 μg ml−1 leupeptin (Sigma)). The lysate was centrifuged 15,000 r.p.m. at 4 °C for 20 min and was separated by SDS–PAGE. Neo1  and glyceraldehydes 3-phosphate dehydrogenase  antibodies were used for western blot analysis. Uncropped western blot images for The dorsal thalamus from Kd value was determined as previously describedNeo1-transfected HEK293T cells were incubated with different concentrations of draxin-AP-conditioned medium for 90 min at room temperature with occasional gentle stirring. Cells were then washed six times with HBAH buffer  and lysed with lysis buffer . Cell lysates were collected and centrifuged to collect supernatant, which was heated at 65 °C for 15 min. An equal volume of 2 × AP buffer  was then added in each tube. Following a 1-h incubation at 37 °C, absorbance was measured at 405 nm.Saturation binding curves and Scatchard analyses of draxin-AP binding to Neo1 were performed and the How to cite this article: Shinmyo, Y. et al. Draxin from neocortical neurons controls the guidance of thalamocortical projections into the neocortex. Nat. Commun. 6:10232 doi: 10.1038/ncomms10232 (2015).Supplementary Figures 1-8.


House dust mites (HDMs) represent one of the most important inducers of respiratory allergies worldwide.We sought to investigate the IgE and IgG reactivity profiles to a comprehensive panel of HDM allergens in children with allergic asthma and to compare them with those of nonasthmatic atopic children.Sera from clinically well-characterized asthmatic children with HDM allergy (n = 105), nonasthmatic children (n = 53), and nonatopic nonasthmatic children (n = 53) were analyzed for IgE and IgG reactivity to a panel of 7 HDM allergens  by means of allergen microarray technology.P = .002; rDer p 2, P = .007; rDer p 5, P = .031; and rDer p 23, P < .001) were significantly higher in asthmatic children than in children without asthma. By contrast, fewer asthmatic children showed IgG reactivity to HDM allergens than nonasthmatic children, but allergen-specific IgG levels were comparable.Asthmatic children with HDM allergy more frequently showed an IgE response to each of the HDM allergens and recognized more allergens than nonasthmatic children with HDM allergy. Furthermore, IgE levels to certain HDM allergens (nDer p 1, The IgE and IgG reactivity profiles to HDM allergens, as well as IgE levels to certain allergen components, differed considerably between children with and without asthmatic symptoms caused by HDM allergy. In fact, asthmatic children were characterized by an expanded IgE repertoire regarding the numbers of recognized allergen components and by increased specific IgE levels. Dermatophagoides were determined to be a major allergen-producing source in house dust,Dermatophagoides pteronyssinus, Dermatophagoides farinae, or both,Exposure and atopic sensitization to indoor allergens, such as those from pets, molds, cockroaches, and house dust mites (HDMs), are known to be strongly associated with the development of asthma.D pteronyssinus,Since 1980, when the first allergen was described for The Immuno Solid-phase Allergen Chip  technology allows determination of a patient's IgE and IgG reactivity profile to a large number of natural and recombinant allergens in a single step by using only minimal amounts of serum.D pteronyssinus allergens Der p 1, Der p 2, and Der p 10. To perform a more profound analysis of sera from patients with HDM allergy, we used the ImmunoCAP ISAC technology to produce an experimental allergen chip, which contains an extended panel of HDM allergens . This microarray was used to measure IgE and IgG responses in 2 groups of clinically well-defined children with HDM allergy with or without HDM-induced asthma and, for control purposes, a group of nonatopic children.The currently available ImmunoCAP ISAC 112 is able to detect IgE and IgG against 112 different allergens, including the www.jacionline.org. Sera from 105 allergic children with HDM-associated asthma were randomly picked from the Multicentre Asthma Genetics in Childhood study. This group was termed asthmatic children with HDM allergy. Sera from 53 children with HDM allergy but without asthma were randomly picked from the International Study of Asthma and Allergies in Childhood Phase II population as age- and sex-matched nonasthmatic group. The latter group was termed nonasthmatic children with HDM allergy. From the same study population, sera from 53 nonatopic children  were randomly picked. The combined use of both study cohorts  and previous allergy testing has been described earlier.Escherichia coli as a His-tagged protein and purified, as previously described.E coli as nonfusion proteins and purified to homogeneity by using ion exchange chromatography, as previously described.nDer p 1 was purified by means of affinity chromatography with mAb 4C1.D pteronyssinus allergens  and several other allergens and allergen-derived peptides (see www.jacionline.org) was produced. Detection of IgE and IgG binding was performed according to the manufacturer's instructions for ISAC 112, with slight modifications. Washing steps were done exclusively with washing solution (no second washing step with water), and the chips were dried by means of centrifugation (1 minute at 1000g and RT). Levels of allergen-specific IgE and IgG antibodies were reported in ISAC standardized units (ISU) with a cutoff of 0.3 ISU. The allergen-specific IgE and IgG levels of the sera from the 53 nonatopic children were tested with the MeDALL allergen chip, which equals the CD chip in technical aspects but comprises additional allergens.For the analysis of the sera from the nonasthmatic and asthmatic groups, a customized version of the ImmunoCAP ISAC microarray termed CD chip  containing a panel of Rat basophil leukemia cells (RBL-2H3)2 testing or the Fisher exact test, depending on the number of observations. Differences in IgE and IgG levels among the 3 groups were analyzed with the Mann-Whitney U test. The correlation between IgE and IgG reactivity to the allergens was evaluated by calculating the Spearman rank correlation coefficient (ρ). A P value of less than .05 was considered statistically significant.IBM SPSS Statistics 20  was used for evaluating the following statistical parameters. Differences in IgE- and IgG-binding frequencies of asthmatic, nonasthmatic, and nonatopic children with HDM allergy were assessed by using multiple χwww.jacionline.org). Sera from 8 children with negative chip results could be tested by means of IgE immunoblotting to D pteronyssinus allergen extract, and 7 showed very weak IgE reactivity (data not shown). Twenty-one percent of the patients were exclusively sensitized to nDer p 1, rDer p 2, or both. Interestingly, 18 children with HDM allergy reacted neither with nDer p 1 nor with rDer p 2. Six of these patients showed an exclusive sensitization to rDer p 23 but not to the other tested HDM allergens (see A). rDer p 5 , rDer p 7 , and rDer p 21  were less frequently recognized, and only a few patients had IgE reactivity to rDer p 10 . In the nonatopic group only 2 children had weak IgE reactivity, one to nDer p 1 (0.37 ISU) and one to rDer p 2 (2.11 ISU), whereas the remaining 51 had no IgE reactivity to any of the 7 tested HDM allergens (data not shown).We found that 149 (94.3%) of the 158 analyzed children with HDM allergy had IgE reactivity to at least one of the 7 spotted HDM allergens . With a combination of nDer p 1 and rDer p 2, 89% of the patients were diagnosed . Interestingly, rDer p 5 was 3-fold more often recognized by asthmatic than nonasthmatic children.The frequencies of IgE recognition of the individual HDM allergens differed considerably between children with HDM allergy with and without asthma. rDer p 2, rDer p 5, and rDer p 7 were significantly more often recognized by asthmatic patients , which was also reflected by a significant difference in total IgE levels between the groups . Finally, we checked whether different sensitization profiles in the groups could be due to differences regarding time points of blood sampling in the seasons of the year, but there were no significant differences , especially not for autumn, where HDM burden is highest in middle Europe.Accordingly, we found that the sum of IgE reactivities to HDM components was significantly higher in the asthmatic than in the nonasthmatic group . Furthermore, the risk of being asthmatic was more than 2-fold higher in patients with IgE reactivity to rDer p 2  and patients with IgE reactivity to rDer p 7 , respectively. The calculated ORs for the other HDM allergens  were statistically not significant.Calculation of the odds ratio (OR) showed that patients with IgE reactivity to rDer p 5 have a more than 4-fold higher risk of asthma than patients without IgE reactivity to rDer p 5  is present, was calculated for the individual allergens. Patients with IgE reactivity to rDer p 5 had a probability of 85% of having asthma. In addition, high positive predictive values were found for rDer p 7 (80%) and rDer p 21 (79%). However, the sensitivities for each of the allergens were low: rDer p 5, 39%; rDer p 7, 37%; and rDer p 21, 29%. The sensitivity could be increased to 57% when the latter 3 allergens were combined.B). Seventy percent of the children in the asthma group reacted to 3 to 6 of the tested allergens, whereas approximately the same percentage (74%) of nonasthmatic children had IgE reactivity only to 1 to 3 HDM allergens . No IgE reactivity was found to several of the unrelated food allergens and food allergen–derived peptides that were part of the array in any of the tested patients ensuring specificity (data not shown).Another interesting finding was that asthmatic children with HDM allergy had IgE reactivity to more HDM allergens than nonasthmatic children , B. SeveP = .002), rDer p 2 (P = .007), rDer p 5 (P = .031), and rDer p 23 (P < .001) were significantly higher in children with HDM allergy with asthma than in those without , followed by rDer p 5  and nDer p 1 .Basophil activation tests were performed to study whether sensitization to a larger number of HDM allergens and increased HDM-specific IgE levels might also have functional relevance. RBL cells expressing the human FcεRI receptor were loaded with serum IgE from 13 asthmatic children from whom serum was available and who were sensitized against a broad spectrum of HDM allergens. Then cells were exposed either to a mix containing only nDer p 1 and rDer p 2 or to a mix containing Der p 1, 2, 5, 7, 21, and 23. At a concentration of 1 ng/mL , the mix containing all allergens induced a significantly higher release of β-hexosaminidase  than the mix containing only nDer p 1 and rDer p 2 in 11 of the 13 patients (see www.jacionline.org). The highest IgG-binding frequencies (100%) were found for rDer p 5 and rDer p 10 in the nonasthmatic group, as well as for nDer p 1, rDer p 7, and rDer p 21 in the nonatopic children. Interestingly, no patient from the nonasthmatic group had shown IgE reactivity to rDer p 10.The IgG recognition frequency of HDM allergens was very high in patients with HDM allergy, as well as in nonatopic subjects . Statistically significant differences among the 3 groups are indicated in We then analyzed the percentages of sera containing IgE and IgG antibodies, only IgG, only IgE or neither IgE nor IgG antibodies specific for each of the HDM allergens . There wP = .019; rDer p 10, P = .014; When patients were grouped into those who only had allergen-specific IgG without specific IgE or those with allergen-specific IgE and IgG antibodies, we noted that for most allergens, allergen-specific IgG levels were significantly higher in the latter group . Althougwww.jacionline.org).Significantly lower HDM allergen-specific IgG levels were found in the nonatopic children compared with the asthmatic and nonasthmatic groups with HDM allergy for all allergens except rDer p 23 (see P < .001) and nDer p 1 . The correlations were less pronounced for rDer p 23  and rDer p 7  and only very weak for rDer p 21  and rDer p 5 . None of the patients with HDM allergy without asthma had IgE reactivity to rDer p 10, but all had IgG reactivity . Only for rDer p 5  and rDer p 21  was the correlation higher in the asthma group (In agreement with the fact that fewer asthmatic patients had allergen-specific IgG responses and higher levels of allergen-specific IgE in the asthma group, the correlation between allergen-specific IgE and IgG levels was weaker for the asthmatic group than for the group of nonasthmatic subjects for most of the tested allergens (nDer p 1: ρ = 0.69, ma group . HoweverIn this study we used a panel of 7 microarrayed HDM allergens to study possible differences in IgE and IgG recognition in clinically well-defined children with HDM allergy with or without allergic asthma. Our study revealed interesting differences regarding IgE recognition in these 2 groups. Asthmatic children with HDM allergy showed a more broadly spread recognition of different allergens, as well as higher allergen-specific IgE levels, compared with the nonasthmatic group.Both high IgE levels and broad recognition of HDM allergens should lead to dense loading of mast cells, basophils, and also antigen-presenting cells with allergen-specific IgE through their IgE receptors and render such patients more sensitive to allergen exposure. In fact, earlier studies performed by measuring HDM-specific IgE levels with allergen extracts also showed that high levels of HDM-specific IgE antibodies are associated with reduced lung function and increased risk of wheezing.One novel aspect of our study is the demonstration that asthmatic children compared with nonasthmatic children were more often sensitized against several allergens, among them the major allergen rDer p 2 and rDer p 5, rDer p 7, and rDer p 21, which were shown to be potent allergens regarding the elicitation of immediate-type allergic reactions.The finding that a mix comprising Der p 1, 2, 5, 7, 21, and 23 induced stronger basophil activation in polysensitized patients than a mix comprising only Der p 1 and Der p 2 suggests that polysensitization might eventually have functional consequences, but this needs to be investigated in clinical studies.H2 pathway.The fact that the IgE response of the asthmatic children was more broadly spread toward several different allergen molecules compared with that of the nonasthmatic children did not seem to be due to different immunogenicity of the individual allergens in the 2 patient groups. On the contrary, we found an inverse situation for allergen-specific IgG recognition in the 2 groups. Nonasthmatic children even more often had IgG reactivity against the individual allergens compared with asthmatic children. Therefore there are several mutually nonexclusive possibilities concerning why asthmatic children have IgE reactivity to more HDM allergens and produce higher allergen-specific IgE levels compared with nonasthmatic children. One possibility is that certain HDM allergens, such as the cysteine protease Der p 1, could act as a kind of “initiator allergen” by disrupting tight junctions, thus reducing epithelial barrier function and leading to increased intrusion of sensitizing allergens.Because the asthmatic children had significantly higher HDM-specific IgE levels, it was not surprising to find that they exhibited also higher total IgE levels compared with the nonasthmatic group because it is known that most of the total IgE consists of allergen-specific IgE.Regarding the naturally occurring allergen-specific IgG responses, we noted that for each of the tested allergens, with the exception of Der p 10, allergen-specific IgG levels were higher in patients who also produced allergen-specific IgE against the same allergen. This observation might be explained by different routes of sensitization because Der p 1, Der p 2, Der p 5, Der p 7, Der p 21, and Der p 23 are mainly present in fecal particles and hence could sensitize preferentially through the respiratory tract, yielding stronger and more frequent IgE responses. By contrast, Der p 10 is mainly present in mite bodies and hence can sensitize through the skin or gut by involving cross-reactivity with homologous food allergens.IgE sensitization to a broad panel of HDM allergens and high IgE levels to certain components are associated with allergy affecting the lower airways in children with HDM allergy.In summary, this study has revealed that the assessment of IgE and IgG reactivities toward a comprehensive panel of HDM allergen components defines complex serologic reactivity profiles that might help to discriminate asthmatic and nonasthmatic children. It will now be interesting to perform longitudinal studies in birth cohorts to answer the question of whether such multiallergen tests might be able to predict the development and eventually persistence of asthma.