Ahr−/−) and wild-type (Ahr+/+) mouse lung fibroblasts (MLFs), we found that Ahr−/− MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr−/− MLFs treated with LY294002—a PI3K inhibitor—although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr−/− MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out ( The bHLH-PAS family is comprised of transcription factors that contain a bHLH domain followed by tandem PAS domains2. In the canonical AhR signaling pathway, activated AhR translocates to the nucleus where it dimerizes with AhR nuclear translocator (ARNT), a class II protein of the bHLH-PAS family, in order to bind to the xenobiotic responsive elements (XREs) on DNA. This classical genomic AhR signaling pathway can be activated by many exogenous and endogenous compounds that share similar structural and chemical properties3. The best characterized AhR ligands are synthetic halogenated aromatic hydrocarbons (HAHs). HAHs are chemicals that consist of benzene rings with one or more halogens  attached. Dioxins are a class of chlorinated aryl hydrocarbons composed of two benzene rings connected through two oxygen atoms, with dioxin being commonly used as the synonym for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a metabolically-inert chemical that is a potent and well-characterized AhR ligand.The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix (bHLH)/Per-ARNT-Sim (PAS) superfamily4. In the nucleus, the AhR exchanges its chaperones for the AhR nuclear translocation (ARNT) and binds to the XRE core sequence (5′-GCGTG-3′) near the promoters of target genes to induce transcription5. Many genes that are targeted by AhR encode for phase I and II proteins such as those in the cytochrome P450 family. After activation, AhR is exported from the nucleus to the cytoplasm and degraded through a proteasome-dependent mechanism. Activation of the canonical AhR signaling pathway is best-known for mediating the toxic effects of TCDD, such as hepatotoxicity and immunosuppression7.The AhR pathway was believed to be primarily involved in the sensing and subsequent metabolization of xenobiotic chemicals. Upon ligand binding, the conformation of the AhR changes, exposing the nuclear localization signal and causing translocation into the nucleus8. This may involve crosstalk with other pathways to produce downstream effects not involved in the canonical AhR-XRE signaling. One of the potential pathways is the AKT serine/threonine kinase (Akt) pathway that is well-known for promoting cell survival, proliferation, and protein synthesis9. The canonical pathway leading to Akt activation is initiated by the binding of growth factors to receptor tyrosine kinases (RTKs)10. Once activated, these receptors activate class IA phosphoinositide 3-kinases (PI3Ks), heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit. Activated PI3Ks then phosphorylate phosphatidylinositol to produce phosphatidylinositol 3,4,5-triphosphate (PIP3)11. This results in the colocalization of inactive Akt and the enzymes that phosphorylate it to the cell membrane, leading to subsequent Akt activation10. Akt has two main activating phosphorylation sites—the threonine 308 residue (T308) which is phosphorylated by phosphoinositide-dependent kinase-1 (PDK1) and the serine 473 residue (S473) which is phosphorylated by mammalian target of rapamycin complex 2 (mTORC2)10. Proteins that negatively regulate activation of the Akt pathway include phosphatase and tensin homolog (PTEN) which dephosphorylates PIP3 and PH domain leucine-rich repeat protein phosphatase (PHLPP) which dephosphorylates Akt12. Reactive oxygen species (ROS) also modulate the PI3K/Akt pathway by negatively regulating the activity of PTEN15.The AhR can also regulate cell physiology processes such as extracellular matrix (ECM) formation, cell cycle progression, and gene expression independent of its activation by ligands16, oxidative stress17 as well as endoplasmic reticulum (ER) stress18, but whether this is due to AhR-dependent control over Akt is not known. Published information on the involvement of the AhR on the Akt pathway is conflicting. Wu et al. found that AhR reduced apoptosis in a mouse hepatoma cell line due to increased Akt activation19 whereas a recent in vivo study found that there was more Akt activity in Ahr−/− mouse liver20. Given our lack of information on the mechanism by which the AhR exerts homeostatic control over cellular processes, we sought to evaluate if AhR promotes the activation of the Akt pathway in lung fibroblasts to upregulate pro-survival mechanisms.Many of the downstream effects of Akt overlap with those of the AhR, including regulation of cell proliferation and survival. We have previously shown that the AhR protects lung cells against apoptosisAll chemicals were purchased from Sigma-Aldrich  unless otherwise indicated. Recombinant mouse platelet-derived growth factor-BB (PDGF-BB) was purchased from STEMCELL Technologies . LY294002 was purchased from Cell Signaling Technology .Ahr+/+ and Ahr−/− mice as previously described21. All animal procedures were approved by the McGill University Animal Care Committee (Protocol Number: 5933) and were carried out in accordance with the Canadian Council on Animal Care and ARRIVE guidelines22. Fibroblasts were cultured in Gibco MEM (Thermo Fisher Scientific) supplemented with 10% FBS (Wisent Bioproducts), 1X Gibco GlutaMAX (Thermo Fisher Scientific), 1X antibiotic–antimycotic solution (Wisent Bioproducts: #450115), and 0.05 mg/ml gentamycin sulfate solution (Wisent Bioproducts). Cells were maintained at 37 °C in humidified air with 5% CO2. Cells used in each experiment were within two passage difference and no fibroblasts exceeding passage ten were used. All cells were seeded at 10,000 cells/cm2, grown to approximately 80–90% confluence and cultured in serum-free medium for 18 h before conducting the experiments unless otherwise indicated.Primary MLFs were derived from 24. Briefly, smoke from one cigarette was bubbled through 15 ml of serum-free medium and the optical density (OD) of the CSE was measured using a SmartSpec Plus spectrophotometer . An OD of 0.65 at 320 nm was considered to represent 100% CSE. CSE was then passed through a Thermo Scientific™ Titan3™ syringe filter with a pore size of 0.45 µm and diluted to 2% CSE using serum-free medium.CSE was generated from filtered research-grade cigarettes (3R4F) obtained from the Kentucky Tobacco Research Council  as previously describedTotal cellular protein was extracted using Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with 1X cOmplet Mini Protease Inhibitor Cocktail (Roche) and 1X PhosSTO Phosphatase Inhibitor Cocktail (Roche). Protein quantification was performed using a Thermo Scientific Pierce Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions and the absorbance was measured with an iMark microplate reader .Twenty micrograms of total cellular protein were mixed with Laemmli SDS sample buffer (Alfa Aesar), subjected to SDS-PAGE, and electro-blotted onto Immun-Blot PVDF membranes . Membranes were blocked with 5% Oxoid skim milk powder (Thermo Fisher Scientific) in 1X PBS and 0.1% Tween-20 (Thermo Fisher Scientific) for one hour at room temperature. After that, membranes were incubated on a rocking platform overnight at 4 °C with primary antibodies against phospho-Akt , total Akt , AhR , phospho-GSK-3β , total GSK-3β , phospho-Akt substrates , p85α , and PTEN . The membranes were then incubated for one hour at room temperature with the antibody against tubulin . The membranes were incubated with the secondary antibody for one hour at room temperature. The secondary antibodies were horseradish peroxidase-conjugated anti-rabbit IgG  and anti-mouse IgG . Detection of protein levels was performed using enhanced chemiluminescence (ECL) and the signals were captured using a ChemiDoc MP Imaging System . Densitometric analysis was performed using Image Lab Software  and target phospho-protein levels were normalized to the corresponding total protein levels. Full length blots are provided in the Ahr+/+ MLF were grown to 60–80% confluency and transfected with 100 nM of small interfering RNA (siRNA) against p85α (PI 3-kinase p85α siRNA (m): Santa Cruz Biotechnology #sc-36218) or non-targeting control siRNA (Control siRNA-D: Santa Cruz Biotechnology #sc-44232) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and siRNA transfection MEM (Santa Cruz Biotechnology) according to manufacturer's instructions. Cells were incubated in the transfection medium for one hour after which serum-containing medium was added. Twenty-four hours later, the cells were serum-starved for 18 h prior to experimentation.Equivalent numbers of fibroblasts were seeded in triplicate in 96-well plates. Cells were allowed to settle overnight then serum-starved for 18 h prior to conducting the experiments. Cells were either treated with 50 μM LY294002 alone or an equivalent volume of DMSO for 24 h. Following the treatments, 10 µl of 5 mg/ml MTT (in 1X PBS) was added to each well and the plates were incubated for 4 h at 37 °C. The plates were then centrifuged, and the media was removed. The precipitate in each well was dissolved by adding 200 µl of DMSO and the absorbance was read with a BioRad microplate reader at 510 nm.https://www.flowjo.com/).Equivalent numbers of fibroblasts were seeded in 6-well plates. Cells were allowed to settle overnight and serum-starved for 18 h prior the experiments. Cells were either treated with 50 μM LY294002 or DMSO for 24 h. Following treatments, cells were detached using Acutase, an equivalent volume of MEM was added, and the cells were collected to assess cell death. For the trypan blue assay, cells were mixed 1:1 with trypan blue (0.4%) and live and dead cells were counted using the Countess 3 FL (Invitrogen). For flow cytometry assessment of cell viability, cells were stained with propidium iodide  in PBS immediately prior acquiring on a flow cytometry. After acquiring, the data were analyzed using FlowJo v10.2 software (Ahr+/+ and Ahr−/− MLFs were cultured in T175 flasks and serum-starved for 18 h before collection. Cells were rinsed and harvested in 20 mM HEPES supplemented with 150 mM NaCl. The cell-containing solution was then centrifuged to remove the HEPES and resuspended in 8 M urea (pH 8) in 20 mM HEPES supplemented with protease and phosphatase inhibitors. The samples were sonicated briefly on ice and cell debris was cleared by centrifugation. The final samples contained pooled protein extracts utilizing cells from two different Ahr+/+ and Ahr−/− mice. Equal amounts of protein were sent to Institut de recherches cliniques de Montréal  for mass spectrometry analysis. Approximately 290 µg of protein from each sample was digested by trypsin and the peptides labeled with tandem mass tag (TMT). The phosphopeptides were enriched using titanium dioxide (TiO2) and liquid chromatography with tandem mass spectrometry (LC–MS–MS) was performed. Peptide and protein identification were carried out using Mascot (Matrix Science) by searching against a Mus musculus reference proteome database . The results were then analyzed by Proteome Discoverer 2.4 (Thermo Scientific). Identified proteins with an abundance ratio adjusted p-value (Ahr−/−/Ahr+/+) less than 0.01 and a Mascot score greater than 20 were selected for further analysis. Peptide sequences of each identified protein were also manually inspected. Information used for functional annotation of each protein was obtained from the Uniprot database. To perform a protein–protein interaction network analysis, the Search Tool for Retrieval of Interacting Genes (STRING) (https://string-db.org) database was employed. Active interaction sources for constructing the network include text mining, experiments, databases, and co-expression. The species was limited to “Mus musculus” and a minimum required interaction score was set to 0.4.https://www.graphpad.com/scientific-software/prism/). Statistical significance between two groups was analyzed by a two-tailed t-test. Statistical differences among the means of more than two groups were determined using one-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparisons test. The interrelationships between more than two independent variables were assessed using two-way ANOVA followed by a Tukey’s post-hoc test unless otherwise indicated. In all cases, values with P < 0.05 were considered significantly different.Statistical analysis was performed using Prism 6  did not affect Akt phosphorylation in Ahr+/+ MLFs . However, CH-223191 did not significantly affect Akt phosphorylation  to boost intracellular GSH levels24 and evaluated Akt phosphorylation. GSH-MEE treatment reduced Akt phosphorylation in Ahr+/+ cells but had no effect on Ahr−/− MLFs . This antibody produced a strong signal near the 45 kDa region in untreated Ahr+/+ and Ahr−/− MLFs  in Ahr−/− MLFs compared to Ahr+/+ MLFs , there was no difference in phosphorylation of Akt between the Ahr+/+ and Ahr−/− cells. The lack of AhR-dependent regulation of Akt phosphorylation by growth factors (including serum) likely means that lower proliferation previously reported in lung fibroblasts caused by AhR deficiency27 is not mediated by the Akt pathway. Moreover, the lack of differentiatial regulation by AhR ligands is in contrast with previous studies showing induction of Akt phosphorylation in cells treated with CSE42. This discrepancy may be due to difference in CSE concentrations used between studies. It has also been reported that components in cigarette smoke, including B[a]P, induce rapid Akt activation44. Specifically, Bölck et al. found that 1 μM B[a]P significantly induced Akt phosphorylation in human immortalized (HaCaT) keratinocytes after 5 min of treatment41. However, the phosphorylation of Akt (S473) from exposure to B[a]P was unchanged in MLFs. One reason that could account for this discrepancy is that the Akt phosphorylation site evaluated by Bölck et al. was T30844, whereas that evaluated in our study was S473. It is therefore possible that these two sites are differently phosphorylated by the AhR depending on the conditions. Collectively, these results lead to the intriguing speculation that under homeostatic conditions in MLFs, the AhR regulates Akt phosphorylation in a way such that it prevents an exaggerated response while still allowing respiratory cells to respond quickly to the changing environment. This notion is further supported by the fact that basal Akt phosphorylation was significantly higher in Ahr−/− cells, suggesting that the AhR exerts homeostatic control over Akt phosphorylation. Although this is inconsistent with previous studies showing that AhR promotes Akt activation45, our result is in accordance with a previous study that demonstrated that knocking-out AhR increased Akt phosphorylation in mouse liver20. The inconsistency between results in these different studies may be due to the fact that some studies have compared the cancer cells with their AhR-deficient counterparts. Specifically, cells used by Wu et al. were a low-AhR expressing variant derived from the parental murine (Hepa1c1c7) hepatoma cells46, which expresses approximately 10% of the wild-type AhR level45, whereas our study has compared primary lung fibroblasts from Ahr+/+ and Ahr−/− mice. This suggests that the regulation of basal Akt activity by the AhR may be cell-type specific and/or be reflective of differences between primary and cancer cells.In response to known activators of either Akt  or AhR  synthesized by eukaryotic cells to protect against oxidative stress48. GSH contains a cysteine residue with an active thiol group, which can act as an antioxidant to reduce reactive oxygen and nitrogen species48. In this study, we used GSH-MEE, a lipophilic molecule that can be hydrolyzed by intracellular esterases to release GSH, thereby increasing intracellular GSH50. We have previously published that GSH-MEE boosts intracellular GSH levels in primary lung fibroblasts to reduce smoke-induced oxidative stress and apoptosis24. However, GSH only decreased Akt phosphorylation in Ahr+/+ cells, suggesting that ROS signaling plays a role in basal Akt activation in Ahr+/+ MLFs, whereas Akt activity in Ahr−/− cells is insensitive to changes in GSH. As GSH-MEE is a lipophilic molecule that is converted to GSH intracellularly through enzymatic activity51, it is also possible that AhR deficiency alters the expression and/or function of these non-specific esterases such that GSH-MEE is uncleaved, thereby reducing its anti-oxidant potential.The mechanism through which AhR regulates basal Akt activity is not known but we speculated could be due to changes in the expression or function of key upstream regulators such as PTEN or PI3K. However, the level of PTEN was not different between Ahr+/+ and Ahr−/− cells. We identified several proteins that showed significant differences and are important in regulating cellular functions controlled by AhR. For example, the AhR regulates cell morphology, focal adhesion, and migration53. We found that many proteins regulating these processes were differentially phosphorylated between Ahr+/+ and Ahr−/− MLFs; the protein with highest phosphorylation due to Ahr ablation was fibrillin-1, a major structural component of microfibrils54 that regulates the storage, release, and activation of extracellular TGF-β1, a cytokine that causes the differentiation of fibroblasts into myofibroblasts. Protein–protein interaction analysis further allowed us to identify potential interactions, some of which may play a role in the regulation of Akt phosphorylation, such as PLA2, Stub1, and Notch2.To take a more comprehensive approach to understanding the AhR, we performed mass spectrometric analysis of differentially phosphorylated proteins between Ahr−/− MLFs by western blot and MS, we suspected that enhanced Akt phosphorylation in Ahr−/− MLFs is associated with cell viability. To test this, we utilized the PI3K inhibitor LY29400225. Basal activation of Akt dependent on the PI3K, as the phosphorylation of Akt was eliminated by LY294002. Using MTT assay, it appeared that LY294002 significantly reduced viability in Ahr−/− MLFs, suggesting that increased Akt phosphorylation is required by Ahr−/− MLFs for cell survival. MTT conversion requires active metabolism via mitochondrial enzymes. Given that we have previously published on the importance of AhR in maintaining mitochondrial health16, we utilized two additional complementary cell viability assays. Using trypan blue exclusion and propidium iodide incorporation with flow cytometry, treatment with LY294002 did not alter cell viability. Thus, LY294002 is affecting the mitochondria without causing increased cell death. The PI3K/Akt pathway influences mitochondrial function, including control over metabolic enzymes15. These new data suggest that an AhR-Akt pathway is involved in the maintenance of mitochondrial health. It is possible that this is occurring through phosphorylation of novel proteins identified by our mass spectroscopy analysis such as FAM49B (Cyrib)55. Furthermore, LY294002 inhibits PI3K by acting on the ATP binding site of the enzyme25 although LY294002 may non-specifically interact with other proteins. For example, LY294002 also binds to and may perturb the functions of casein kinase 2 (CK2), GSK-3β, and mammalian target of rapamycin (mTOR)56, which can be a limitation in the interpretation of data obtained from this study. Moreover, LY294002 can bind to the AhR and antagonize its activation by TCDD57. Although the binding of LY294002 to AhR does not activate its DNA-binding ability at the concentrations used in this study57, LY294002 may still interfere with the physiological function of AhR that could confound interpretation of the results. It is also noteworthy that LY294002 eliminated basal Akt phosphorylation but not that of GSK-3β. Although GSK-3β is one of the best-established targets of Akt26, these data suggest that there are additional signaling mechanisms that regulate GSK-3β phosphorylation in MLFs.Although we did not detect upregulated phosphorylation of well-characterized Akt substrates in Ahr−/− MLFs was associated with dysregulation of the ECM (Fig. In conclusion, we report for the first time that the AhR controls the phosphorylation of Akt under basal conditions but not in response to growth factors, CSE, or AhR ligands. We also show that basal Akt activation in MLFs was dependent on the PI3K pathway. Our findings raise intriguing questions on how AhR regulates Akt-related pathways in mammalian cells. Nevertheless, the finding that absence of the AhR results in differential phosphorylation of a number of proteins, including fibrillin and fibronectin, gave rise to the speculation that increased Akt phosphorylation in ECM Fig. . Given tSupplementary Information.


Sceloporus virgatus, affected by the Horseshoe 2 Fire in the Chiricahua Mountains, Arizona, USA. The condition‐dependent female ornament was significantly smaller the month following the fire than 2 years prior and was both smaller and less orange on the burned site relative to a nearby unburned site. These patterns are similar to those found in a previous experimental study examining the response of the ornament to corticosterone manipulations. Yet, in the current study, corticosterone levels were not different in lizards on the burned and unburned sites. Perhaps glucocorticoid levels already returned to baseline, or do not adequately track environmental change. Females tended to be smaller and lighter on the burned site than the unburned site; however, the year after the fire, body condition was higher for females on the burned site, indicating a rapid recovery and potential long‐term benefits in response to low‐intensity fires in this fire‐adapted ecosystem. We found that the lizards adjusted energy allocation away from sexual signaling and growth in response to low‐intensity fires. As fires and fire management are likely to increase in response to changing fire regimes across the globe, it will be important to consider behavioral and physiological responses of impacted species, as well as population‐, community‐, and ecosystem‐level responses.Controlled low‐intensity fires are commonly used in ecosystem management for both habitat restoration and wildfire management. Animals in those ecosystems may respond to fire by shifting energy allocation away from reproduction and growth, and toward maintenance. Stress‐induced shifts in energy allocation may affect the expression of condition‐dependent sexual signals, which are sensitive to energetic and physiological trade‐offs mediated by glucocorticoids. Here, we examine the effect of fire on ornament expression, corticosterone, and other phenotypic traits in a population of striped plateau lizards,  We show that environmental stress resulting from low‐intensity fire reduced the expression of a condition‐dependent ornament. Female striped plateau lizards had smaller and less orange dewlap ornaments in the month following the Horseshoe 2 Fire in 2011 than 2 years prior, and on the burned site relative to a nearby unburned site even in the absence of detectable differences in stress hormone levels. We also consider the effects of the fire on body size, body condition, and ectoparasitic mite loads. The long‐term site is 515 m long and 1.7 km SW of the Southwestern Research Station (SWRS), the unburned site is 565 m long and 1.9 km NW of SWRS, and the two sites are 2.1 km apart from each other. The width of each site varies with topography and includes the plateau above the creek and the surrounding slopes. In 2011, we determined the percent of the long‐term study site burned by laying transects perpendicular to the creek bed (from the embankment to the outer limit of the site) every 20 m along the creek and continuously scoring the ground or vegetation under the transect tape as burned or unburned. To compare the abiotic conditions of the burned and unburned sites, we placed HOBO data loggers  on burned and unburned ground on the burned site and on unburned ground on the unburned site for 9 days in 2011, rotating their placement every other day to the sighting location of a randomly selected female. Data were logged every 15 min, and for each 24‐hr period, we determined minimum, average, and maximum temperature and relative humidity. To compare the canopy cover of the two sites in 2011, we took photographs of the canopy in each cardinal direction at randomly selected locations , measured as described below, and released to the exact site of capture. Five individuals were measured in both 2011 and 2012; we randomly selected one observation from each female to include in analyses. Measurements include body size , body mass, ectoparasitic mite load (counted under 8× magnification), and reproductive state . All females  were gravid except for three small individuals  that were still vitellogenic. Body condition was calculated as the residuals from a regression of body mass and SVL3. In addition, the right throat patch was photographed under full shade using an Olympus C‐5050 ZOOM 5‐megapixel digital camera and was matched to Munsell color chips (Munsell/MacBeth/Kollmorgen Corporation) under full shade and a standardized light source (Pelican Super SabreLite). Ornament size was determined from photographs using Photoshop to automate the selection of orange pixels and ImageJ to determine the area of the selected pixels. A “color score” was calculated from the Munsell data of hue, chroma, and value following the methods of Burley et al.,  were run in triplicate with a 1:60 dilution and 2% steroid displacement buffer. Intra‐assay variation was 4.0%, and inter‐assay variation across two assay runs was 3.1%.Blood samples were used to compare corticosterone levels of females on the burned and unburned site approximately 1 month after the burnout operation on the long‐term study site. Samples were collected within 4.4 min of an individual being sighted (mean time = 154.4 ± 5.1 s) and were held in a cooler on ice for up to 8 hr before processing. After centrifugation, plasma was frozen for storage and transport to the University of Puget Sound for analysis using a Corticosterone Enzyme Immunoassay Kit (Enzo Life Sciences #ADI‐900‐097) optimized for 2.1We compared abiotic conditions between locations  using general linear models, with date as a repeated factor for all variables except canopy cover. We also used general linear models to determine (a) whether female phenotype was affected by site and by year, including 2011 and 2012 only, and (b) whether the phenotype of a subset of large females on the long‐term study site differed before, year of, and 1 y after the fire. The latter analysis included only large females (SVL ≥ 65 mm) because that is all that had been measured in 2009. Body condition was calculated independently for this subset of data, and significant results were followed by pairwise Tukey's HSD tests. For statistical models for the ornamental traits, which are our primary interest, possible covariates included SVL, body condition, and mite load; we used AIC to select which, if any, covariates were included in our final model, and present results for all remaining model covariates.To meet model assumptions, orange area and mite load were log‐transformed, Munsell scores were transformed to the fourth power, and minimum temperature data were transformed to the tenth power. Analyses conducted on orange area and on relative orange area  led to similar conclusions; only analyses on orange area are presented in detail. All analytical statistics were performed using RStudio  and we determined that 83% of the substrate was burned by the fire. The burned ground had higher average temperature and lower minimum relative humidity than did nearby unburned ground on the same site, but did not differ from unburned ground on the unburned site  were significantly longer than the unburned stretches , but in the subsequent year, females on the burned site tended to be in higher condition than those on the unburned site . Mite load was significantly higher in 2012 than in 2011 on both sites , but was not significantly affected by year  nor a site X year interaction . Female body size was a significant covariate in this model, with larger females having larger ornaments . When relative ornament size was used instead, body size remained in the final statistical model and was marginally significant . Color score was also significantly lower on the burned site relative to the unburned site  and was lower in the year of the fire relative to the subsequent year . The interaction term was not significant , though females on the burned site in the year of the fire tended to have the lowest color score , year , and their interaction , though females on the burned site in the year of the fire tended to have the highest mean reflectance. Final models for both reflectance and PC1 included covariates, as follows. Mean reflectance was marginally related to mite load , with heavier mite loads associated with lower reflectance. PC1 was related positively to body size , negatively to body condition , and nonsignificantly to mite load . In contrast, PC2 was lower on the burned site than on the unburned site  and lower in the year of the fire than in the subsequent year  indicating reduced reflection of orange‐red and UV wavelengths in response to stress; the interaction term was not significant , and body size was a significant positive covariate in the model . PC3 was the same on the two study sites , but significantly lower (indicating both reduced red and blue wavelength reflection) in the year of the fire than the following year ; there was no significant interaction term  and no remaining covariates.We further assessed four aspects of ornament color using spectrometry Figure : mean re3.3We compared a subset of the largest one‐third of females (SVL ≥ 65 mm) from the long‐term study site measured in 2011 (year of fire) and 2012 (year after fire), to a cohort of females sampled there in 2009 (preburn) as part of Weiss . Only onF2,27 = 9.55, p < 0.001; Figure p = 0.078) and in the subsequent year (p < 0.001). There was no difference in ornament size in 2011 and 2012 (p = 0.801). Body condition and mite load were both included as covariates in the final model, but neither contributed significantly. Patterns were nearly identical when relative ornament size was used instead of absolute ornament size. Color score of large females did not vary between years , females on the two study sites did not differ in microhabitat location  or rate of tail damage, a potential indicator of predation threat . Stress effects of fire may also have been ameliorated by increased prey availability. Though we did not directly measure prey nor foraging behavior, the S. virgatus diet is composed primarily of Coleoptera, Formicidae, and Diptera . A comparison between sites could help isolate the effect of the fire itself. In the year of the fire, female body condition was lower on the burned site than on the unburned site; although not statistically significant, the pattern suggests a biologically relevant effect of the fire on fitness‐related traits. Interestingly, in the year following the fire, females on the burned site had little change in condition, whereas females on the unburned site had a large reduction. It is possible that changes in the unburned site reflect continued impact of drought, whereas females on the burned site were buffered from that further reduction. That buffering may have been due to positive changes in habitat structure and/or prey abundances 1 year following the fire ; Data curation ; Formal analysis (lead); Funding acquisition ; Investigation ; Methodology ; Writing‐original draft (lead); Writing‐review & editing (lead). Robert M. Brower: Conceptualization (supporting); Data curation ; Funding acquisition ; Investigation ; Methodology .


Adjacent segment disease (ASD) in the lumbar spine is a possible consequence in segments adjacent to a fusion. As the number of lumbar fusions in the United States increases, the rates of ASD will continue to climb. There are several treatment options for ASD with open decompression and extension of the fusion being common. However, need for exposure and removal of existing instrumentation can lead to increased operative times resulting in increasing blood loss and infection risk. The purpose of this paper is to describe a case report for unilateral cortical trajectory screw instrumentation, allowing for posterior instrumentation without having to remove the existing pedicle screws in the setting of ASD. Our technique can be done with standard c-arm fluoroscopy without the need for navigation. Adjacent segment disease (ASD) in the lumbar spine is a possible complication in segments adjacent to a lumbar fusion. As the number of lumbar fusions performed in the United States has increased, the rates of ASD have also climbed –5. ASD cOne alternative is stand-alone anterior or lateral lumbar interbody fusion ; howeverOur patient is a 77-year-old man with a previous history of L4-5 transforaminal interbody fusion (TLIF) and posterior instrumentation with pedicle screws done in 2007 at another institution. He presented to our office with complaints of recurrent low back and right-sided buttock and lateral leg pain for months. His preoperative MRI demonstrated a right-sided facet cyst at L3-4 along with lateral recess stenosis. Transforaminal epidural steroid injections only provided temporary relief, and so surgical intervention was offered. Preoperative CT scan  showed aIntraoperatively, exposure is only needed laterally to the level of the pars at both levels and no further. There is no need for wide exposure beyond the pars laterally. In fact, the previous instrumentation was not seen within the operative bed. The C-arm was then brought in, and a perfect AP X-ray of the L4 pedicle was obtained. The start site for the cortical screw in terms of medial to lateral start site is then marked and the cranial-caudal start site estimated as well. Lateral C-arm X-ray is then used to confirm the cranial-caudal start site and angle. Based on the preoperative CT scan, we aimed to be just distal to the existing pedicle screw. The pedicle is then drilled aiming parallel to the pedicle screw on the lateral view but heading out slightly laterally on the AP. This is slightly different from the description of standard cortical screws where the screw is aimed cranially as the existing pedicle screw has to be adjusted for. The walls are then probed and tapped, and the appropriate screw was inserted . The L3 Adjacent segment degeneration and symptomatic disease are a potential consequence of lumbar fusion surgery. The potential mechanisms of ASD development have been suggested to be increased biomechanical stress and motion at adjacent levels, particularly, the level superior to the fusion , 20. TheCortical bone trajectory screws were first described in the lumbar spine in 2009 by Santoni et al. . The pla


Axon guidance proteins play key roles in the formation of neural circuits during development. We previously identified an axon guidance cue, named draxin, that has no homology with other axon guidance proteins. Draxin is essential for the development of various neural circuits including the spinal cord commissure, corpus callosum, and thalamocortical projections. Draxin has been shown to not only control axon guidance through netrin-1 receptors, deleted in colorectal cancer (Dcc), and neogenin (Neo1) but also modulate netrin-1-mediated axon guidance and fasciculation. In this review, we summarize the multifaceted functions of draxin and netrin-1 signaling in neural circuit formation in the central nervous system. Furthermore, because recent studies suggest that the distributions and functions of axon guidance cues are highly regulated by glycoproteins such as Dystroglycan and Heparan sulfate proteoglycans, we discuss a possible function of glycoproteins in draxin/netrin-1-mediated axon guidance. The proper function of the nervous system relies on appropriate patterns of connectivity among an enormous number of neurons. A key process in the formation of neural circuits is the navigation of neuronal axons to their targets, which is regulated by axon guidance molecules . In the We previously identified a novel axon guidance cue, draxin, that is highly expressed in the developing central nervous system (CNS) . We showin vitro (draxin knockout (KO) mice show defects in many axonal tracts including the spinal cord commissure, anterior commissure, corpus callosum, internal capsule , and hippocampal commissure  family, draxin shares no homology with any known proteins . Draxin in vitro . In addimmissure . Thus, ddraxin KO mice clearly demonstrate that draxin plays an essential role in axon guidance in the spinal cord and the brain. Yet, how draxin regulates axon guidance in vivo is still poorly understood. Through cell binding assays, we previously showed that draxin binds the netrin-1 receptors Dcc, Neo1, Unc5s, and Down’s syndrome cell adhesion molecule (DSCAM)  domain of Dcc, which is separate from the netrin-1-binding domain  . Based og domain . Furtherg domain , thalamug domain , and dorg domain  requiresg domain . On the g domain . Draxin g domain . Netrin-g domain . From thCommissural axon guidance has been extensively studied in the developing spinal cord . CommissBmp7 and Gdf7 mutant mice  of the ventricular zone (VZ)  . Recentlone (VZ) . Furtherone (VZ) . Interesnetrin-1 hypomorphs  of the corpus callosum in humans is associated with various neurological disorders such as language dysfunction and abnormalities in social interaction . A largeand mice . Loss-ofcallosum . Althougal axons . In thesDuring brain development, callosal pioneering axons originate in the cingulate cortex, and cross the midline in a region initially separated by the interhemispheric fissure (IHF) . SubsequDCC pathogenic variants that display agenesis of the corpus callosum  and indusium griseum (IG) . Netrin-G region , and Dccal axons . In vitr midline . Further midline . During  midline .draxin is expressed in midline glial structures including the GW and the IG -tagged Slit2 to the basement membrane is absent. Thus, it is possible that Dystroglycan directly or indirectly regulates the distribution of draxin protein in the basement membrane of the spinal cord. HSPGs, components of the basement membrane, are also possible candidates to regulate the distribution of draxin protein. HSPGs were shown to interact with extracellular ligands such as those of the Wingless/Wnt and HH families and control their distributions . This fact suggests that HSPGs might serve as low affinity receptors for draxin and participate in the conversion of draxin-induced attraction into repulsion.Although the contribution of HS to draxin-dependent axon guidance is unclear, we suggest that HS might be involved in the concentration-dependent effects of draxin on cortical and thalamic axons . NeuriteMAP1B, which encodes a microtubule-associated protein, have axon guidance defects in the corpus callosum and the hippocampal commissure /Akt signaling pathway and activation of glycogen synthase kinase-3β (GSK-3β). Thus, it is likely that GSK-3β dependent MAP1B pathway is essential for repulsive draxin signaling. In contrast to draxin repulsion, molecular events in draxin attraction are still unclear. Further investigations are needed to elucidate the intracellular mechanisms underlying the bimodal effects of draxin.It was shown that mice deficient for mmissure . These p KO mice . Meli etdraxin KO mice suggest that draxin has multiple roles in hippocampal development  because of inappropriate ipsilateral projections of the corticospinal tract from the hindbrain (draxin KO mice are viable and fertile, even though they have drastic axon guidance defects in the brain. It will therefore be of great interest to investigate whether mutations in the DRAXIN gene are directly linked to human neurological disorders in future studies.Studies of axon guidance signaling are critical to advance not only our knowledge of axon guidance mechanisms in the developing CNS, but also our understanding of human neurological disorders caused by axon guidance defects. Multiple human neurological disorders such as intellectual disability, autism spectrum disorders, and schizophrenia are known to be associated with developmental errors in axonal pathfinding . It was indbrain . CMM areBoth authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PTEN, CTNNB1 (β-catenin), and CSC-related β-catenin target genes, is inversely regulated by AMBRA1, suggesting a negative regulatory role of AMBRA1 in the expression of ALDH1B1-regulated genes. We found that the K27- and K33-linked ubiquitination of ALDH1B1 is mediated via the cooperation of AMBRA1 with other E3 ligases, such as TRAF6. Importantly, ubiquitination site mapping revealed that K506, K511, and K515 are important for the K27-linked ubiquitination of ALDH1B1, while K33-linked ubiquitination occurs at K506. A ubiquitination-defective mutant of ALDH1B1 increased the self-association ability of ALDH1B1, suggesting a negative correlation between the ubiquitination and self-association of ALDH1B1. Together, our findings indicate that ALDH1B1 is negatively regulated by AMBRA1-mediated noncanonical ubiquitination.Activating molecule in Beclin-1-regulated autophagy (AMBRA1), a negative regulator of tumorigenesis, is a substrate receptor of the ubiquitin conjugation system. ALDH1B1, an aldehyde dehydrogenase, is a cancer stem cell (CSC) marker that is required for carcinogenesis via upregulation of the β-catenin pathway. Although accumulating evidence suggests a role for ubiquitination in the regulation of CSC markers, the ubiquitination-mediated regulation of ALDH1B1 has not been unraveled. While proteome analysis has suggested that AMBRA1 and ALDH1B1 can interact, their interaction has not been validated. Here, we show that AMBRA1 is a negative regulator of ALDH1B1. The expression of ALDH1B1-regulated genes, including  Activating molecule in Beclin-1-regulated autophagy (AMBRA1) is a highly intrinsically disordered protein. As with other intrinsically disordered proteins . AMBRA1 Recently, AMBRA1 has emerged as a tumor suppressor that mediates the degradation of proto-oncogene c-Myc  and D-ty+-dependent enzymes and catalyze the oxidation of aldehydes to their respective acids. ALDH1B1, which is one of the ALDH isozymes, is reported to be strictly expressed in the SC compartments of normal human colon and is highly expressed in human colonic adenocarcinoma [CTNNB1, Akt, and Notch1 [Aldehyde dehydrogenases (ALDHs) are members of a superfamily of NAD(P)arcinoma . In a clarcinoma , the uprd Notch1 . Negativd Notch1 . Althougd Notch1 , few stuAccording to the Biological General Repository for Interaction Datasets , ALDH1B1 has been identified as one of the AMBRA1-interacting proteins via affinity purification-mass spectrometry analysis of Flag-AMBRA1-transfected HEK293T human embryonic kidney cells . HoweverA recent study showed that the interactome of AMBRA1 contains ALDH1B1 ; howeverThus, we next questioned whether AMBRA1 affects the interaction between ALDH1B1 and its interaction partners, including itself. According to a structural modeling study of ALDH1B1, ALDH1B1 is capable of interacting with another ALDH1B1 . HoweverPTEN reflects ALDH1B1 enzyme activity [PTEN through miR-106a-5p in mouse embryonic palatal mesenchymal cells [PTEN transcription in keratinocytes [PTEN via either way, we questioned whether ALDH1B1 regulates PTEN transcription in HCT116 cells. Among the available colorectal cancer cell lines, HCT116 cells were selected for studying ALDH1B1 enzyme activity because these cells constitutively and highly express ALDH1B1, while they show low ALDH1A1 expression [PTEN, irrespective of whether ALDH1B1 was stably or transiently expressed RNAs targeting two different coding sequences of ALDH1B1, PTEN transcription was enhanced  PCR. The data showed that, in contrast to ALDH1B1, AMBRA1 affected the PTEN mRNA level  mRNA levels. Based on this previous report, we determined whether the suppressive effect of AMBRA1 on ALDH1B1 self-association would lead to a reduction in CTNNB1 mRNA levels. The overexpression of AMBRA1 reduced the CTNNB1 mRNA level in parental HCT116 cells, but not in ALDH1B1-knockdown HCT116 cells  or seven Lys residues  of ubiquitin, and each polyubiquitination linkage type has a distinctive conformation and a unique role . Among tIn summary, we verified that AMBRA1 negatively regulates the self-association of ALDH1B1 via K27- and K33-linked ubiquitination. AMBRA1 downregulates the CSC-associated Wnt/β-catenin pathway via ALDH1B1. Moreover, our data revealed that ALDH1B1 is ubiquitinated by TRAF6 in an AMBRA1-independent way through K29-linked ubiquitin chains, and by DDB1 through K27- and K33-mediated ubiquitin chains.Many questions are left after this study. The function of the K29-linked ubiquitination of ALDH1B1 remains to be elucidated. Whether the K29-mediated ubiquitination of ALDH1B1 by TRAF6 requires another substrate receptor, whether TRAF6 can directly ubiquitinate ALDH1B1 without a substrate receptor, and whether AMBRA1 determines the ubiquitination linkage type as well as the specific substrate of the E3 ubiquitin ligase remain unknown. Finally, the detailed mechanism of K27- and K33-linked ubiquitination to inhibit the self-association of ALDH1B1 remains to be unraveled.HCT116 human colorectal cancer cells and the human embryonic kidney cell lines HEK293T and HEK293FT were used. Among several colorectal cancer cell lines, HCT116 was selected for the following reasons. According to a previous study , ALDH1A12 and 37 °C) were used.HCT116 cells were cultured in RPMI-1640 medium  supplemented with fetal bovine serum  and 1× penicillin-streptomycin solution . HEK293T and HEK293FT cells were cultured in Dulbecco’s modified Eagle’s medium  with the same supplements. For ALDH1B1- and AMBRA1-knockdown HCT116 cells, 2 μg/mL puromycin  was added to the medium as the selection agent. For HCT116 cells transfected with pCMV-Myc-ALDH1B1, 600 ng/mL G418  was added to the medium for the selection of stably ALDH1B1-overexpression cells. For all cell culturing, standard conditions , which is a mammalian expression vector, to tag proteins with the 3×Flag epitope at the N-terminus, and pCMV-3Tag-2  to tag proteins with the 3×Myc epitope at the N-terminus. cDNA, which was reverse transcribed from RNA extracted from HCT116 cells using oligo-dT primers , was used as the template to amplify the coding sequences of target genes. The primer sets used are listed in Opti-MEM  and Lipofectamine 2000 transfection reagent  were used for transgene transfection. A mixture of plasmids and transfection reagent (1:2) was prepared in 1 mL of serum-free Opti-MEM. After incubation for the liposomes to entrap DNA plasmids, we carefully added the mixture into culture medium. For lentiviral infection, cells were cultured in medium containing the lentiviral particles and polybrene at a final concentration of 8 μg/mL . After 6 h, the culture medium was replaced with fresh medium. For cell selection, appropriate drugs were added 24 h later.AMBRA1- and ALDH1B1-depleted HCT116 cells were generated using shRNA-based gene silencing. First, shRNAs were cloned into a pLKO.1 TRC cloning vector . The shRNAs used, targeting the coding sequences and 3′-untranslated regions, are listed in ALDH1B1 into pCMV-3Tag-2 and transfected the plasmid into HCT116 cells. After 48 h, the cells were selected on G418 (600 ng/mL). At least 10 days later, the expression efficiency was evaluated by RT-qPCR and western blotting with anti-Myc antibody.To construct a HCT116 cell line stably overexpressing ALDH1B1, we inserted 3VO4, 1 mM PMSF, and 1× PI ). Proteins were collected by centrifugation  and quantified using Protein Assay Dye Reagent Concentrate  and a Sunrise Microplate Reader . The total proteins were separated by molecular mass using electrophoresis and transferred to polyvinylidene fluoride membranes . The membranes were blocked using skim milk  or bovine serum albumin  and then incubated with primary antibodies under agitation at 4 °C overnight and then secondary antibodies at room temperature for 1–2 h. For detection, we used an ImageQuantTM LAS 4000 instrument , and ImageJ  was used for quantification.Harvested cells were resuspended in radioimmunoprecipitation assay buffer (1% Nonidet P-40 (NP-40), 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) supplemented with protease inhibitors  supplemented with protease inhibitors. Proteins were collected by centrifugation and incubated with anti-c-Myc agarose affinity gel antibody  or anti-Flag affinity gel  at 4 °C overnight under rotation. Then, the beads were washed with lysis buffer five times. Protein complexes were eluted by boiling with Laemmli SDS sample buffer  and analyzed by immunoblot analysis.2, 50 mM Hepes, pH 7.4, and 0.5% sodium deoxycholate) supplemented with 0.1% SDS, 10 mM N-ethylmaleimide , and protease inhibitors. The proteins were collected and immunoprecipitated using an anti-c-Myc agarose affinity gel antibody and immunoblotted using an anti-HA antibody.The ubiquitination of ALDH1B1 in the cells was investigated using a cell-based ubiquitination assay. After treating the cells with 10 μM MG132  for 6 h, they were harvested and lysed using a ubiquitination assay buffer  on a 7300 Real-time PCR system . Dissociation curves were generated to test the presence of off-target amplification products or contaminants. The primers used are listed in Total RNA was extracted using TRI reagent  and chloroform  and reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit  with oligo-dT primers. qPCRs were run using SYBR Premix EXt-test. p < 0.05 was considered significant.Data are reported as means ± standard deviations (SDs). For pairwise comparisons, we used the two-tailed Student’s This study revealed that AMBRA1 acts as a negative regulator of ALDH1B1 by controlling its ubiquitination. The K27- and K33-linked poly-ubiquitination of ALDH1B1 is catalyzed by AMBRA1 in cooperation with E3 ligases, such as TRAF6. The AMBRA1-mediated ubiquitination of ALDH1B1 results in reduced self-association of ALDH1B1, leading to downregulation of the CSC-associated Wnt/β-catenin pathway. This study provided the first evidence of the important role of AMBRA1-mediated noncanonical ubiquitination in the negative regulation of the CSC marker ALDH1B1. Thus, this study may contribute to the development of CSC-selective therapeutics by identifying the AMBRA1-ALDH1B1-Wnt/β-catenin axis as a novel target of CSC function.


The future availability of experimental evidence is critical for clarifying the mechanism by which ligands prevent phosphorylation and should be the priority of future T2DM-focused research. Following this, the properties of ligands that enable them to block phosphorylation can be improved upon to generate ligands tailored for blocking phosphorylation and therefore restoring insulin sensitivity. This would represent a significant step forward for treating T2DM. This review summarizes current knowledge of the roles of PPARγ in T2DM as well as the effects of synthetic ligands on the modulation of these roles. We hypothesize potential factors that contribute to the reduction in recent developments and summarize what has currently been done to shed light on this critical field of research.A promising approach for treating type 2 diabetes mellitus (T2DM) is to target the Peroxisome Proliferator-Activated Receptor γ (PPARγ) transcription factor, which regulates the expression of proteins critical for T2DM. Mechanisms involved in PPARγ signaling are poorly understood, yet globally increasing T2DM prevalence demands improvements in drug design. Synthetic, nonactivating PPARγ ligands can abolish the phosphorylation of PPARγ at Ser273, a posttranslational modification correlated with obesity and insulin resistance. It is not understood how these ligands prevent phosphorylation, and the lack of experimental mechanistic information can be attributed to previous ambiguity in the field as well as to limitations in experimental approaches;  The Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-modulated transcription factors belonging to the nuclear receptor superfamily of proteins and have critical roles in controlling many processes in the human body. The PPARs are sorted into three subtypes: PPARα, PPARβ/δ, and PPARγ. They differ in their ligand specificity, transcriptional activity, and tissue expression profiles. Of the three subtypes, PPARγ has attracted the greatest research efforts for its roles in a number of diseases , 2, and PPARγ binds to a diverse range of endogenous ligands, which act as indicators of the cell’s metabolic state. Endogenous ligands of PPARγ bind to the ligand-binding domain (LBD) of the protein and include metabolites, such as fatty acids and eicosanoids , 11, 12,e.g., Steroid Receptor Coactivator (SRC) or Nuclear Receptor Coactivating Proteins (NCoA), or corepressors, such as Nuclear Receptor Corepressor (NCoR) or Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT)  . Coactivr (SMRT) .Many different genes are under the transcriptional control of PPARγ , which aPPARγ activity is vital to the development and prevalence of type 2 diabetes mellitus (T2DM) due to its various roles in metabolism, particularly glucose homeostasis, which is dysregulated in T2DM. One of the main functions of PPARγ is to control adipogenesis in white adipose tissue; in fact, PPARγ is both necessary and sufficient to drive fibroblastic precursors into adipocytes , 6. AdipIn the absence of a ligand, PPARγ-controlled genes have a basal level of expression and this level can be altered by synthetic ligands binding to PPARγ . Full agA well-studied full agonist of PPARγ is Rosiglitazone (Avandia), an effective insulin sensitizer that was generously prescribed to treat T2DM; yearly sales of Avandia reached US $3.3 billion in 2006 , until iIn light of this, research attention was turned to partial agonists of PPARγ, which showed fewer side effects compared with TZDs . DespiteIt is apparent from research undertaken with full agonists, partial agonists, and nonactivating ligands of PPARγ that the insulin-sensitizing properties are consistent across these classes of compounds, irrespective of their level of transcriptional effects on the receptor . This suPosttranslational modifications (PTMs) of PPARγ, such as phosphorylation, deacetylation, ubiquitination, and SUMOylation, are adding to the complexity of its modulation . Of signThe PPARγ phosphorylation site that is of greatest clinical relevance is at Ser273 (Ser245 in PPARγ isoform 1 nomenclature), located within the LBD of PPARγ . CriticaThere is a well-studied correlation between high levels of PPARγ phosphorylated at Ser273 with increased obesity and insulin resistance. In order to investigate the mechanism behind this, a fibroblast cell line harboring PPARγ ΔS273A was established as a useful tool for understanding the effects of abolishing Ser273 phosphorylation . The autet al.,  catalyzes this PTM , and moret al.  simulations to study the conformational dynamics of the full-length PPARγ and full-length RXRα complex bound to DNA . Lemkul a et al.  and testa et al.  showed aa et al. , samplesin silico approach was to use crystal structures of PPARγ LBD and Cdk5/p25 kinase to predict the binding interaction between the two proteins   . The autclature) . This hyclature) . This wa (et al. , who fouet al. (Another group also sought to use protein–protein docking to extract information that may be helpful in deciphering the mechanism of ligands blocking phosphorylation . The majet al. , who notIn each of the attempts to characterize the mechanism of ligands preventing phosphorylation of Ser273, a consensus conclusion was drawn, which is that ligands binding to PPARγ stabilize regions of the LBD to make them less available for energetically plausible binding with a kinase.There are a number of challenges that have been hampering the development of effective antidiabetic ligands of PPARγ, and research efforts should focus on overcoming them to advance understanding of PPARγ modulation and signaling. An example of these challenges is the ambiguity of the kinase responsible for phosphorylating PPARγ, which is potentially causing confusion and lessened productivity in this field. The apparent complexity of the signaling pathways involved in T2DM is also a challenge to the development of safe and effective inhibitors of PPARγ phosphorylation. Some knockout studies have provided some surprising results, which have, in some cases, raised more questions than they have answered, and this level of complexity could likely take decades to elucidate in order to overcome this challenge. Given the increasing incidence of T2DM, it is imperative that these challenges are overcome and research efforts continue to develop understanding of the mechanism of blocking phosphorylation by antidiabetic ligands.The failure of TZDs as a result of poor mechanistic understanding highlights how critical it is to understand molecular mechanisms of potential drugs before they progress through the drug discovery pipeline. The effectiveness of TZDs and other PPARγ-targeting compounds at restoring insulin sensitivity certainly merits continued research efforts in the development of these compounds. This is especially in light of the advantages of nonactivating ligands of PPARγ, which maintain the capacity to block phosphorylation independently of transcriptional activation.It is critical to understand how ligands of PPARγ block Ser273 phosphorylation so that future drug design efforts can harbor this capacity and optimize compounds at the molecular level to yield drugs that will be very effective at blocking this phosphorylation. It is already understood that increased transactivation of PPARγ will result in dysregulated gene activation, which manifests as side effects and so drug design efforts have focused on reduced transcriptional capacity in the form of antagonists and inverse agonists. Improvements have been made in the affinity, potency, and pharmacokinetic profile of developing PPARγ ligands. The remaining property of these compounds left to understand and optimize is perhaps the most important one—their mechanism of restoring insulin sensitivity.Current research efforts indicate that ligands of PPARγ are not in direct contact with the site of phosphorylation, or with the kinase, but instead cause subtle structural changes in the global dynamics of PPARγ to make the protein less favorable to interaction with kinases. This is most likely achieved by ligands closely associating with residues comprising the ligand-binding pocket, including the Ω-loop, to make them unavailable for interaction with kinases, which require flexibility in this loop to achieve an induced-fit mechanism of binding to PPARγ. This has been exhibited by predominantly computational methods, and research should focus on experimentally validating this model to develop a well-established mechanism of blocking phosphorylation by ligands. Successful completion of this will yield safe and effective PPARγ ligands that will increase insulin sensitivity in patients by blocking the phosphorylation of Ser273.It is critical for the final piece of the puzzle—experimental evidence for the mechanism of how PPARγ ligands block Ser273 phosphorylation—to be robustly investigated in order to inform future drug design efforts to lower the incidence of T2DM.The authors declare no conflicts of interest.


Predictions during language comprehension are currently discussed from many points of view. One area where predictive processing may play a particular role concerns poetic language that is regularized by meter and rhyme, thus allowing strong predictions regarding the timing and stress of individual syllables. While there is growing evidence that these prosodic regularities influence language processing, less is known about the potential influence of prosodic preferences  on neurophysiological processes. To this end, the present electroencephalogram (EEG) study examined whether the predictability of strong and weak syllables within metered speech would differ as a function of meter (trochee vs. iamb). Strong, i.e., accented positions within a foot should be more predictable than weak, i.e., unaccented positions. Our focus was on disyllabic pseudowords that solely differed between trochaic and iambic structure, with trochees providing the preferred foot in German. Methodologically, we focused on the omission Mismatch Negativity (oMMN) that is elicited when an anticipated auditory stimulus is omitted. The resulting electrophysiological brain response is particularly interesting because its elicitation does not depend on a physical stimulus. Omissions in deviant position of a passive oddball paradigm occurred at either first- or second-syllable position of the aforementioned pseudowords, resulting in a 2-by-2 design with the factors foot type and omission position. Analyses focused on the mean oMMN amplitude and latency differences across the four conditions. The result pattern was characterized by an interaction of the effects of foot type and omission position for both amplitudes and latencies. In first position, omissions resulted in larger and earlier oMMNs for trochees than for iambs. In second position, omissions resulted in larger oMMNs for iambs than for trochees, but the oMMN latency did not differ. The results suggest that omissions, particularly in initial position, are modulated by a trochaic preference in German. The preferred strong-weak pattern may have strengthened the prosodic prediction, especially for matching, trochaic stimuli, such that the violation of this prediction led to an earlier and stronger prediction error. Altogether, predictive processing seems to play a particular role in metered speech, especially if the meter is based on the preferred foot type. Spoken language is based on »quasi-regular« properties, exemplified by physiological and articulatory processes such as the vibration pattern of the vocal folds or the repetitious sequence of consonants and vowels . It is nRegularities in spoken language have a particular relation to predictions, because they allow for these predictions to be sharpened . Aside fMetrical prosodic structure in speech is of general relevance for segmentation, timing, stress, and lexical access . ViolatiExperiments investigating the neurophysiological bases of these processing consequences of regular or irregular prosody rely on event-related potentials (ERP) of the human electroencephalogram (EEG). Most of the aforementioned studies focused on a violation response that has been established in the early eighties as electrophysiological index of a semantic context effect , 1984. ISeveral studies have shown that ease of processing is not only determined by suitable semantic context but also by regular prosody , triggered by the repeating standard . As the The latter study as well as previous experiments on long-term memory effects on the MMN provide the basis of our assumptions here. We hypothesize that the omission MMN between 100 and 200 post-stimulus onset  is not oParticipants were twenty native speakers of German, recruited from the participant database of the Max Planck Institute . The sample size was based on previous studies with similar designs . All parN = 10) or a strong final syllable (N = 10). Carrier-sentences and pseudowords were spoken by a phonetically trained female speaker and recorded with 44.1 kHz temporal and 16 bit amplitude resolution in a silent recording chamber of the Max-Planck-Institute for Empirical Aesthetics in Frankfurt (Germany). From the entire set of 20 recordings, we selected those gu-syllables that had the most comparable pitch changes between strong and weak versions and showed the least difference in intensity. We decided to use stimuli that approximate typical trochaic and iambic disyllabic words without differing too much in acoustic terms, for any change of acoustic properties would modulate the omission MMN. We arrived at four syllables from one trochaic and one iambic pseudoword, of which the weak syllables had a very comparable pitch contour, differing from the strong counterparts by about 35 Hz in average pitch height. Final full-word stimuli were cross-spliced in that the original strong syllable from the selected trochaic pseudoword was combined with the weak syllable from the selected iambic pseudoword, resulting in a trochaic cross-spliced test stimulus. Vice versa, the weak syllable from the trochaic pseudoword was combined with the strong syllable of the iambic pseudoword, resulting in an iambic cross-spliced test stimulus. All syllables were trimmed to 250 ms with the phonetic software PRAAT, using the overlap-add algorithm. This was done in order to avoid MMN asymmetries that arise solely by differences in stimulus or stimulus part durations. Longer stimuli in deviant compared to standard position elicit a smaller MMN than vice versa, i.e., shorter stimuli in deviant compared to standard position ,” with “gugu” either pronounced with a strong initial syllable .Omissions were realized as syllable omissions. All omissions were created by truncating the cross-spliced pseudowords at their respective mid-points. For instance, an omission in first-syllable position of a trochee resulted in a weak syllable that originally stemmed from an iamb. In total, due to two foot types and two positions, four truncated pseudowords realized the four types of omissions.The stimulus material was arranged in a typical oddball paradigm, where stimuli could occur in standard or deviant position, distributed over several blocks. Blocks as displayed in All stimuli were presented with a constant inter-stimulus interval of 300 ms. This means that inter-stimulus differences were 300 ms (measured from the end of the second syllable of one disyllabic word to the beginning of the first syllable of the next disyllabic word). This translates into a Stimulus Onset Asynchrony (SOA) of 800 ms. Note that deviants with word-initial omissions effectively resulted in an SOA of 1,050 ms measured from the beginning of the standard immediately before the deviant and the beginning of the truncated deviant syllable.Stimuli were presented over open-field loudspeakers placed symmetrically 1 m in front of the participants. Participants were seated in electrically and acoustically shielded EEG-cabins. Next to the loudspeakers, a flat-screen was placed 1.2 m in front of the participants. This screen was used to display a silent movie during the passive oddball paradigm.After EEG-setup, participants passively listened to the 12 blocks of standard-deviant trains. There was no task except the request to ignore the sounds as best as possible, while watching a silent movie (without subtitles). After each block, the experimenter allowed for a short break. In the middle of the experiment, the break was longer and the air in the EEG cabin was refreshed. Each block lasted for about 5 1/2 min; the entire experiment in the cabin about 65 min.Continuous EEG was recorded from 64 Ag/AgCl electrodes, arranged on a nylon cap following the extended 10–20 system . EEG sigElectroencephalogram raw data were analyzed within fieldtrip , runningt-tests between single-trial amplitude values for standards and single-trial amplitude values for deviants, separately for the first-syllable and the second-syllable omission conditions. We thereby obtained uncorrected by-participant t-values for all time points and all electrodes. These t-values were subsequently tested against zero using dependent-sample t-tests at the second, i.e., group level, of our cluster-analysis. We estimated type I-error controlled cluster significance probabilities (at p < 0.05) by a Monte-Carlo non-parametric permutation method with 1,000 randomizations. The resulting matrix of t-values (electrodes × time points) was then analyzed between 100 and 200 ms post word onset for the first-syllable omission condition, and between 350 and 450 ms post word onset for the second-syllable omission condition. These time windows represent the expected temporal location of the omission MMN, measured from stimulus onset . In the first-syllable omission condition, this led to a cluster of 20 electrodes, showing significant standard-deviant differences between 130 and 180 ms post-stimulus onset . This resulted in mean MMN values for each participant, electrode, omission position and meter. Additionally, within the two time windows of the omission MMN, we determined the peak amplitude and the time point (latency) of this peak amplitude. Peak amplitudes were selected automatically by determining the minimum value of the deviant-standard difference in the respective time windows, and by manually inspecting the plausibility of the peaks. The automatic approach performed well, and only in three cases manual adjustment was necessary.T2 test against zero (with df = 19).Cortical sources of the omission MMN were estimated using Variable Resolution Electromagnetic Tomography VARETA; . The VARF-values that were estimated by the lmerTest package , FOOT TYPE , ELECTRODE  and the random effect SUBJECT in a full-factorial design .Omission MMN mean amplitudes, peak amplitudes and latencies were then submitted to linear-effects mixed models (LMMs), calculated with the statistical software R . Results are reported as mixed-effects analysis of variance (ANOVAs) with  package , using tPOSITION (first vs. second syllable) and FOOT TYPE , topographies of omission MMNs showed typical fronto-central distributions, with sources in left and right temporal areas, including primary and secondary auditory cortex, planum temporale and parts of superior and middle temporal gyrus  = 38.97, p < 0.001; peak amplitude: F = 20.57, p < 0.001], but not trochees , showed higher amplitudes for omissions in second-syllable, compared to omissions in first-syllable position. All effects and interactions were independent of the electrodes.For both mean and peak amplitudes, omission MMNs were larger for omissions in the second syllable  than in the first syllable . The interaction of the effects plitudes . NotablyPOSITION, as seen from the decomposition of the interaction of the effects of POSITION and FOOT TYPE Omissions in both first- and second-syllable position resulted in robust omission MMNs. This replicates the findings for omissions of speech sound sequences shorter than syllables  and exte(b)Omissions in second-syllable position resulted in a generally enhanced omission response compared to omissions in first-syllable position. This, however, depended on foot type and only held for iambs.(c)FOOT TYPE indicated that first-syllable omissions resulted in larger MMNs for trochees than for iambs, and that second-syllable omissions resulted in larger MMNs for iambs than for trochees. This pattern corresponds to the weight-carrying syllable in the two foot types, with trochees consisting of a weight-carrying syllable in first position and with iambs consisting of a weight-carrying syllable in second position.Within first-syllable and second-syllable position, the main effect of (d)The latency results suggest that an omission in first-syllable position was detected earlier for trochees than for iambs. Together with the amplitude patterns, trochees appear to imply stronger prosodic expectations, possibly caused by their preference in German . The fouThe omission MMN has been identified as prediction error response to rare omissions in tone sequences whose inter-onset intervals would not exceed a specific temporal window of integration of about 125–150 ms , 1998. SThe difference between omissions in first- and second-syllable position in our experiment may—at first sight—be based on differences in temporal predictions. Second-syllable omissions are characterized by a violation of the word-internal timing. In all disyllabic pseudowords, the onset-to-onset interval of the two syllables is 250 ms. In addition to the prediction that the syllable in second position is a repeated version of the syllable in first position, there is also a strong temporal prediction that the onset of the second syllable is 250 ms after the onset of the first syllable. Temporal predictions in audition are particularly fostered by regular acoustic contexts, such as provided by oddball paradigms . Tavano per se, but rather a strong prediction of when strong syllables occur in either trochaic or iambic words. In iambic words, the strong syllable appears in second position, thus, the omission of the second syllable should result in a stronger prediction error, if the omission MMN is sensitive to prosodic properties such as syllable weight. This is supported by the results of our experiment, where indeed second-syllable omissions in iambs resulted in stronger MMNs than first-syllable position omissions. The same, complementary pattern, held for trochees: Here, omissions in first-position resulted in stronger omission MMNs than omissions in second-position. Put differently, the omission MMN is not only sensitive to syllable omissions and their temporal position, but also to the prosodic properties of these syllables, following the different foot types. This partially replicates the findings of A more plausible interpretation of the differences between first- and second position is based on the interaction of the effects of position and foot type. This interaction indicates that the position effect crucially depends on foot type: Only for iambs, the omission of the second syllable resulted in a larger omission MMN. That is, there is not a position effect Finally, when looking at the MMN latencies, our patterns of results suggest that trochees take a specific role in that the omission of their (strong) first syllable results in an earlier MMN than the omission of the (weak) first syllable of iambs. Hence, the omission of a strong syllable in first position results in a particularly salient prediction violation. Of course, it is impossible to base this effect on foot type because foot type and the position of strong syllable are confounded. To disentangle these effects, future work is necessary. However, in combination with the amplitude data, the conclusion seems warranted that trochees have a specific influence on the omission response in that this response is not only elicited at earlier latencies but also with a stronger amplitude when the strong syllable is omitted. Note that the omission of the strong syllable in iambs led to an even stronger MMN, indicating that at least the amplitude pattern does not depend on whether the syllable occurred word-initially or word-finally. Therefore, we conclude that the particular pattern elicited by trochees reflects their preferred status in German prosody . FurtherAudition benefits from local and global regularities, both temporally and phonologically . Regularities can generate strong predictions, whose violations lead to well-known electrophysiological responses. We here demonstrated the feasibility of the omission MMN to quantify foot type-based differences in prediction violations. This research can mark the starting point for further studies more concretely looking at the interplay of predictive processing and aesthetic evaluation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Max-Planck-Society. The participants provided their written informed consent to participate in this study.KH was involved in research question identification, study planning, protocol preparation, data analysis, data interpretation, and manuscript editing. MS was involved in research question identification, study planning, protocol preparation, data analysis, data interpretation, manuscript writing, editing, and reviewing. Both authors reviewed and approved the manuscript prior submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The neonatal Fc receptor (FcRn) is known to mediate placental transfer of IgG from mother to unborn. IgE is widely known for triggering immune responses to environmental antigens. Recent evidence suggests FcRn-mediated transplacental passage of IgE during pregnancy. However, direct interaction of FcRn and IgE was not investigated. Here, we compared binding of human IgE and IgG variants to recombinant soluble human FcRn with β2-microglobulin (sFcRn) in surface plasmon resonance (SPR) at pH 7.4 and pH 6.0. No interaction was found between human IgE and human sFcRn. These results imply that FcRn can only transport IgE indirectly, and thereby possibly transfer allergenic sensitivity from mother to fetus. IgG is one of the most abundant serum proteins and the most abundant immunoglobulin found in human serum10. Mutational analysis have led to the identification of IgG1-Fc variants influencing IgG binding to FcRn. IgG1-MST-HN has been found to exhibit increased binding to FcRn11, whereas IgG1-IHH cannot bind FcRn12.The neonatal Fc receptor (FcRn) is a mostly intracellularly expressed10. In contrast to IgG, IgE is the immunoglobulin with the lowest abundance in serum. Next to its role in the defense of parasites13, IgE is known for its involvement in immune reactions against environmental antigens, causing type I hypersensitivity15. There are two main IgE Fc receptors, the high affinity FcεRI and the low affinity FcεRII (CD23). Cross-linking upon binding of antigens to FcεRI-bound IgE or binding of IgE ICs on the surface of e.g. basophils and mast cells has been reported to initiate cellular immune responses14. CD23 on the other hand exists in both in a trimeric membrane-bound and soluble mono- and trimeric forms, regulating IgE synthesis and homeostasis16. Membrane-bound CD23 is expressed on B cells and intestinal epithelial cells, where it also controls IgE synthesis and mediates transfer of IgE-ICs to the intestinal lumen, respectively17.IgG can mediate a non-cellular response by engaging the complement system as well as it can bind Fc gamma receptors (FcγRs) and cross-link them on effector cells in the form of IgG immune complexes (ICs), triggering cellular effector functions18, transport of other isotypes such as IgA and IgM is generally not considered relevant, most likely passive, as only a small fraction of what is found in maternal sera can be found in cord blood19. However, FcRn involvement has been reported for the transfer of tolerance to food allergens from mother to offspring in mice20 as well as for the transfer of IgE in anti-IgE IgG/IgE ICs in mice21 and humans22. A recent study suggested FcRn-dependent placental transport of IgE from mother to offspring in mice23.Whereas it is clear that the active transport of IgG across the placenta to the unborn is FcRn-mediatedIn this study we investigated the binding of human IgE and IgG variants to human sFcRn using SPR aiming to complement the already published cellular data from a physicochemical point of view.25, as described previously27. Linear DNA strands encoding for the soluble FcRn α-chain with a C-terminal BirA deca-Histidine (His) tag and ß2-microglobulin were ordered accordingly and cloned separately into pcDNA3.1 expression vectors, as described elsewhere28. In brief, expression vectors and DNA inserts were digested with EcoRI and NheI FastDigest restriction enzymes (Thermo Scientific). The expression vector backbone was isolated by gel purification using a 1% UltraPure agarose (Thermo Scientific) gel with 1:10.000 SYBR Safe (Invitrogen). DNA was extracted from the gel using the NucleoSpin Gel and PCR clean-up kit (Macherey–Nagel) according to the manufacturer’s protocol. The DNA fragments were isolated using the same kit but without prior gel purification.Linear DNA strands encoding for mutated IGHG1*03 and IGHE*02 Fc-regions were ordered from Integrated DNA Technologies and cloned into a pcDNA3.1 expression vector containing anti-biotin heavy chain variable regions obtained fromThe DNA fragments were ligated into the pcDNA3.1 backbone overnight at 16 °C using T4 DNA Ligase (New England Biolabs) in 1 × T4 DNA Ligation buffer (New England Biolabs) with a molar ratio of insert to vector of 3:1. 5 µL of the ligation reaction was transformed into 50 µL DH5α competent cells (Thermo Scientific) by heat shock.The cells were plated on LB-agar plates containing 50 µg/mL ampicillin (Thermo Scientific) and incubated overnight at 37 °C. Colonies were picked and grown in 2 mL LB medium containing 50 µg/mL ampicillin (Thermo Scientific) overnight at 37 °C, shaking at 180 RPM. DNA was isolated from the bacterial culture using the NucleoSpin Plasmid EasyPure kit (Macherey–Nagel) according to the manufacturer’s protocol and sequenced.Sequence-confirmed DNA was used for transformation of DH5α competent cells as described above and colonies were picked and grown in a 5 mL preculture. This was subsequently used to inoculate 200 mL LB medium containing 50 µg/mL ampicillin (Thermo Scientific), after which the culture was grown overnight at 37 °C, shaking at 180 RPM. DNA was isolated using the NucleoBond Xtra Maxi kit (Macherey–Nagel) according to the manufacturer’s protocol and again sequenced.26. In brief, 31.35 µg of the heavy chain vector, 37.65 µg light chain vector (pcDNA3.1 anti-biotin VLCL25) and 31 µg pSVLT/p21/p27 mix29 were added to 6.66 mL opti-MEM (Thermo Scientific) per 100 mL of transfection cell culture. 300 µL Polyethylenimine (PEI) MAX  was added, the mixture was immediately vortexed and incubated for 20 min at room temperature. 100 mL of HEK293F cells (Thermo Scientific) at 1*106 cells/mL in fresh FreeStyle 293 Expression Medium (Thermo Fisher Scientific) were transfected with the mixture and incubated at 37 °C at 8% CO2 and shaking. After 4 h, 100 units/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific) were added to the culture. Human sFcRn was produced as described previously28, using equimolar amounts of both the soluble FcRn α-chain and ß2-microglobulin expression vectors. The culture supernatants were harvested 6 days after transfection by spinning down the cells twice for 5 min at 3.100 × g and filtering through a 0.45 µm syringe filter (Whatman).Antibodies were produced as described previously30, or a HisTrap HP column (sFcRn) . Fractions containing the antibodies or sFcRn were combined and concentrated using a 10 K MWCO Pierce Protein Concentrator PES (Thermo Scientific). Antibodies were fractionated by HPLC-SEC using an AKTA UPC-900, P-920 and Frac-950  with a Superdex 200 10/300 GL column . Monomeric fractions were combined, antibodies were dialyzed to 5 mM sodium acetate (pH 4.5) and human sFcRn to 1xPBS (Fresenius Kabi) using either a 10 K MWCO Slide-A-Lyzer dialysis cassette (Thermo Scientific) overnight at 4 °C or using a 7 K MWCO Zeba Spin desalting column (Thermo Scientific) according to the manufacturer’s protocol. Protein concentrations were measured using a Nanodrop 2000c spectrophotometer (Thermo Scientific), adjusted to 1 mg/mL and aliquoted to 20 µL working stocks. Working stocks were stored at − 20 °C until assayed.Anti-biotin IgGs were purified from culture medium with AKTA prime  by affinity chromatography using either a 5 mL HiTrap HP protein A (IgG1-WT and -MST-HN) or protein G (IgG1-IHH) column , as described previouslyAnalytical HPLC-SEC runs were performed using an Agilent 1260 Infinity II HPLC system (Agilent) coupled to a SDP-20A UV/Vis detector (SHIMADZU), a miniDAWN (Wyatt Technologies) and an Optilab (Wyatt Technologies). 16.67 µg of each molecule were assayed on a Superdex 200 10/300 GL (Cytiva) in 1xPBS (Fresenius Kabi) at a flow speed of 0.75 mL/min.31 or 0.25% (w/v) ß-Mercaptoethanol in NuPAGE™ LDS Sample Buffer (Thermo Scientific) for non-reduced and reduced conditions, respectively. Samples were loaded on a NuPAGE™ 4–12% Bis–Tris Gel (Invitrogen) and run with MOPS SDS running buffer (Thermo Scientific) for 10 min at 100 V followed by 45 min at 120 V. The gel was stained overnight in Blue-Silver solution  and 20% methanol (Thermo Scientific) and thoroughly destained in distilled water.Each antibody was tested in SDS-PAGE under reducing and non-reducing conditions. Samples were incubated for 5 min at 70 °C or 95 °C in the presence of 20 mM Iodoacetamide (to prevent reduction during denaturation)32 and IgE-containing culture supernatant were diluted in 2 × Native Tris–Glycine Sample Buffer (Life Technologies) and run for 90 min at 150 V on a NuPAGE 3–8% Tris–Acetate Gel (Thermo Scientific). The gel was transferred to a iBlot2 NC Mini Stack (Thermo Scientific) and blotted using an IBlot2 Gel Transfer device (Thermo Scientific). The membrane was blocked for 30 min using 1 × Western Blocking Reagent (Roche), followed by incubation with 1:500 anti-hIgE (MH25-1) (Sanquin) in the same solution for 1 h. After extensive washing, the membrane was incubated with 1:500 polyclonal goat anti-mouse HRP (Dako) for 1 h in the same blocking solution. After another extensive washing step, the blot was developed using Pierce ECL Western Blotting Substrate (Thermo Scientific) according to manufacturer’s instructions.IgE-containing serum sample3PO4 pH 2.4. KD values were calculated by fitting a Langmuir 1:1 binding model to a Rmax = 700, as described previously33, using Scrubber software version 2 (BioLogic Software) and excel. IgE culture supernatant was confirmed to contain anti-biotin IgE by injection of 100 nM of anti-hIgE (MH25-1) (Sanquin) or soluble FcεRI α-chain (soluble FcεRI)  in 1xPBS containing 0.075% (v/v) Tween (80) at pH 7.4 or pH 6.0 after antigen capture of antibodies.Affinity measurements to human sFcRn in SPR were performed using an IBIS MX96 (IBIS Technologies) device and a Continuous Flow Microspotter (Wasatch Microfluidics). BSA-biotin (ITK Technologies) was spotted at four concentrations each on a SensEye G Easy2Spot (SensEye) in 10 mM sodium acetate at pH 4.5 with 0.075% (v/v) Tween (80) in 2 × dilution series starting at 60 nM. Anti-biotin IgG or anti-biotin IgE was injected at a concentration of 100 nM or 2 × diluted culture supernatant, respectively. Titration of human sFcRn was performed by injecting seven concentrations of human sFcRn, from 15.63 nM to 1000 nM (2 × dilution series) in 1xPBS containing 0.075% (v/v) Tween (80) at pH 7.4 or pH 6.0. The sensor was regenerated between the cycles by two subsequent injections of 20 mM Tris–HCl, 150 mM NaCl pH 8.8 and 20 mM H32. We then employed our SPR platform to test antigen binding and further validate the molecular identity of the IgE in the culture supernatant. As shown in the schematic overview in Fig. Anti-biotin IgG1 variants were tested in HPLC-SEC in order to confirm their integrity. All molecules showed expected sizes and were monomeric  or food allergens to offspring. Transfer of such protective IgG and IgG-ICs has been reported to occur via placental passage and via the mother milk, respectively, both in a FcRn-dependent manner42. Possibly, some placental transcytosis of IgE molecules takes place40. Evidence has been reported that this happens in a FcRn-dependent manner in humans22 and in mice23. Two recent studies implied a role of maternal IgE in priming allergic responses in offspring, providing evidence for such indirect FcRn-dependent placental transport in mice23 and suggested a direct FcRn-mediated transport of IgE43. It is unclear to which extent the high amounts of antigen-specific IgE administered to the parental animals during pregnancy in these studies23 – more than 100-fold higher than naturally occurring total IgE levels in mice44 – can be extrapolated to more physiological concentrations, as recently noted elsewhere45.The presence of IgE in cord blood has been reported in humans, albeit at low concentrations compared to paired maternal blood22. The presence of such complexes in serum is controversial46. Next to anti-IgE IgG/IgE ICs, FcRn-mediated placental passage of antigen-specific IgE could also occur in the form small ICs formed by IgG and IgE binding the same antigen. Interestingly, a study by Weil et al. provides indirect evidence for this scenario in the context of parasitic infection. Although they found remarkable levels of parasitic IgE in cord blood, often exclusively antigen-specific but generally ~ 50–100 times less than found in maternal blood, the data was interpreted as an antigen-specific IgE response originating from the fetus. However, such IgE levels could also be interpreted as evidence for ICs formed by IgG and IgE bound to the same antigen36, but do not favor the hypothesis of IgE being commonly transported across the placental barrier.If direct FcRn-mediated transport of IgE does not occur, are there other ways FcRn might play a role in IgE transport across the placenta? One possibility is FcRn-mediated placental passage of complexes of IgE with IgG, forming anti-IgE IgG/IgE complexes40, comparable to or even lower than relative levels of IgA or IgM, respectively19, not suggestive of an active, directly FcRn-mediated placental transport as described for IgG19.Invariably, the relative total IgE concentrations found in cord blood compared to matched parental samples are very low22 with binding data to human sFcRn in SPR at both pH 7.4 and pH 6.0. We conclude that it is very unlikely that human FcRn directly mediates placental transcytosis of human IgE, but rather through ICs containing IgG, possibly as anti-IgE IgG/IgE ICs, as suggested elsewhere47.Our results complement previously published data showing no binding of hIgE to hFcRn when overexpressed on MDCK cells by FACSThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information.