The climate varies due to human activity, natural climate cycles, and natural events external to the climate system. Understanding the different roles played by these drivers of variability is fundamental to predicting near-term climate change and changing extremes, and to attributing observed change to anthropogenic or natural factors. Natural drivers such as large explosive volcanic eruptions or multidecadal cycles in ocean circulation occur infrequently and are therefore poorly represented within the observational record. Here we turn to the first high-latitude annually-resolved and absolutely dated marine record spanning the last millennium, and the Paleoclimate Modelling Intercomparison Project (PMIP) Phase 3 Last Millennium climate model ensemble spanning the same time period, to examine the influence of natural climate drivers on Arctic sea ice. We show that bivalve oxygen isotope data are recording multidecadal Arctic sea ice variability and through the climate model ensemble demonstrate that external natural drivers explain up to third of this variability. Natural external forcing causes changes in sea-ice mediated export of freshwater into areas of active deep convection, affecting the strength of the Atlantic Meridional Overturning Circulation (AMOC) and thereby northward heat transport to the Arctic. This in turn leads to sustained anomalies in sea ice extent. The models capture these positive feedbacks, giving us improved confidence in their ability to simulate future sea ice in in a rapidly evolving Arctic. The Arctic sea ice decline observed in recent decades, while robustly linked to anthropogenic forcing3, includes a contribution from internal variability4 and volcanic activity5. While the short-term response of sea ice to volcanic forcing can be understood in terms of reduction of incoming radiation, atmospheric reorganisation and changes in surface ocean temperature6, which can be examined in the observational record8, the multidecadal to centennial response is likely to be mediated through large-scale ocean dynamics such as the inflow of warm water to the Arctic within the upper limb of the AMOC12. Modelling studies have proposed a number of mechanisms through which volcanic activity can lead to decadal and longer changes in AMOC strength15, but agreement about how ocean circulation has changed on these timescales remains elusive. Understanding Arctic sea ice variability on multidecadal timescales therefore not only helps to better explain recently observed changes and potential predictability, but also to uncover the mechanisms of multidecadal variability in the wider climate system.Sea ice is one of the most dynamic components of our climate system. Changes in sea ice amplify energy imbalance in the polar regions, and may have significant impacts on mid-latitude weather and extremes16. This low-salinity layer is made up of very fresh Polar Mixed Layer waters at the surface and the more saline Arctic Halocline waters below16  Seas are characterised by a number of stratified water-masses with contrasting temperature and salinity properties. The position of these water masses is tightly coupled to that of the sea ice edge Fig. . The larw16 Fig. . Warm reMOC Fig. . Where te17 Fig. . Within urs Fig. .Figure 118  record has been constructed spanning the last millennium from aragonite samples drilled from shell growth bands belonging to an Arctica islandica bivalve chronology20 . The North Iceland bivalve δ18O timeseries, which is derived from aragonite precipitated in apparent equilibrium with the ambient seawater salinity and temperature21, displays marked multidecadal variability  at a given temperature, according to the empirical equation of Grossman and Ku21. Four distinct water-masses can be seen in δ18Owater-space across the East-West GIN Seas transect, mapped out by calculating the δ18O of aragonite in equilibrium with seawater at observed temperatures and salinities22, described herein as the δ18Oequil. 23 included simulations of the last millennium, forced by time varying land-use and greenhouse gas concentrations, orbital parameter changes, solar irradiance variability and volcanic aerosol concentration change24. Radiative forcing over the pre-industrial component of the last millennium (850 Common Era (CE) to 1849 CE) was dominated by volcanic and solar activity24. We find remarkable agreement between the PMIP3 multi-model mean GIN-Seas sea ice extent , and the annually resolved North Iceland Shelf δ18O recorded in the bivalve shells  follows a large increase in recorded sea ice off North Iceland27  indicate that the variability constrained by the bivalve δeas Fig. . Consistgth Fig. . The reding Fig. . A latiting Fig.  can be ling Fig. . The redSea Fig. . The mecAccurate near-term prediction, attribution of climatic events, and quantification of weather extremes relies on the ability of climate models to simulate internally and externally-driven variability on a range of timescales. Here we have presented a marine proxy-tested model reconstruction of multidecadal Arctic sea ice variability over the last millennium and identified an important contribution of natural external forcing to this variability. We have shown that climate models are capable of capturing the timing of this variability, despite the forcing being integrated by, and the response arising through, complex coupled atmosphere, sea ice and ocean processes. These results give us confidence that models are capable of simulating the oceanographic feedbacks which are likely to occur in response to anthropogenically driven sea ice decline.22 was converted to a synthetic δ18O  simulations. The model output used was from CCSM434, CSIRO-Mk3L-1-235, GISS-E2-R36, HadGEM2-ES38, MIROC-ESM40, MPI-ESM-P41, MRI-CGCM342 and bcc-csm1-143.The first ensemble member of all PMIP3 last millennium model simulations with the required variables, and compliant netcdf files were used, other than that of HadCM344, and the sea ice fraction extracted for the region 45W-25E, 0N-90N and between the years 950CE and 1850CE, and averaged from monthly to yearly means. The area-weighted average timeseries was calculated for each model, this was linearly detrended, then the multi-model mean timeseries calculated.Model output was interpolated onto a regular 1 by 1 degree grid using the Climate Data Operator remapbil function18O timeseries presented in Fig. p-value of the correlations. Significance is therefore defined here as where the correlation coefficient exceeds  the correlation coefficient of at least 95% of 100 randomly generated timeseries of the same length which have undergone the same smoothing. The synthetic timeseries were generated with a uniform distribution between 0 and 1. It should be noted that this significance test is not directly analogous to a p-value, and the precise outcome is conditional on the spectral characteristics of the synthetic timeseries. The timing of the partitioning of the modelled sea ice timeseries into three periods  simulations. For sea ice and salinity analysis these models were CCSM4, CSIRO-Mk3L-1-2, HadGEM2-ES, MIROC-ESM, MPI-ESM-P, MRI-CGCM3, bcc-csm1-1, and for AMOC analysis  these were bcc-csm1-1, CCSM4, CSIRO-Mk3L-1-2, FGOALS-gl45, MIROC-ESM, MPI-ESM-P and HadGEM2-ES.The first ensemble member of all PMIP3 last millennium model simulations with the required variables, and compliant netcdf files were used, other than that of HadCM344, and the calculated Atlantic stream function data interpolated onto common depth levels using the Python scipy RectBivariateSpline function. High and low sea ice years were defined as where the multi-model-mean sea ice timeseries  was greater than 1 standard deviation and less than −1 standard deviation from the mean respectively, but excluding the 10 years following a volcanic eruption with a global total stratospheric sulfate aerosol injection greater than 15Tg46. A subset of each 3D  model field for each variable was selected, containing data from only the high and low sea ice years respectively. These fields were averaged along the time dimension, and the difference for each model between the high and low sea ice composites calculated. The multi-model mean was calculated from these composite difference fields.Model output was interpolated onto a regular 1 by 1 degree grid using the Climate Data Operator remapbil functionSupplementary Information.


Quantitative determination by ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS) showed higher conversion yields (%) for naringin and rutin esters using acyl donors with 8C and 10C. Rutin decanoate had higher partition coefficients (0.95), and naringin octanoate and naringin decanoate showed greater inhibitory effects on XO . Kinetic analysis showed significant differences (p < .05) between the flavonoids before and after acylation regarding Km values, whereas the values for Vmax were the same, implying the competitive nature of XO inhibition.Studies have reported that flavonoids inhibit xanthine oxidase (XO) activity; however, poor solubility and stability in lipophilic media limit their bioavailability and applications. This study evaluated the kinetic parameters of XO inhibition and partition coefficients of flavonoid esters biosynthesised from hesperidin, naringin, and rutin  Acylation of 3-OH group of a new isomer of mesquitol  as the catalyst, in a bid to obtain compounds with greater capacity to inhibit XO than the untreated flavonoids. The partition coefficients and the kinetic parameters of the XO inhibition (Vmax and Km) were assessed for both the untreated flavonoids and their acylated derivatives.Our research group previously reported the enzymatic acylation of hesperidin using as acyl donor a medium chain fatty acid (decanoic acid) catalysed by CALB in a two-phase system containing [bmim]BFCALB immobilised in acrylic resin  (Novozyme 435), xanthine oxidase from bovine milk, xanthine, allopurinol, hesperidin, naringin, and rutin standards and hexanoic, octanoic, decanoic, lauric, and oleic acids were obtained from the Sigma-Aldrich Chemical Co., St. Louis, MO. All the solvents and reagents were of analytical, spectrometric, or chromatographic quality.,The enzymatic synthesis of acylated derivatives of flavonoids was performed according to a previously described method−5) (peak area) + 112.28 and rutin/naringin concentration (μg/ml) =  (6 × 10−5) (peak area) + 256.71 and the data were expressed in percentage yields (%).The chromatographic separation was achieved using an Acquity UPLC system  equipped with a Waters UPLC BEH column  at a temperature of 30 °C. A 3 μl of each sample was injected and the gradient applied used two mobile phases – (A) ultrapure water with 1% of formic acid and (B) methanol, starting with 5% of B, increasing to 100% B in 8 min, maintained until 8.50 min, and finally returning to the original conditions and stabilising at 10 min. Detection was in negative ions mode using an Acquity TQD electrospray ionization-mass spectrometer  in the following conditions: capillary – 3000 V, cone – 30 V, source temperature 150 °C, and desolvation temperature 350 °C. Quantification of the compounds was achieved using standard curves obtained by injecting standards of the flavonoids in concentrations of ranging from 30 to 300 μg/ml. The conversion yields were calculated from the ratio between the concentration of the acylated derivatives and the initial concentration of flavonoids before the acylation reaction. Calibration curves for each flavonoid were obtained using standards in methanol. The calculations were based on the following equations: hesperidin concentration (μg/ml) = (4 × 10k) were analysed to determine their degrees of lipophilicity. In test tubes, 2.0 ml of a solution of the sample with a concentration of 50 μM were added to 2.0 ml of octanol saturated with water. The mixture was shaken for 1 min and then centrifuged for 15 min at 3000 rpm. After filtering through 0.22 μm polyethylene filter with a PTFE membrane , the compound concentration was determined for each phase by UHPLC–MS. The partition coefficient was obtained using the equation:r = ratio of the volumes of the oily and the aqueous phases.The partition coefficients of the untreated flavonoids in octanol/water . The samples were pre-incubated at 37 °C for 10 min. The XO solution  was added to the reaction mixture and the flasks were incubated at 37 °C for 20 min. The enzymatic reaction was interrupted by adding 25 μl of 3.2% HCl. The absorption of the samples was measured using an ELISA reader Epoch, BioTek  at 290 nm. The uric acid production was calculated from the differential absorbance with a blank solution without xanthine oxidase. The control was a solution containing xanthine and XO. Allopurinol, a specific XO inhibitor, was used as a positive control. XO inhibitory activity was expressed as the percentage inhibition of XO  by using the following equation:Vmax (maximum reaction rate) and Km  were calculated and the type of inhibition kinetics was identified using the Origin 8.6 program and the rectangular hyperbola model. The Michaelis–Menten equation linearized by Lineweaver–Burk was used to determine Vmax and Km by plotting a graph, that is 1/V against 1/[substrate concentration], and estimated by the intercept and slope respectively. Reaction rate (expressed as uric acid concentration in μmol/min) was obtained from the uric acid standard curve . Those concentrations associated to 50% inhibition (IC50) were also calculated.The enzymatic kinetics trials were made with those compounds that showed the highest percentage inhibition (XOI) of XO activity. p values was .05 or less.The experiments were performed in triplicate ANOVA one way was used to analyse statistical significance followed by Turkey (Bonferroni) test using Origin Pro 8  statistics software. The results are presented as mean ± SD and statistics were considered significant when the ,O-β-D-glucopyranoside) can be efficiently substituted at 6″-OH by acetate or by C4- to C16-aliphatic acids by CALB. Shorter dicarboxylic acids (C2 to C4) were not substrates for the lipase and did not react at all, while the enzyme has accepted C5- to C12-dicarboxylic acidThe highest conversion yields were obtained for hesperidin, followed by rutin and naringin. Acylation of rutin and naringin was more favourable with the shorter chain hexanoic (6C), octanoic (8C), and decanoic (10C) fatty acids. Lauric (12C) and oleic (18C) acids are poor substrates for the acylation of rutin and naringin albeit they efficiently esterified the hesperidin molecule. Earlier studies have reported that the highest CALB-catalysed biosynthesis yields of acylated derivatives of naringin and rutin are obtained using fatty acids with up to 10CVmax, Km, and IC50) and to determine their partition coefficients in octanol/water (k).There was a considerable increase in the inhibitory effect on XO activity associated to three of the acylated derivatives, naringin octanoate, naringin decanoate, and hesperidin decanoate , compared to the untreated flavonoids . Compared to allopurinol, which strongly inhibited XO (89.67%), the derivatives biosynthesised with naringin and hesperidin showed a moderate, but rather promising, activity. For the rutin derivatives, however, the acylation reaction did not enhance the inhibition of XO activity. In the light of those results, the three acylated compounds were selected to assess the kinetic parameters involved in XO inhibition (Two concentrations (45 and 90 μM) of the inhibitors hesperidin decanoate, naringin octanoate, and naringin decanoate and three concentrations of xanthine  were used to determine the kinetic parameters. The reactions were performed in the presence and absence of the inhibitor to obtain the Michaelis–Menten curves and the Lineweaver–Burk double reciprocal plots as shown in Km and Vmax values obtained , expressed as 1/Vo (y) plotted against 1/[S] (x) produces a straight line for which the slope gives the value of Km/Vmax, the intercept on the 1/Vo axis is equal to 1/Vmax and the intercept on the 1/[S] axis is equal to −1/Km.The response of the acylated flavonoids naringin octanoate, naringin decanoate, and hesperidin decanoate in inhibiting XO activity is directly proportional to their concentrations and 90 μM is the concentration with the greatest effect in reducing the reaction rate (uric acid concentration formed/minute). The results were analysed by means of the Lineweaver–Burk double reciprocal plots and the obtained  by graphVmax values show no significant alterations and that the Km  were higher in the presence of inhibitors when compared to the control, then it can be supposed that there is a competitive, reversible inhibition reaction mechanism of the untreated and acylated flavonoids. The competitive inhibitors are those most similar to the substrate and they, therefore, occupy the active site. The occupation by the inhibitor thus prevents the substrate from connecting to the active site of the enzyme. Whenever [S] greatly exceeds [I], the probability of an inhibitory molecule connecting to the enzyme is minimized and the reaction will show a normal Vmax value. That effect on the increase in the apparent Km, together with the absence of any effect on the Vmax, is the diagnosis for competitive inhibition and it is readily revealed in the double reciprocal plots. To compare the inhibitory efficiency of the compounds in regard to XO, the Vmax/Km ratios provide better evidence. As seen in the results, the Vmax/Km ratios were higher when acylated flavonoid derivatives were used as inhibitors compared to similar untreated flavonoids.Given that the ,That finding is in agreement with an earlier study that evaluated the inhibitory effect of apigenin, quercetin, myricetin, genistein, and isovitexin on XO activity and reported that they all performed as competitive inhibitors50 values are displayed in x when y = 50 (IC50). It can be seen that the acylation reaction led to a reduction in the IC50 for the three acylated derivatives. Naringin octanoate showed the greatest inhibitory effect on XO activity (IC50 = 110.35 μM), followed by naringin decanoate (IC50 = 117.51 μM) and hesperidin decanoate (IC50 = 198.96 μM), whereas the standard inhibitor of xanthine oxidase  had an IC50value of 14.67 μM.The XOI (%) as a function of flavonoid concentration and the IC50 values of 27 and 61 μM, respectively)50 values of saturated esters with the decrease in the carbon chain length Higher inhibition activities were reported for isoquercitrin oleate (C18:1), which presents an unsaturation in the acyl group, when compared to its saturated analogue  and the flavonols  are capable of inhibiting the activity of this enzyme even when they are only present in low concentrationsBased on the structure of flavonoids, a method for predicting ICk) was measured for all prepared compounds as a basic empirical determination of hydro- or lipophilicity . That high partition coefficient of quercetin in an octanol/water system when compared to its glycosylated form has already been reportedThe variety of substituents on the flavonoid molecules largely influence their physicochemical properties such as dipole moment or hydrophobicity, and thus determine the partitioning into lipid membranesFlavonoid acylation, as has been described, provides a useful tool for flavonoid ester formation with improved characteristics. The results suggest that selective enzymatic synthesis of acylated flavanone derivatives catalysed by CALB lipase may represent a new approach to the production of XO competitive inhibitors with greater lipophilicity. The most potent XO inhibition was observed in naringin derivatives with octanoic and decanoic acids (naringin octanoate and naringin decanoate). The results provide the basis of the kinetics of the interaction mechanisms of acylated flavonoid derivatives with XO that may lead to the development of potential new drugs for XO inhibition. This approach also enables the use of flavonoid fatty acid esters in oil-based systems. Thus, these acylated derivatives are promising candidates to be used in pharmaceutical, cosmetic, and nutritional preparations for preventive and/or therapeutic purposes.


The mitotic checkpoint  is a signaling pathway that ensures faithful chromosome segregation. Mitotic checkpoint proteins inhibit the anaphase-promoting complex (APC/C) and its activator Cdc20 to prevent precocious anaphase. Checkpoint signaling leads to a complex of APC/C, Cdc20, and checkpoint proteins, in which the APC/C is inactive. In principle, this final product of the mitotic checkpoint can be obtained via different pathways, whose relevance still needs to be fully ascertained experimentally. Here, we use mathematical models to compare the implications on checkpoint response of the possible pathways leading to APC/C inhibition. We identify a previously unrecognized funneling effect for Cdc20, which favors Cdc20 incorporation into the inhibitory complex and therefore promotes checkpoint activity. Furthermore, we find that the presence or absence of one specific assembly reaction determines whether the checkpoint remains functional at elevated levels of Cdc20, which can occur in cancer cells. Our results reveal the inhibitory logics behind checkpoint activity, predict checkpoint efficiency in perturbed situations, and could inform molecular strategies to treat malignancies that exhibit Cdc20 overexpression. Cell division is a fundamental event in the life of cells. It requires that a mother cell gives rise to two daughters which carry the same genetic material of their mother. Thus, during each cell cycle the genetic material needs to be replicated, compacted into chromosomes and redistributed to the two daughter cells. Any mistake in chromosome segregation would attribute the wrong number of chromosomes to the progeny. Hence, the process of chromosome segregation is closely watched by a surveillance mechanism known as the mitotic checkpoint. The molecular players of the checkpoint pathway are well known: we know both the input , and the output . However, we do not exactly know the path that leads from the former to the latter. In this manuscript, we use a mathematical approach to explore the properties of plausible mitotic checkpoint networks. We find that seemingly similar circuits show very different behaviors for high levels of the protein targeted by the mitotic checkpoint, Cdc20. Interestingly, this protein is often overexpressed in cancer cells. For physiological levels of Cdc20, instead, all the models we have analyzed are capable to mount an efficient response. We find that this is due to a series of consecutive protein-protein binding reactions that funnel Cdc20 towards its inhibited state. We call this the funneling effect. Our analysis helps understanding the inhibitory logics underlying the checkpoint, and proposes new concepts that could be applied to other inhibitory pathways. Cdc20 becomes active, chromosome segregation takes place and now has a high chance of being executed correctly , a second where Cdc20 and MCC2 compete for APC/C [(ii) competitive inhibition], and finally a third which is the combination of the first two [(iii) combined model]. We showed that all three models displayed the same behavior when Cdc20 is limiting compared to APC/C and Mad. However, their behavior differs radically when Cdc20 is in excess. While the sequential inhibition model (i) can buffer extra Cdc20, competitive inhibition (ii) and combined model (iii) fail to do so. Interestingly, the very presence of free MCC2 suffices to turn the robust sequential model into a system sensitive to Cdc20 overexpression.In particular, we analyzed three networks: one where APC/CCdc20 than for free Cdc20 in order to efficiently avoid APC/CCdc20 formation. Our analysis confirms that allowing free MCC2 formation creates sensitivity to high levels of Cdc20. Noticeably, however, our data show that free MCC2 formation is compatible with a mitotic checkpoint response for a large range of measured Cdc20 levels in physiological conditions , this topological difference between the activating and inactivating branches of the mitotic checkpoint pathway results in Cdc20 accumulation in the inhibitory pathway Click here for additional data file.S1 TableWe omitted studies that reported: (i) Mad1 values higher than Mad2 (not leaving any free Mad2), (ii) values that could not easily be converted to molar concentrations, or (iii) stoichiometries that were widely different from other studies. Both absolute (abs.) and relative (rel.) amounts are shown. Relative amounts are normalized to Mad3. Values of free Mad2 are calculated as Mad2 − Mad1. References can be found in (PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigD = 0.01 (A) and KD = 0.1 (B) on the Mad-normalized scale. Analytical solutions for low Cdc20 correspond to eqs 23–26  and eqs 45–49 (competitive inhibition and combined model); analytical solutions for high Cdc20 are obtained from eqs 29–31  and eqs 61–63 (competitive inhibition and combined model) in Comparison between the analytical solutions for the simplified models (dashed lines) and the numerical solution for the full models Click here for additional data file.S3 Fig(A) Numerical simulations for the steady state concentrations of APC/CMCC2 and APC/CCdc20 for different total Cdc20 concentrations in the competitive inhibition model (ii), when assuming either the same or different KDs for APC/CCdc20 and APC/CMCC2 formation . (B) Robustness of model behavior under parameter variations. The ratio of APC/CCd20 to APC/CMCC2 at Cdc20total = 1 and Cdc20total = 4 was evaluated for 1000 randomly generated parameter sets. In each set, parameters were independently drawn from a log-normal distribution set-value times 10) that roughly varies between 0.5 and 2 times the set-value. (C) Numerical simulations for the steady state concentrations of APC/CMCC2 and APC/CCdc20 for different total Cdc20 concentrations in all three models, either assuming a combined species Mad, which combines both Mad2 and Mad3 (solid lines), or assuming sequential Mad2 and Mad3 binding to Cdc20 (dashed lines).(TIF)Click here for additional data file.S4 FigNumerical simulations for the steady state concentration of each species, dependent on the total Cdc20 concentration for each of the networks (i), (ii), and (iii); similar to (TIF)Click here for additional data file.


Thus, inappropriate or unregulated activation of mast cells and basophils through antigenic crosslinking of FcεRI can have deleterious, sometimes deadly, consequences. Accordingly, FcεRI has emerged as a viable target for the development of biologics that act to inhibit or attenuate the activation of mast cells and basophils. At the forefront of these strategies are (1) Anti-IgE monoclonal antibody, namely omalizumab, which has the secondary effect of reducing FcεRI surface expression, (2) Designed Ankyrin Repeat Proteins (DARPins), which take advantage of the most common structural motifs in nature involved in protein-protein interactions, to inhibit FcεRI-IgE interactions, and (3) Fusion proteins to co-aggregate FcεRI with the inhibitory FcγRIIb. This review presents the published research studies that support omalizumab, DARPins, and fusion proteins as, arguably, the three most currently viable strategies for inhibiting the expression and activation of the high affinity FcεRI on mast cells and basophils.Allergies and asthma are a major cause of chronic disease whose prevalence has been on the rise. Allergic disease including seasonal rhinitis, atopic dermatitis, urticaria, anaphylaxis, and asthma, are associated with activation of tissue-resident mast cells and circulating basophils. Although these cells can be activated in different ways, allergic reactions are normally associated with the crosslinking of the high affinity Fc receptor for Immunoglobulin E, FcεRI, with multivalent antigen. Inflammatory mediators released from cytoplasmic granules, or biosynthesized  Allergic disease refers to a variety of disorders that include seasonal allergies, atopic dermatitis, urticaria, life-threatening anaphylaxis reactions to food, and allergic asthma. Curiously, the incidence of allergic disease has increased dramatically in recent decades, and continues to rise in developed countries. Allergies and asthma are among the most prevalent chronic diseases worldwide , 2. The βγ2, is expressed predominantly on tissue-resident mast cells and circulating basophils , FcεRI, with multivalent antigen . High afasophils . Howeverasophils , bronchiasophils , 7, Langasophils , 9, dendasophils , 11, monasophils , and eosasophils , neutropasophils –16.βγ2 expressed on mast cells and basophils is predominantly involved in signal transduction leading to mast cell and basophil activation or degranulation, whereas αγ2 on antigen presenting cells is mostly involved in IgE-FcεRI internalization.Binding of IgE to FcεRI on mast cells and basophils enhances FcεRI expression –21. It iThe role of FcεRI as the primary activator of mast cells and basophils leading to the release of allergic/inflammatory mediators resulting in IgE-mediated immediate hypersensitivity reactions and allergic inflammation is well-documented . Accordiin vitro study with in situ-matured mast cells from human skin demonstrated that IgE-dependent enhancement of FcεRI on human skin mast cells was both prevented and reversed by omalizumab  is a humanized anti-IgE mouse monoclonal antibody that is FDA-approved for the treatment of mild to severe allergic asthma and chronic spontaneous urticaria –26. Omalalizumab . In thisalizumab , 33. Thealizumab . Human sThe efficacy and safety of omalizumab as treatment against allergic asthma and urticaria has clearly been demonstrated, including as an add-on therapy with traditional treatments such as glucocorticoids , 24. TheOmalizumab as an adjunct to allergen immunotherapy (AIT) against IgE-mediated food allergy and allergic asthma is also currently under investigation –50. The Other anti-IgE antibodies have also been developed and tested including Ligelizumab (QGE031), Quilizumab (MEMP1972A), XmAb7195, and MEDI4212 that might provide additional opportunities for anti-IgE therapy in the future . To dateDARPins (designed ankyrin repeat proteins) are a class of small (14–21 kDa) binding proteins comprised of a varying number of stacked ankyrin repeat domains , which a4-Ser]4. A bispecific DARPin (30/85) was identified as being capable of simultaneously binding FcεRIα at both epitopes with affinity for FcεRIα greater than that of IgE. In in vitro studies, DARPin 30/85 blocked IgE binding to FcεRI, and inhibited IgE-induced degranulation of human FcεRIα-transfected RBL-2H3 cells to a similar extent as omalizumab. In a similar study  biosynthesis , had reached the primary end point in two Phase III trials for the treatment of neovascular age-related macular degeneration (AMD). In two trials, Abicipar pegol demonstrated non-inferiority to the approved anti-VEGF ranibizumab (Lucentis®). Of significant concern, however, was a significantly greater incidence of ocular inflammation with Abicipar pegol than Lucentis®. Allergan is expected to file Abicipar pegol with the FDA in early 2019. Thus, whether DARPins are safe and efficacious in humans is currently being determined.in vivo study utilizing passive and active immunization of mice determined that FcεRI-FcγRIIb crosslinking contributed significantly to the inhibition of IgE-mediated anaphylaxis by IgG blocking antibodies particularly under low concentrations of IgG blocking antibody 3 linker 3 linker. mGE was shown to inhibit IgE-dependent degranulation and cytokine production from wild type but not FcγRIIb-deficient mice BMMCs. mGE also inhibited in vivo passive cutaneous and systemic anaphylaxis in mice, with extended protection. Conversely, mGE treatment increased FcγRIIb phosphorylation and its association with SHIP and SHP1/2 phosphatases.Over the years, Fcε-Fcγ fusion proteins to co-aggregate FcεRI and FcγRIIb have been investigated. One of the earliest bi-functional fusion proteins that was engineered, termed GE2, is comprised of the hinge-Cγ2-Cγ3 domains of the human IgG Fc and Cε2-Cε4 domains of human IgE Fc connected by a 15 amino acid  allergen in Rhesus monkeys that were naturally allergic to the D. farina allergen. In a later study, Mertsching et al.  was reported to have an IgE-binding affinity 5.3-fold greater than omalizumab, and 400 times greater binding affinity for FcγRIIb due to mutations in its Fc region . XmAb7194-Ser)3 linker to a human IgG1 Fc region. DE53-Fc, as it was named, was shown to not be anaphylactogenic, and inhibited allergen-induced activation of basophils in whole blood samples from allergic donors. In a subsequent study (4 biosynthesis in human primary basophils and huFcεRIα-expressing mouse BMMCs in vitro, and decreased in vivo passive systemic anaphylaxis induced in huFcεRIα transgenic mice. This study demonstrated that FcγRIIb-mediated inhibition of degranulation requires direct ligation with FcεRI, and that DARPins, at least D11_E53, could safely be applied to animals to inhibit anaphylaxis.DARPins have also been used to co-aggregate FcεRI and FcγRIIb. Eggel et al.  generatent study , a DE53-nt study  generateThe dramatic increase in prevalence of allergies warrants additional research to develop new strategies and therapies to treat allergic disease. At the forefront are the anti-IgE monoclonal antibody omalizumab, DARPins, and fusion proteins that directly or indirectly alter FcεRI expression and activation. In order to maximize the use of omalizumab, additional clinical studies are needed to identify allergic diseases against which omalizumab could be effective beyond asthma and spontaneous urticaria. The development of newer anti-IgE antibodies could also have an impact. The development of DARPins hold the promise of targeting FcεRI or IgE with greater specificity and better efficacy than monoclonal antibodies without the hurdles associated with development of humanized monoclonal antibodies. As potential clinical therapeutics, DARPins also have the potential to reach a broader population since allotypic differences associated with the use of monoclonal antibodies might not factor in their development. However, safety issues regarding immunogenicity due to anti-DARPin antibodies and unwanted effects due to inhibiting positive effects of mast cell activation must be considered. Whether DARPins can supersede monoclonal antibodies remains to be determined. Harnessing the inhibitory properties of FcγRIIb to inhibit FcεRI with fusion proteins also shows promise as evidenced in pre-clinical studies with non-human primates. It is hoped that these strategies will lead to therapeutics that provide relief to the millions of people worldwide suffering from allergic disease.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Changes in Earth’s orbit set the pace of glacial cycles, but the role of spatial variability in the insolation forcing of global ice volume remains unknown. Here, we leverage the intrinsic dynamical information in empirical records to show that ice volume responded to summer energy at high northern latitudes, as predicted by Milankovitch theory. However, the external forcing of ice volume encompasses insolation signals with a wide range of orbital frequency content, and cannot be fully accounted for by a unique time series. Southern mid-latitude insolation forcing coincides with the position of the subtropical front and the westerlies, which have been implicated in Quaternary climate changes. Dominant forcing modes at northern mid-latitudes are anti-phased with the canonical Milankovitch forcing, consistent with ice volume sensitivity to latitudinal insolation gradients. This periodicity is clearly visible in global sea level reconstructions2 and is also mirrored in other climate indices such as global sea surface temperature3. Because Earth’s rotational axis and the shape of its orbit vary with similar frequencies as the glacial cycles, the orbital changes have been dubbed a pacemaker of Quaternary glacial-interglacial climate variability1. Hypotheses for the origin of the recurrent ice ages include obliquity, precession and combined orbital pacing of deglaciations by various mechanisms12. The extent to which latitudinal insolation acted as a dynamical forcing of late Pleistocene ice volume variability, however, has not been directly quantified.Earth’s climate and global ice volume oscillated in tune with changes in orbital geometry during the Quaternary period. Evidence of this relationship is derived from geochemical indicators in marine microfossils found in deep ocean sediment cores. Isotopic ratios in foraminiferal shells (tests) show that Northern Hemisphere ice sheets grew and receded periodically13. We test this prediction by quantifying the dynamical response of global ice volume to latitudinal insolation forcing. To achieve this, we target a recent reconstruction of global sea level (GSL)2 spanning the past 800,000 years. The advantage of using GSL over stacked benthic isotope records (e.g.14) is that it explicitly records global ice volume changes, and that it minimises bias due to temperature-driven fractionation and differences in oxygen isotopes between the Atlantic and Pacific basins2.In the Pleistocene, large paleo-ice sheets were located in the Northern Hemisphere and summer ablation is considered a key factor controlling ice sheet size. Milankovitch theory predicts that if insolation controls continental ice sheet dynamics, then northern latitude summer insolation plays a central role17. Hence, if latitudinally varying insolation was a dynamical influence on changes in ice volume, then information about this insolation forcing should be recoverable from the GSL record of ice volume variations. To test this assertion, we use a model-free time series analysis method, convergent cross mapping (CCM)19. This method is based on state-space reconstruction from time delay embedding, and measures the extent to which a forcing time series can be predicted from a response time series. CCM can detect causal coupling in non-linear dynamical systems18, making the method well suited for studying climate dynamics, where non-linear interactions are ubiquitous. We use CCM to test if GSL can predict latitudinal insolation reconstructions. If GSL significantly predicts the insolation time series at a given latitude, then there is empirical support for local insolation at that latitude contributing to the dynamical forcing of ice volume.The GSL time series does not simply record a response to insolation forcing, but contains information on greenhouse gases, ocean circulation, and other processes in the climate system of which ice volume itself is an active component. However, dynamical systems theory shows that one can reconstruct the dynamics of a system of unknown complexity from an observed time series of a single variable20 and varies with latitude and the choice of the threshold value21.Ice sheets are most sensitive to insolation during summer melting season. Choosing a meaningful insolation metric is thus crucial for investigating climate system responses to insolation forcing. Summer energy is defined as the sum of daily insolation over days of the year exceeding a specified insolation intensity thresholdWm−2 to 500 Wm−2 in 25 Wm−2 increments. Integrating summer insolation over low thresholds yields time series representing longer summer periods, including the full annual insolation. Higher thresholds, on the other hand, yield time series representing peak summers . A necessary condition for statistical detection of a causal response of ice volume to insolation forcing at a given latitude is that the prediction of summer energy time series from GSL is significant beyond their shared frequencies. This condition is tested by a null ensemble of amplitude-adjusted Fourier transform (AAFT) surrogates, which are constructed by randomising the data in a way that retains the autocorrelation function, or, equivalently, the periodogram of the original forcing time series. In order to resolve causal directionality, we compute CCM over a range of negative and positive time lags, and require that CCM skill must be stronger for negative lags  than for positive lags . If both the surrogate test and the lag test pass, there is statistical evidence of dynamical forcing of sea level by insolation at that latitude.CCM analysis yields three connected regions of positive prediction skill in latitude-threshold space Fig. . These tWm−2  is also predicted by GSL. At these thresholds, successful prediction occurs in both hemispheres 45–55° (included in NH2 and SH).Further, we detect significant prediction of summer energy by GSL in two continuous clusters in latitude-threshold space, one in each hemisphere . Thus, the detected couplings in our study capture not only direct, linear responses of ice sheets to specific insolation peaks, but also nonlinear, lagged effects which include modulation by other climatic factors such as greenhouse gases.We re-iterate that CCM does not make assumptions about properties or mechanistic behaviors of the ice sheets, or their interaction with other climate system components. Our approach only targets the intrinsic dynamical information in the observed GSL record, and estimates the strength of dynamical coupling over the entire 800 kyr interval covered by the GSL reconstruction. This way of looking at dynamical causality is fundamentally different from an event-based view27. However, Antarctic climate variability can also be explained by a local response to the duration of the Antarctic summer28. Both local and remote forcings might thus influence a given geographical region. Because of the uncertainty inherent in ice sheet reconstructions, the boundary separating regions of direct insolation influence from regions of indirect influence cannot be precisely resolved. The NH1 cluster represents mostly direct insolation forcing effects, but likely also some indirect effects for the southernmost forcing signals. Similarly, the northernmost forcing signals in NH2 may represent the direct effect of annual insolation on ice sheets at these latitudes.Causal pathways from local insolation to global ice volume vary with latitude. Different forcing scenarios overlap in their relative ratio of obliquity to precession variability Figs  and 3 anOur analyses indicate that Northern Hemisphere ice sheets respond to local summer insolation, but that insolation in the SH cluster Fig.  also con29. Variation in the dust supply to surface waters drives natural iron fertilisation and regulates the intensity of the biological pump30. This process could influence ocean stratification and venting of CO2 from the deep oceans, resulting in global climatic impacts. We also note that the 40–60°S latitudinal band coincides with the position of the oceanic subtropical and sub-Antarctic front systems and the mid-latitude westerlies31. It has been proposed that the position of the subtropical front relative to the southern tip of Africa modulates glacial climate through regulation of heat and salt exchange between the Indian and Atlantic oceans by the Agulhas leakage, affecting the strength of the Atlantic meridional overturning circulation33. In addition, the position of the westerlies and Southern Ocean sea ice dynamics may regulate venting of deep water and the release of stored CO2 to the atmosphere, which in turn affects ice sheets in the Northern Hemisphere34.There are several climatic processes operating at southern mid-latitudes that might be significantly influenced by local insolation. For example, the Patagonian ice sheet resides at these latitudes, and it has been hypothesized that Patagonian ice sheet dynamics affect the flux of dust over the Southern Ocean and Antarctica28, it is less affected by albedo and elevation feedbacks35. Its influence on global sea level on the time scales considered here could thus be negligible compared to the Northern Hemisphere ice sheets, which show much larger variability over the same time span.Our analysis shows no statistically significant effect of southern high latitude summer energy on global sea level. The response to direct insolation forcing at high southern latitudes may be too small in comparison to that of northern hemisphere ice sheets for it to be detectable beyond our chosen null hypothesis. This result may be understood in terms of the difference between the Arctic and Antarctic ice sheets. Because the Antarctic ice sheet mostly loses mass along the continent edges36, greenhouse gases34, changes in vegetation37, dust38 and glacial isostasy39. The explicit role of different mechanisms in the insolation-climate link, however, cannot be resolved with the global data sets used here. For regions in the Northern Hemisphere south of the maximal extent of the late Pleistocene ice sheets, the influence of local summer energy forcing on ice volume must necessarily be indirect. Overall, insolation forcing in the NH2 cluster is overall more precession dominated compared to further north in NH1  of negative lags (past affects future) had to exceed that of positive lags (future affects past):45, but limited our interpretation to causal directionality. Our lagged causality test  as the integer dimension strictly larger than twice the box-counting dimension17 of the reconstructed attractor, and required the false nearest neighbour (FNN) rate to be less than 0.01. We used the tseriesChaos47 and fractaldim48 R packages to estimate the minimal FNN  in the range q in the expression ρ is the median CCM predictive skill and L is the library size. The value of q was found through linear regression of the logarithmic transformation of the same equation, or CCM requires that the correlation between predicted and observed values increases with increasing library size18. Therefore, to claim significant forcing, we establish a distribution in the form of an ensemble of amplitude-adjusted Fourier transform (AAFT) surrogate time series50. These surrogates are randomized realizations of the original time series that preserve both the histogram and the frequency power spectrum (i.e. autocorrelation) of the original insolation forcing time series. Rejecting the null hypothesis thus implies that the dynamical coupling between GSL and the insolation cannot be fully accounted for neither by noise properties nor by shared frequencies.The upper limit of the CCM skill for a given analysis is determined by the coupling strength between the variables, but also by process noise51 where the ρccm of the data had to exceed the 99th percentile ρccm of the surrogate ensemble. We used 400 surrogates, and verified the results at selected threshold-latitude configurations using 1,000 surrogates.Rejection of the null hypothesis for each driver-response pair involved passing a one-sided rank-order test2 is aligned with the LR04 stack14, which is tuned to an orbitally forced ice sheet model. We constructed an alternative age model following the approach of ref.22, wherein a filtered GSL (band-pass filtered using a 22 ka Gaussian filter) was tuned to an equivalently filtered composite δ18O record from U/Th dated Chinese speleothems24. GSL was then aligned with the speleothem record by tiepoints determined from peaks and troughs in the band-pass filtered versions. The speleothem record goes back to 640 kyr; age control for the older parts of the GSL record was obtained by linear interpolation to a tie point on the LR04 stack at 787 kyr, close to the Brunhes-Matuyama boundary. In an effort to obtain an orbitally independent chronology, we constructed another GSL age model by tuning a North Atlantic SST composite proxy record to the speleothem record. The GSL record was then matched to the benthic δ18O records of the respective North Atlantic archives. This approach utilizes the close connection between millennial-scale North Atlantic climate and the intensity of the Asian Monsoon documented in several studies54. In the interval from 0 to 332 kyr we used the GICC05/NALPSpeleo chronology from ODP site 98323 with a benthic δ18O record from ref.55. In the interval from 332 to 553 kyr we used the SST record from IODP site U131356 and the corresponding benthic δ18O record57. In the interval older than 553 kyr, we used the abundance of the polar planktonic foraminifer Neogloboquadrina pachyderma sinistral and the benthic δ18O records from ODP site 98058. Age control beyond the reach of the speleothem composite was obtained by matching the ODP 980 data to the Epica Dome C methane record59 placed on a modified AICC2012 gas age chronology. The AICC201260 was modified by matching the methane record to the composite speleothem record from 554 to 627 kyr, and to two tie points at 783 and 792 kyr determined by Ar/Ar dated tephras and a δ18O record from an Italian lacustrine sediment sequence61, utilizing the close relationship between the atmospheric methane concentration, North Atlantic climate and Asian Monsoon intensity (e.g.63). Age models were constructed with Oxcal 4.364, using the P sequence65 and variable k66 options.We explored three different chronologies for the GSL record Fig. . The orihttps://github.com/kahaaga/Haaga_et_al_insolation.Code to reproduce all analyses and figures is available at


While there are several different animal models for use in the characterization of spinal fixation, none have emerged as a definitive model for comparative studies in spinal fixation methods. The purpose of this study is to establish morphometric data of porcine vertebrae and to characterize the feasibility of pedicle screw fixation in porcine spines for potential comparative human study.Four spines from 45 to 50 kg Hanford minipigs were cleaned of soft tissue and analyzed by computed tomography and dual-energy x-ray absorptiometry. Two 5 × 30-mm pedicle screws were placed in each vertebra and tested to failure using a combined moment-load protocol.2 (range 1.139–1.016). Force-to-failure demonstrated mean 1171.40 N (+ 115.34).Pedicle widths were measured from L6-T5. Widths ranged from 7.15 mm (T6) to 9.24 mm (T14). Posterior cortex to anterior cortex depth ranged from 25.9 to 32.6 mm. Mean bone mineral density was 1.0665 g/cmOur baseline morphometric and compositional data demonstrate that porcine vertebrae can serve as a useful model for comparative studies due to their similar pedicle widths and bone mineral density to the human vertebra. This biomechanical data could provide a baseline comparison for future studies. This study also suggests that the minipig could be a suitable model for comparative studies due to similarities in pedicle width and bone mineral density to the human vertebrae. There is no generally accepted single animal model for spinal fixation methods. Several different study animals have been used in the characterization of spinal fixation, including sheep , 2, cow If the minipig is to be a useful model for spinal fixation methods, measurements of bone mineral density (BMD) and pedicle screw force to failure in normal porcine spines would be necessary for baseline comparison to future fixation experiments. This leads to the current investigation into the feasibility of using the minipig to assess spinal fixation strategies.Many previous authors assessing the biomechanics of pedicle screw fixation used pure axial pullout to assess screw interface, but there are many questions about the clinical validity of these simple loading schemes. We have described previously a complex test method that simulates complex in vivo loading for pedicle screw fixation analysis .The goals of this pilot study are twofold: to establish morphometric and compositional data of the minipig thoracic and lumbar vertebrae for potential comparative human study and to characterize the feasibility of pedicle screw fixation in normal minipig spines for comparison with future studies.Four porcine spines, cleaned of soft tissue, were procured from the Sinclair Research Laboratory Farm . The spines were from adult male Hanford miniature swine aged 14 to 15 months, weighing 45 to 50 kg, which were sacrificed for reasons unrelated to this particular study. The lumbar regions of the whole vertebral columns were scanned with dual-energy x-ray absorptiometry (DXA)  to determine BMD. Specimens were then imaged with quantitative computerized tomography  to generate vertebral morphometry. Using Amira  visualization software, transverse pedicle isthmus width and distance from posterior to anterior cortex along the pedicle axis (AC depth) were measured for each vertebra.The vertebrae were separated from connective tissue for instrumentation. A small starting hole (approximately 3 mm) in the posterior cortex was created on each side with a rongeur, exposing cancellous bone. A pilot hole was made with a tapered Lenke probe. A titanium 5 × 30-mm (Synthes USS) fixed pedicle screw  was inserted into each pilot hole Fig.  and a 5.Θ = 45, and stabilized superiorly to prevent bending. The specimen was held in a custom apparatus which clamped the vertebral body similar to the complex loading previously described   Table . The lon2 ±.062 g/cm2 In each case, the failure loads were so high that the screws bent either just previous to or simultaneously with screw failure. Mean force to failure was 1171.40 N (± 115.43) Table . Once we2 (+ 0.110), Lunar 1.200 g/cm2 (+ 0.120), Norland Europe 1.085 g/cm2 (+ 0.115), and Norland US 1.164 g/cm2 (+ 0.162) [2 (± .0462 g/cm2), comparable to adult human reference BMD reported in the literature. This similarity is a favorable factor in determining whether the minipig would be a suitable study model. Our findings also agree with others who have investigated porcine BMD.The mean and standard deviation of BMD values for young normal adults have been previously published in the literature. Simmons et al. reported the adult human reference BMD for each of the principal DXA manufacturers. Reported values were as follows: Hologic 1.079 g/cm+ 0.162) . The adu2 [2 in live, anesthetized, 60 kg pigs [Lee et al. found similar BMD to our study in their in vitro porcine model of osteoporosis with a mean BMD of 1.189 ± .05 g/cm2 . Mitchel kg pigs . The sim kg pigs . In a se kg pigs . This raAlong the same lines, some animal model research has focused on coated pedicle screws that may improve fixation at the bone-implant interface , 24–26. The pedicle morphometry data in our study demonstrate that instrumentation with adult human-sized pedicle screws is possible. Lumbar and thoracic vertebrae had similar pedicle isthmus widths to the human widths measured by Zindrick et al. . Mid to The vertebrae in our study demonstrated an extremely high force to failure. In this pilot study, the titanium rod would often bend before screw failure. By providing enhanced rod reinforcement and defining failure as the relative movement of the screw suggesting displacement within the trabecular meshwork, we were able to achieve consistent force to failure data. Aerssens et al. found that porcine bone had a higher bone mineral content and higher yield stress necessary for trabecular compression than commonly seen in humans . In a huOur study has several limitations. The high failure loads exhibited in this study were quite notable, raising the question of whether mechanical findings in the healthy minipig would be directly predictive of implant behavior in humans. These data could be the result of differences in the microarchitecture of the porcine spine or a function of the smaller vertebral bodies. However, even if the force to fixation failure is consistently higher than in the adult human, the relative patterns of fixation between healthy bone and induced osteopenia/osteoporosis may still be very instructive. Another limitation is the shorter AC depth in the mid to upper thoracic vertebrae which precluded pedicle screw instrumentation as our smallest available screw length was 30 mm. Since porcine spines contain up to 15 thoracic and up to 6 lumbar vertebrae, we found 8 to 10 vertebrae from each spine in our study that would accommodate a pedicle screw, more than adequate to conduct studies in spinal fixation. Given the morphometric variation based on spinal levels, it would seem very important that any future studies specify exactly which porcine vertebrae would be used in order to draw conclusions about spinal fixation in humans. A final limitation to this pilot study was the small sample size of four porcine spines. Although our failure data and morphometric analysis in this pilot generated consistent measurements, a larger study with additional specimens may provide additional detail beyond our initial characterization.This pilot study suggests that the minipig could be a suitable model for comparative studies due to similarities in pedicle width and BMD to the human vertebrae. These measurements and biomechanical data could provide a useful reference for future studies of spinal fixation methods considering the minipig as a model.


In many sensory systems, different sensory features are transmitted in parallel by several different types of output neurons. In the mouse olfactory bulb, there are only two output neuron types, the mitral and tufted cells (M/T), which receive similar odor inputs, but they are believed to transmit different odor characteristics. How these two neuron types deliver different odor information is unclear. Here, by combining electrophysiology and optogenetics, it is shown that distinct inhibitory networks modulate M/T cell responses differently. Overall strong lateral inhibition was scarce, with most neurons receiving lateral inhibition from a handful of unorganized surrounding glomeruli (~5% on average). However, there was a considerable variability between different neuron types in the strength and frequency of lateral inhibition. Strong lateral inhibition was mostly found in neurons locked to the first half of the respiration cycle. In contrast, weak inhibition arriving from many surrounding glomeruli was relatively more common in neurons locked to the late phase of the respiration cycle. Proximal neurons could receive different levels of inhibition. These results suggest that there is considerable diversity in the way M/T cells process odors so that even neurons that receive the same odor input transmit different odor information to the cortex. However, it is still unclear how neurons that share similar inputs vary in their output information, and which mechanisms govern this process.In many sensory systems, sensory information is processed locally and then transmitted to higher cortical regions in parallel by different cell types. In the mammalian olfactory bulb (OB) the two projection neurons, the mitral and tufted cells (M/T cells), are also believed to form two parallel streams of information. This notion has been supported by recent findings showing that mitral and tufted cells differ in some of their odor response properties and project to distinct cortical regions6.There are at least two possible ways the response of M/T cells can be dissociated: lateral inhibition and back projection modulation. Recent work has shown that back projections can decorrelate mitral cell odor responses, but less in the case of tufted cells17. These lateral inhibition circuits can modulate M/T cell odor responses so that each cell type carries a different component of the odor stimulus that reflects the response of its main glomerulus and the surrounding glomeruli that also responded to the odor. To better understand how this takes place, the frequency, strength and spatial distribution of lateral inhibition and the way it differentially affects different cell types need to be determined. Despite extensive research in this field, these parameters are not fully known. For example, it is currently unknown how many surrounding glomeruli each M/T cell receives inhibitory inputs from, and whether each of these connections is strong enough to change the neuron’s activity. It also remains unclear whether all projection neurons receive lateral inhibition and whether there are differences in the level of lateral inhibition between cell types.Lateral inhibition is a prominent motif in many sensory systems, in which neurons receive inhibition from adjacent cells at the same processing level. In the mammalian OB, M/T cells receive their main excitatory input from one glomerulus via their apical dendrite and can receive inhibitory input from several surrounding interneurons in the glomerular, granular and the external plexiform layersin-vivo in anesthetized animals using odor stimulation combined with imaging and electrophysiology techniques. These have led at times to contradictory conclusions regarding strength, frequency and spatial distribution. One study found that M/T cells exhibit a patchy center-surround organization reminiscent of lateral inhibition organization in the retina in which inhibitory glomeruli tend to cluster together close to the excitatory glomerulus18. By contrast, more recent studies have reached the opposite conclusion that M/T cells receive inhibitory inputs from neurons randomly scattered or semi-organized over the OB surface20. However, odor typically excites several glomeruli and may also directly inhibit some glomeruli due to receptor-odorant interactions or local circuits; hence, using odors to elucidate the inhibitory contribution of each glomerulus and their spatial organization is an indirect form of assessment. In-vitro studies typically cannot examine all spatial connections21. Thus, to date there is no direct estimation of the amount, frequency and spatial extent of lateral inhibition in the olfactory bulb in-vivo.Several studies have attempted to characterize lateral inhibition 23 we used mice that express Channelrhodopsin2 (Chr2) in the olfactory sensory neurons (OSNs) such that optical stimulation resembles odor activation24. We found that M/T cells can be divided into at least three functional groups based on the amount and strength of lateral inhibition they receive. These results support a model in which there are several classes of output neurons, each of which receives different levels of surround inhibition, which provides a mechanism for sending different odor features to the cortex in several parallel pathways.Here we used precise optogenetic stimulations in a transgenic mouse strain to systematically characterize the frequency, strength and spatial distribution of lateral inhibition in different cell types in the mouse OB. Unlike a previous optogenetic approach that used mice expressing Channelrhodopsin2 (ChR2) in M/T cellsAll surgical and experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Bar Ilan University guidelines for the use and care of laboratory animals in research, and were approved and supervised by the Institutional Animal Care and Use Committee (IACUC).Twenty-three transgenic male and female mice aged 3 to 6 months were used. The animals were housed in a group cage and received no experimental treatment except genotyping. They were maintained in a reverse light/dark cycle and all experiments were performed during their dark cycle.24.We used the Omp-ChR2 transgenic mouse line, in which Channelrhodopsin is expressed exclusively in OSNsAnimals were first anesthetized with ketamine/medetomidine  and then fixed in a stereotaxic frame. The bone overlying the dorsal OB was removed. Additional anesthesia was administered as needed . The animal’s body temperature was maintained at 37 °C using a homeothermic blanket system (Harvard Apparatus).Ketamine is a known antagonist of NMDA receptors, which might hinder inhibition by granule cells. It is therefore possible that the result here reflects inhibitory circuits that are less affected by ketamine .The spiking activity of neurons was recorded extracellularly using tungsten electrodes . Neural signals were amplified and filtered at 300–5,000 Hz (AM-Systems 1800), digitized at 40 kHz  and stored on a computer. Spike signals were sorted offline using MClust software in MATLAB (written by A.D. Redish).25. M/T cells are the largest and most abundant neurons at these depths and therefore, as in previous studies, blind recordings at 200–400 µm will mostly result in recordings of M/T cells32 as we also recently demonstarted26. It is unlikely that we recorded granule cells as previous studies have noted that granule cells are not visible to extracellular electrodes35. To further verify that we were not recording granule cells, we lowered the electrode to the granule cell layer (~500 µm) and found that no neural activity could be recorded. Furthermore, there was no response to strong light stimulation when the electrode was in the granule cell layer. That said, we cannot completely overrule the possibility that some recorded neurons are not M/T cells.M/T cells are the sole projection neurons in the olfactory bulb (OB) and are located at a depth of ~200–400 µm from the OB surfaceThe electrode was lowered with a micromanipulator  perpendicular to the OB surface. Neurons were recorded from the dorsal bulb at a depth of ~200–400 µm.36. In brief, M/T cell spike activity was recorded extracellularly in anesthetized mice while optogenetically activating the surrounding glomeruli in the exposed dorsal surface of the OB. Precise spatial control of optical stimulation was achieved by projecting two-dimensional light patterns onto the dorsal surface of the OB using digital micromirror technology (duration 100 ms) coupled to an optical imaging system. This resulted in an image where each projected pixel corresponded to ~11 µm. Optical stimulation was controlled with the MATLAB psychophysical toolbox.Optical stimulation of the OB has been described in detail in38, optical stimulation was randomly applied with respect to respiration cycles, with typically 20 repetitions of each spot stimulation. On average 98.34 ± 52.99 spots were stimulated for each recorded neuron.To avoid possible bias due to respiration phase39 and ranged from 10–20 mW/mm2 as measured by a photometer (Thorlab PM100D). The expression of Chr2 in the OMP-Chr2 mice varies which result in different light power required to elicit a clear response near the recording electrode. Spot sizes ranged from 55 to150 µm . We scanned the full extent of the exposed dorsal OB divided into an N by M non-overlapping spots. N and M were determined by the size of the craniotomy. The result did not change when we only used experiments with the same spot sizes (e.g. 110 µm).The light intensity was set to obtain excitatory responses40 activation of axons of passage is more common when stimulating the anterior parts of the OB. To avoid this, in a few neurons in which there was a clear response from the anterior OB parts we tended to focus our light stimulation on the more posterior parts of the OB and ignored the anterior parts.Finally, due to the spatial organization of the axons of passage Data from all animals tested were included in the analysis. Spots were considered inhibitory or excitatory if the mean firing rate across all trials was significantly (P < 0.05) higher or lower than the mean baseline activity 200 ms before light onset. We termed spots that caused a significant reduction in firing rate as strong inhibitory spots. Although statistical significant does not necessarily imply large normalized effect size because statistical significant depends also on the sample size and variance, since in our experiments all spots were stimulated for a similar number of times the P values obtained are comparable to each other and can be used to assess the normalized effect size. Therefore a significant inhibitory spot is likely to have relatively stronger inhibitory effect than a non-significant inhibitory spot  we were likely to have several false positive detections. To estimate the number of false positive detected spots we repeated the same analysis but shuffled the spike count before and after light stimulation of each spot for all trials and recomputed the heat map and the rate of spots that evoked a significant change in firing rate. This shuffling procedure assumes that light stimulation has no effect on the evoked firing rate.36. This is because we used a smaller spot size in the range of the size of a glomerulus and scanned the whole dorsal OB in the current study. Peri-event time histograms (PETHs) were smoothed using a Gaussian filter (s.d.: 20 ms).The rate of significant lateral inhibition spots reported in this study is slightly lower than what we reported in2. To calibrate our respiration measurements with those conducted in2 we compared the respiration signals obtained by a thermocouple, a piezoelectric sensor  and a pressure sensor (1 INCH-Dx-4V-MINI). The piezoelectric and pressure sensor signals were very similar, with 5.9 ± 2.1 ms jitter (mean ± s.d). The thermocouple signal, however, was ~26.3 ± 8.6 ms before the pressure sensor signal. We therefore shifted our respiration estimates by 26.3 ms to be consistent with the beginning of respiration as defined in2.We recorded respiration using a thermocouple. Because the transition between inhalation and exhalation was the most salient feature in the respiration recording trace, we defined a respiration cycle as being from the beginning of exhalation to the beginning of the next exhalation. The beginning of respiration was thus the same as inTo compute the neurons’ preferred phase we aligned all respirations to the beginning of exhalation and constructed a PETH. The time of the PETH peak relative to the mean respiration duration was computed and used as the neuron’s preferred phase. We considered a neuron to be phase-locked only if the spike time distribution was significantly different from a uniform distribution  and if the peak firing rate was higher from the minimum by at least three spikes/sec. These thresholds detected all phase-locked neurons. Changing the threshold parameters (e.g. setting P < 0.05 or not setting a three spikes/sec difference limit) did not change the results.Not all recorded neurons were locked to the respiration phase. However, the result of this study did not change if we restricted our analysis to only neurons that were significantly locked to respiration and we therefore did not exclude neurons from our analysis when the neuron preferred phase was not relevant to the analysis.41. About 3.6% of the light stimulated spots that were not adjacent to the recording electrode also showed significant excitation. This however was expected from estimates of the false discovery rate  which indicated that activation via axons of passage was infrequent in our setup .We first characterized the lateral inhibition input of M/T cells in a relatively systematic and comprehensive way. We extracellularly recorded the spiking activity of M/T neurons in the OB while sequentially optically stimulating all exposed glomeruli . We found that contrary to what might be expected, inhibitory responses were quite rare. First, in ~29% (38/132) of the recorded neurons none of the light-stimulated spots elicited a significant reduction in baseline firing rate. Second, on average, only 5.3% ± 8.6% (mean ± s.d.) of the surrounding light-stimulated spots elicited a significant inhibition . However, some of the light-activated spots evoked weak inhibition that might not have crossed this sharp and somewhat arbitrary threshold (P = 0.05). Thus, this procedure is biased towards detecting spots that exert a relatively strong inhibition and is more likely to fail when there is weak inhibition.Examining more carefully the mean reduction in baseline activity caused by the none-significant inhibitory spots . This method provides a stronger statistical power as it pools all average spot responses and therefore can capture small reductions in baseline activity as long as it is caused by a substantial number of spots. To see this, consider the response map example shown in Fig. To estimate the number of surrounding glomeruli that elicited inhibition (weak or strong) we computed the percentage of spots that caused any reduction in baseline firing rate. We found that the distribution of the percentage of spots that caused any reduction in baseline activity was normally distributed around 62% and could reach up to 80% in a few M/T cells Fig. . The perWhile on average significant lateral inhibition was not common there was substantial variability in the number of spots that elicited significant inhibition. Specifically, a large group of recorded neurons did not receive significant inhibition from any of the surrounding glomeruli whereas a small group received inhibition from as much as 20–51% of the surrounding glomeruli Fig. . Five ne42. Tufted cells seem to fire during the first half of the respiration cycle (π/8 to π radians), while mitral cells tend to fire during the second half of the respiration cycle (π to π/8 radians). Consistent with studies that have examined neurons’ preferred phases in a large number of M/T cells43 we also observed that the recorded neurons’ preferred sniff phases were widely distributed across the sniff cycle .We first noticed that all neurons receiving significant lateral inhibition from many spots (e.g. five neurons receiving inhibition from more than 25% of the surrounding spots) and almost all neurons receiving relatively dense levels of surround inhibition (6 out of 7 neurons receiving inhibition from more than 20% of the surrounding spots) were phase-locked to a relatively narrow band in the first half of the respiration cycle . This may imply that the differences we found are not between mitral and tufted cells but rather between early and late phase spiking cells. The differences between these two neuron types cannot be explained by differences in their baseline firing rates because the EP and LP baseline firing rates were similar .These findings suggest that the percentage of surrounding glomeruli eliciting strong lateral inhibition is more variable in EP cells. A subset of EP cells receives inhibition from a large number of surrounding glomeruli whereas LP neurons tend to receive strong inhibition from only a relatively small number of surrounding glomeruli Fig. .We next examined whether EP and LP cells differed in their level of weak inhibition. To address this, we focused on neurons that have a small number of significant inhibitory spots . This allowed us to examine neurons which mostly receive weak inhibition or no inhibition. Comparing these neurons’ percent of spots that caused any reduction in baseline activity and their preferred phase showed that LP cells tended to have more spots that caused a reduction in baseline activity  or comparing the percent of spots that caused any reduction in baseline activity (i.e. weak and strong inhibition). We found that inhibition similarity was independent of the distance between the neurons as even proximal neurons could receive dissimilar amounts of inhibitory inputs  which might miss few strong inhibitory changes that are highly variable or include weak inhibitory changes that have low variance. Therefore one should interpret ‘strong’ as relating to the normalized effect size . Furthermore, Fig. 45 and/or activate cortical processing. Thus, the group of neurons that do not receive any weak or strong lateral inhibition can signal the brain that an odor has been detected without any attenuation or delays in their firing. Once the presence of an odor is detected, decorrelating the responses can improve the animal’s ability to identify and discriminate odors, as was shown in46. This may be the role of the EP cells and some LP cell that receive strong lateral inhibition. Finally, the group of LP cells that receive weak lateral inhibition from many surrounding glomeruli could act to maintain responses across several odor concentrations. Increasing odor concentration typically increases the neuron’s firing rate but also increases the response of the surrounding glomeruli. When there are many inhibitory connections, the increase in response from the increase in odor concentration can be canceled out or attenuated by inhibition from the surrounding glomeruli, thus resulting in a similar response to several odor concentrations.The existence of several types of lateral inhibition circuits suggests that lateral inhibition plays several roles in addition to decorrelating odor responses. One possible role of the subgroup of neurons that do not receive lateral inhibition could be to detect the existence of an odor. Quick detection at a low odor concentration is crucial because it can direct the animal towards (or away) from an odor source and can be used to immediately change sniffing strategy24. To estimate the possible contribution of activation of axons of passage to our analysis, we compared the reduction in spike count caused by all the stimulated spots anterior to the excitatory spots to those posterior to the excitatory spots. The reduction in firing rate caused by stimulating the posterior spots did not differ significantly from the reduction caused by stimulating the anterior spots . All this suggests that under the experimental conditions reported here, the effect of activating axons of passage on neural activity was negligible.Our results are based on optogenetic stimulations. Although optogenetics is well suited to revealing the contribution of specific glomerulus compared to odor or electrical stimulation, it has several limitations. Similar to electrical stimulation, optogenetic stimulation of the OSNs may activate axons of passage; hence, activating one glomerulus can cause the activation of other more posterior glomeruli. While this may have occurred here, it is unlikely to have significantly biased our results. This is because only very few spots outside the primary spatial zone elicited a statistically significant excitation in M/T cells , indicating minimal fiber stimulation. This is consistent with a previous study also reporting that the threshold for activating M/T cells was much lower in glomeruli than in axons of passageAnother limitation of light  stimulation is that light may only reach parts of a glomerulus or can hit several neighboring glomeruli. This can result in insufficient activation or activation of several glomeruli. While possible, we consider that this problem did not skew our results substantially due to the relatively large number of surrounding spots tested  which should have averaged out instances of activation of too many or too few glomeruli.20. We also found that proximal neurons could receive completely different levels of inhibition . However, it is worth noting that previous examination of lateral inhibition under urethane anesthesia also reported low rates of lateral inhibition22. This might suggest that the effect of granule cells on M/T cell baseline firing rates is not very strong. Consistent with these results, recent studies reported that silencing granule cells did not substantially affect M/T cells activity49. However, direct examination of granule cells effect on M/T cells in a wake mice is required to better understand their role in lateral inhibition and other odor processing tasks.Finally, our experiments were conducted in ketamine-anesthetized animals. Ketamine is a known antagonist of NMDA receptors, which might hinder inhibition by granule cellsThus overall, despite extensive research and seminal discoveries, the mechanisms by which odors are encoded in the OB are still elusive. Our results show that lateral inhibition can differentially shape the responses of the different OB projections neurons into at least three possible parallel streams. These results have important implications for the ways in which odors are encoded in the OB, how different odor features are transmitted in parallel to the cortex, and how the cortex processes odor information.


The nutritional traffic light label (NTLL) has become one of the most used Front of Package labels (FOP’s) around the world, for its simple and easy to understand graphical system. In Ecuador, this labelling system has recently been implemented; then, this research aims to evaluate the use and knowledge of NTLL and its effectiveness as a public health promotion strategy.In a cross-sectional study at two different urban supermarkets in Quito-Ecuador, a survey was conducted in 73 participants to inquire about knowledge, perspectives and purchasing habits regarding the NTLL. Objective data obtained from pictures of the participants’purchase was compared with subjective data obtained from the survey. For categorical variables, Chi square or Fisher’s Exact test were used and variables with a statistical significance at α = 0.1 were included in multivariate logistic regression models.p = 0.007) or knowledge level (p = 0.001) were found. A significant association was also found between the refered use of the NTLL and the shopping influencing factor (p = 0.02). In the multivariate analysis an association between knowledge of the NTLL and observed use was found only when adjusted for the supermarket (p = 0.038).88.7% of participants knew about the NTTL. 27.4% reported using the NTLL, while 28.4% of participants were observed to really use it. Significant associations between self-knowledge of the NTLL and education level (This study found that the level of knowledge of the NTLL in the studied population was relatively high; however, both the referred and the observed use of the NTLL were low. Use and knowledge of the NTLL were associated with the socioeconomic and educational status of the participants. Thus, the change in nutritional patterns needs additional strategies to put the NTLL before the brand once customers make their purchases. Obesity prevalence worldwide almost doubled between 1980 and 2014, making it a serious public health problem , a four item Likert’s scale  for the question did you based your purchases on the NTLL? and three dicotomic (use/do not use) questions about self use of NTLL. In Ecuador, the NTLL consists of a scheme that values three characteristics or nutrients of the products: fats, sugars and salt. Each one of them is represented by one of 3 colors defined as: high, medium and low content . In the current investigation, every product purchased by a participant was classified as “healthy” (no single red light on its label) or “non-healthy”.The second part of the study was an observation of the participant’s purchasing pattern, which was done by taking individual photographs of the NTLL of each of the products, once they were registered by the cashier and prior to their package. Then, this data was analyzed as the objective part of the study to classify participants in levels depending on the amount of healthy (no red labels) products, as follows: “high” (> 74%); “medium” (50–74%); “low” (25–49%) and very low (< 25%) of their shopping list. Finally, global objective use of the NTLL was considered if at least 50% of the shopping list was “healthy”.p-value less than 0.05 was considered as significant.The survey questions, as well as the information collected from the photographs were recorded and analyzed using the IBM SPSS Statistics, v20. Continuous variables (e.g.age) are presented as mean and standard deviation (SD). For categorical variables, Chi square or Fisher’s Exact test were used depending of the count. Finally, variables with a statistical significance at at α = 0.1 were included in multivariate logistic regression models adjusted for possible confounding factors  to obtain OR and a n = 41) and 90.4% (n = 66) had a high educational level (college or university).The mean age of the participants was 38.4 SD 10.5 years old, the majority were female  of participants indicated they knew about the NTLL. It is important to note that the majority  of participants mentioned they did not check the NTLL to do their purchase and for 78.1% (n = 57) the NTLL did not influence their purchase. In the same sense, 36.1% (n = 26) participants mentioned that their purchase is based on the NTLL “very few times”, 30.6% (n = 22) “sometimes” and only 9.7% (n = 7) indicated that they “always” based their purchase on it. Finally, 72.6% (n = 53) of the participants do not use the NTLL.Regarding subjective data, the majority . Of those, healthy products (no red lights) were categorized in “low” and “medium” amount in 61.6 and 24.7% of the total, respectively. On the other hand, red lights  products were found at the “medium” and “low” levels with 60.3 and 20.5% of products, respectively.88.7% of the participants indicated knowing about the NTLL. Most participants (52.1%) had a high level of knowledge of the NTLL, 32.9% had an average level of knowledge and only 15.1% had a low level of knowledge.Analysis of the knowledge of the NTLL versus the subjective and objective variables, showed a significant association between knowledge of the NTLL and educational level. Thus, 91.8% of those who defined themselves as knowing the NTLL, showed to have a higher educational level.p = 0.02) with respect to the referred use. In fact, 87.8% of the participants who answered NO to the referred use of the NTLL mostly take into account brand of the product when making their purchases. Similarly, 57.1% of the participants who reported NOT using the NTLL also referred taking into account the price of the product at the time of their purchases.Referred (subjective) use of the NTLL was almost equal to the observed use of it . Only “purchase factor” had a significant association  association between “does NTLL influence your purchase” and the observed use of the NTLL (OR 3.39 95% CI 1.06–10.79). In addition, an association (p = 0.04) was found between “NTLL knowledge” and observed use when it was adjusted by the supermarket (OR 4.26 CI 95% 1.08–16.76). No associations were found in the other crosses or with any of the other adjusted models.Multivariate logistic analysis showed a significant  knew about the NTLL, however only 52.1% had a high level of knowledge of it. In this sense, nutritional labels are designed to provide simplified and concise information to customers, but it is still on the consumer hands, to correctly interpret and understand such information, being that an essential requirement for the final buying decision .A previous study to evaluate the understanding of nutritional labels showed that approximately two thirds of the participants were able to identify the caloric content and daily values of the label , and theRelated to our results, the European Food Information Council reported that only 1 in 4 consumers looked for and used the NTLL while doing their shopping. In addition, observation of the NTLL by the consumer is in most cases by accidental exposure to the NTLL information in a product , 23.The present study found that regarding the referred or subjective use of the NTLL, 59.7% participants did not look at it when making their purchase, which correlates to the 78.1% saying the NTLL did not influence their purchase. In addition, 72.6% of participants do not use the NTLL based on subjective data while 71.6% of participants do not use it based on the objective data. It was found also that for healthy products, most people bought medium and low amounts; however, this variable did not show significant relevance regarding knowledge of the NTLL.Our study sought to discriminate differences in the use and understanding of the NTLL by making a socieconomic stratification, for which we conducted surveys in two different supermarkets. We also differentiated and analyzed for possible confounding factors such as gender, age and educational level. The present study did not find any association between socioeconomic status (supermarket), gender or age when compared with the knowledge and use of the NTLL. However, an association was found regarding the knowledge of NTLL and the educational level, which tends towards the highest level of education related to a positive knowledge of the NTLL.In agreement with our results, a study in Spain showed based on subjective data that 41.4% of the participants knew about the NTLL, 61.5% of which stated that the NTLL seemed useful and 31.4% referring a usual use of it. In addition, the study also showed that 18.6% of the participants correctly understood the NTLL information .Finally, in the case of Ecuador, there is only one previous study showing that the subjective use of the label was generally low, but higher among mestizos compared to indigenous people, owing mainly to differences in education and limited health and nutrition related knowledge in the indigenous population , similarOur study has several strenghs, among them, the contrast between reported and observed used of the NTLL, but we also recognize its main limitation is the lack of generalizability of the findings at a country-level.In this urban population, the level of knowledge of the NTLL was relatively high, however, the observed use of the NTLL was low and it was associated with the socioeconomic and educational status of the participants. This study puts in evidence that a successful strategy implementation does not only depends on good regulation, but also requires customer acceptance and involvement. It would be interesting to replicate this model in any other country with NTLL in place.