Spinning up a highly complex, coupled Earth system model (ESM) is a time consuming and computationally demanding exercise. For models with interactive ice sheet components, this becomes a major challenge, as ice sheets are sensitive to bidirectional feedback processes and equilibrate over glacial timescales of up to many millennia. This work describes and demonstrates a computationally tractable, iterative procedure for spinning up a contemporary, highly complex ESM that includes an interactive ice sheet component. The procedure alternates between a computationally expensive coupled configuration and a computationally cheaper configuration where the atmospheric component is replaced by a data model. By periodically regenerating atmospheric forcing consistent with the coupled system, the data atmosphere remains adequately constrained to ensure that the broader model state evolves realistically. The applicability of the method is demonstrated by spinning up the preindustrial climate in the Community Earth System Model Version 2 (CESM2), coupled to the Community Ice Sheet Model Version 2 (CISM2) over Greenland. The equilibrium climate state is similar to the control climate from a coupled simulation with a prescribed Greenland ice sheet, indicating that the iterative procedure is consistent with a traditional spin‐up approach without interactive ice sheets. These results suggest that the iterative method presented here provides a faster and computationally cheaper method for spinning up a highly complex ESM, with or without interactive ice sheet components. The method described here has been used to develop the climate/ice sheet initial conditions for transient, ice sheet‐enabled simulations with CESM2‐CISM2 in the Coupled Model Intercomparison Project Phase 6 (CMIP6). We describe a computationally tractable, iterative procedure for spinning up a coupled Earth system‐ice sheet modelEquilibrium state from the iterative procedure is similar to a more expensive traditional model spin‐up with prescribed ice sheetsThe procedure is used for developing initial conditions for transient, fully coupled simulations in the Coupled Model Intercomparison Project phase 6 This initial condition should be free of any residual drift related to the numerical equilibration of simulated climate components, both in terms of their internal states and in terms of fluxes between components. Development of such initial conditions represents a major task that is common to all modeling centers and typically requires a substantial fraction of each center's computing resources. Including an interactive ice sheet component into global coupled model frameworks adds considerable complexity to coupled model spin‐up procedures.A primary challenge arises because ice sheets have large thermal and dynamic inertia and thus respond slowly to imposed climate forcing at the whole‐ice sheet scale, notwithstanding dynamic processes at the ice sheet margins and in ice streams that can respond comparatively quickly. Standalone ice sheet model simulations of the GrIS typically reach steady state in the order of 10,000 years when run under a constant forcing protocol All model components are allowed to evolve and equilibrate in a coupled fashion to account for bidirectional feedback processes, including ice sheet/climate feedbacks that regulate ice sheet evolution.(ii)The final result is an internally consistent climate state where all model components are in statistical equilibrium .(iii)The equilibrium climate, aside from ice sheet conditions, is similar to that obtained using a standard spin‐up technique with a prescribed ice sheet geometry. Where differences arise, they can be attributed to the presence of a dynamic ice sheet.(iv)The technique is computationally feasible for model equilibration, given available computing resources.Building on the concept of asynchronous and periodic synchronous acceleration, here we present a novel iterative procedure for spinning up a coupled climate model state, targeted at computational requirements, configurations, and designs of the CESM2. This method is well suited for computationally expensive models with an interactive ice sheet component, because of uniquely long ice sheet thermal and dynamic interia. The method is designed to satisfy the following goals and requirements as well as possible: Our method satisfies these goals in the course of developing a feasible ice sheet/Earth system modeling and analysis workflow within CESM2. Given commonalities between CESM2 and other high‐complexity ESMs .The CESM is a state‐of‐the‐art, global ESM, primarily developed and administered at the National Center of Atmospheric Research (NCAR). CESM2  has adopted the Beljaars et al.  form draThe Community Land Model (CLM5) . Bare ground is prescribed under the modern observed GrIS extent, while tundra is prescribed around the periphery, in regions of Greenland that are ice free at a given time. When CLM5 is run interactively with CISM2, CLM5 snow accumulation that exceeds the 10 m maximum allowed snow depth over the region that overlaps the CISM2 spatial domain  is added to the top of CLM5's snowpack, and an equivalent amount at the bottom of the snowpack is converted to ice and passed as a positive SMB to the ice sheet. Melting of the CLM5 ice column in the ice sheet domain is communicated to CISM2 as a negative SMB, with meltwater runoff  routed from CLM5 to the ocean   as liquid runoff. In POP2, solid runoff is melted instantaneously using energy from the global ocean, and the resulting salinity and heat anomalies are spread diffusively in the surface ocean, following a normal distribution with a maximum radius of 300 km. This treatment of solid runoff also applies to the default CESM2 setup without interactive ice sheets. MOSART is currently not capable of complex subglacial hydrology, so basal meltwater is instead sent directly to the ocean model using nearest neighbor mapping. While this is not an exact emulation of more complex subglacial hydrological routing, we consider it adequate, given the small magnitude of basal fluxes and the general tendency of subglacial hydrology to route water to nearby ice sheet margins. In the simulations discussed here, because any floating ice is immediately calved, no ice shelves develop, and basal melting of floating ice shelves is thus not considered. Freshwater and salinity are conserved in the coupled model configuration. However, eustatic sea level change is not explicitly simulated by changes in the land‐ocean distribution.By default, CISM2 is configured to run synchronously with other climate components. However, it can also be configured to run asynchronously, where the ice sheet evolves several years for each CESM2 model year. In this configuration, the ice sheet model cycles repeatedly over 1 year of SMB forcing during the accelerated period. In this case, fluxes from the ice sheet to the ocean are accumulated and averaged over the accelerated period. This methodology violates freshwater and energy conservation in the model but conversely reduces instabilities and artificial drift in the ocean model that could emerge if the accumulated freshwater flux from an accelerated ice sheet was sent to the ocean at a greater rate.All model components are dynamically coupled and exchange state information via a coupler that conservatively interpolates fields between the different model domains. The atmosphere (CAM6) and land (CLM5) models run on a 0.9° 2.1compsets, are configured by a combined user interface/control system called the Common Infrastructure for Modeling the Earth . The method described here relies on the iterative use of multiple model configurations, each of which is described below.CESM2 supports a variety of model configurations and forcing protocols, ranging from standalone experiments with individual modeled Earth system components , to coupled simulations that include all Earth system components, where the model climate is determined internally under imposed planetary boundary conditions. These model configurations, commonly referred to as component sets or 2.1.1The BG component set consists of the coupled model with a two‐way interactive ice sheet, hereafter referred to as “fully coupled.” In using the term “fully coupled,” it is nonetheless recognized that further and more detailed coupling is possible—for example, in the context of the ice sheet/Earth system, bidirectional ocean‐ice sheet coupling at marine ice sheet interfaces, or via global gravitational impacts of ice sheets on sea level. This is the most comprehensive way of running CESM2 at present, where all components are interactive and the entire simulated system responds to imposed boundary conditions, such as the top of the atmosphere radiation fluxes, greenhouse gases, topography, and other planetary boundary conditions. This is also the most computationally expensive type of simulation Table , though 2.1.2As part of the methodology for this study, a new model configuration (JG component set) was introduced. This component configuration is similar to the fully coupled model configuration, but with CAM6 replaced by a computationally inexpensive data atmosphere model where the atmospheric fluxes are prescribed. We note that this configuration would be unavailable in ESMs, which, for underlying structural reasons, cannot operate without an interactively coupled atmosphere. With an optimized processor layout, throughput  is more than four times greater than for the fully coupled model configuration, and the computational cost is reduced by a factor of three Table .This model configuration allows for two‐way coupling between CISM2 and all other model components except for the prescribed data atmosphere. Most importantly, this allows GrIS meltwater runoff and calving to interact with long‐timescale ocean processes such as the Atlantic Meridional Overturning Circulation (AMOC) during convergence to steady state. Similar to standalone ocean model simulations, we apply a weak  restoring of the sea surface salinity field to suppress spurious drift in the overturning circulation from runaway feedbacks with a prescribed atmospheric state  can be run as a free‐standing  model within the CIME infrastructure using the T component set. In this configuration, SMB and surface temperature on multiple elevation classes in each land grid cell are downscaled to CISM2 to account for elevation feedbacks as the ice sheet evolves. Typically, the surface forcing is provided by a previous run with an active land model. The computational cost of the ice sheet‐only configuration is low compared to the coupled model configurations described above, allowing for multimillennial simulations within a wall‐clock day  is time invariant. In standard model configurations without interactive ice sheets, this is acceptable. However, it introduces an inconsistency when running with interactive CISM2—particularly multicentennial or multimillennial simulations—as ice sheet topography changes over time. Updating the CAM6 topography continually at run time is currently not practical, as information about subgridscale topography variance and ridge orientation (used in orographic drag and gravity wave parameterizations in the planetary boundary layer) are derived from a high‐resolution global data set using algorithms that are not included or optimized for runtime CAM6 operation.(i)At the completion of each run segment, atmosphere and ice sheet (GrIS) states are written to standard restart files.(ii)The ice sheet topography is extracted from the CISM2 restart file, regridded to a 30‐s grid (approximately 1‐km resolution) and overlaid onto the GMTED2010 data set The CAM6 topography generation routine The new global, smoothed CAM6 topography and subgrid roughness fields, which incorporate the altered CISM2 topography, are reinserted into the CAM6 restart and topography files.(v)CESM2 is then automatically resubmitted.Thus, as part of the experiments presented here, an off‐line tool was developed that periodically updates the topography boundary condition in CAM6, to include changes in the ice sheet topography. Since this topography updating routine is not officially supported in the CESM2 model distribution, we briefly outline the workflow in the following:3The primary objective of spinning up (or equilibrating) an ESM is to generate an internally consistent coupled Earth system state, where all model components are in a statistical equilibrium . This state reflects an internal balance among all intercomponent fluxes, states, interactions, and feedbacks. The length of a model spin‐up is practically determined by the longest equilibration time of included Earth system components. In the absence of ice sheets, ESM equilibration times are typically determined by abyssal ocean and deep soil conditions, which can carry traces of past climate in dynamic, thermodynamic, and geochemical conditions for several millennia. Inclusion of an interactive ice sheet component complicates the exercise further, since ice sheets can carry a dynamic and thermodynamic memory spanning well over 10 kyr. This makes ice sheets the Earth system component with the longest spin‐up timescale in coupled ESMs, by a wide margin.In contrast to ice sheets, the atmosphere has almost no memory and adjusts quickly to altered conditions. At the same time, atmospheric models are often the most computationally expensive parts of coupled models. In CESM2, for example, the atmosphere component (CAM6) accounts for about 70% of the total coupled model cost at the standard 1° resolution (based on the optimized layout in Table (i)u and v) wind, as well as 3‐hourly surface temperature, pressure, precipitation, and near‐surface  potential temperature, specific humidity, density, and elevation.A fully coupled simulation section  is run f(ii)Next, the simulation continues using the model configuration where the interactive, coupled atmosphere model is replaced by the high‐frequency data archive section  generate(iii)Steps (i) and (ii) are repeated for as many iterations as required.(iv)As a final step, the coupled model is run for 100 years with synchronous climate‐ice sheet coupling to remove any residual inconsistencies resulting from the iterative procedure and provide a basis for assessing internal consistency and equilibration.The workflow of the spin‐up procedure is as follows , as developed in the long ice sheet simulation of Fyke et al. , after iGiven a constant preindustrial forcing protocol, the time needed to spin up the GrIS is expected to be roughly equal to the average ice sheet residence time—that is, the time an average ice parcel spends in the ice sheet before returning to the ocean or atmosphere via calving, runoff, or sublimation. Given the modern GrIS volume of 2.93 5To demonstrate the applicability and viability of the spin‐up procedure, we now demonstrate that (i) the climate simulated by the procedure behaves predictably throughout the iterative spin‐up, including transitions between the coupled and data atmosphere simulation segments; (ii) the model climate converges toward an equilibrium state sufficiently similar to the piControl simulation with a prescribed GrIS geometry, excepting physically realistic differences resulting from the presence of a coupled ice sheet; (iii) the final ice sheet state is consistent with prior climate history; and (iv) no significant biases in the final simulated GrIS state result from the spin‐up procedure itself, as opposed to biases inherent to the CESM2 climate or CISM2 representation of ice sheet physics. We explore each of these findings in the following sections.5.1Large‐scale oceanic conditions are sensitive to spin‐up methodology section . Thus, sThe top panel in Figure The meridional heat transport  conditions, as represented by piControl.5.3GrIS geometry and mass balance evolution during the course of the iterative spin‐up are shown in Figure Figure Ice sheet area also increases rapidly in the first decades of the simulation Table . This exThe initial mass imbalance corresponds to a global mean sea level decrease of about 1.3 mm/year. In comparison, the residual drift at the end of the simulation (end of the last fully coupled segment) is 0.03 mm/year, with a standard deviation of 5.4The final GrIS area is ns Table , with thns Table –5c. Consns Table  by arounIn broad agreement with previous studies  typically occur with a periodicity of up to subdecadal timescales. Hence, there is a possibility that sampling every 10 model years could result in aliasing of major climate modes in the atmosphere data archive. Moreover, running the coupled model for only 1 year after advancing the overall climate/ice sheet state by 10/100 years (respectively) could introduce inconsistencies in the long‐term climate evolution. Specifically, although the atmosphere is quick to adapt to changes in planetary boundary conditions, it can still take several months for full adjustments to abrupt changes in surface conditions, notably the ice sheet topography.In contrast, the philosophy adopted when designing our simulation procedure was to (1) capture the model's internal climate variability by prescribing high‐frequency atmospheric data over a standard climatological period of three decades; (2) regain internal consistency  with reasonable periodicity by running relatively short data atmosphere segments .7(i)The iterative spin‐up procedure alternates between a fully coupled and a computationally cheaper configuration where the atmospheric component is replaced by a data model. By periodically regenerating the atmospheric forcing, the data atmosphere remains adequately constrained to respect coupled conditions, ensuring that the broader coupled model state does not drift unrealistically.(ii)The simulated climate at the end of the spin‐up procedure is similar to the preindustrial control climate from a 1,200‐year fully coupled spin‐up simulation with a prescribed GrIS, indicating that the climate in our iterative procedure converges toward a climate state that is consistent with a traditional spin‐up approach despite a significant computational savings.(iii)The iterative method is an order of magnitude faster and cheaper than a traditional “brute force” synchronous spin‐up method, and significantly faster and cheaper than other accelerated/asynchronous spin‐up methods.(iv)The iterative method presented here could enable faster and cheaper spin‐ups for computationally expensive coupled climate and ESMs, with or without interactive ice sheets.In this study we have described and demonstrated a computationally efficient iterative procedure for spinning up coupled ice sheet/ESMs. We test its applicability by spinning up the preindustrial climate in CESM2, including a two‐way interactive ice sheet model (CISM2) over Greenland. We summarize our methods and conclusions as follows:The preindustrial ice sheet/Earth system state achieved via the new spin‐up method will provide initial conditions for transient simulations with CESM2‐CISM2 in support of ISMIP6 coupled historical and future simulations .The simulations in this study were produced with the publically released version of CESM2.1.1, which is open source software (freely available at


Protein activity is often regulated by ligand binding or by post-translational modifications such as phosphorylation. Moreover, proteins that are regulated in this way often contain multiple ligand binding sites or modification sites, which can operate to create an ultrasensitive dose response. Here, we consider the contribution of the individual modification/binding sites to the activation process, and how their individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable conformational free energy contributions from distinct sites, and associate a so-called activation parameter to each site. Our analysis shows that the ultrasensitivity generally increases as the conformational free energy contribution from one or more sites is strengthened. Furthermore, ultrasensitivity depends on the mean of the activation parameters and not on their variability. In some cases, we find that the best way to maximize ultrasensitivity is to make the contribution from all sites as strong as possible. These results provide insights into the performance objectives of multiple modification/binding sites and thus help gain a greater understanding of signaling and its role in diseases. Multisite protein modification is ubiquitous in gene regulation and signal transduction, often in the form of multisite phosphorylation. Many models of multisite ultrasensitivity are available in the literature, but they usually assume that all sites contribute equally to the activation of the multisite target. In this work, we relax this assumption and carry out computational and mathematical analysis of a multisite system in which the conformational free energy contribution varies across sites. We find that the ultrasensitivity of the system tends to increase (with some exceptions) when the conformational free energy contributed by any given site is strengthened. Our analysis predicts that all active sites should have approximately the same conformational free energy contribution, a property observed in proteins with unstructured modification domains and bulk electrostatics. We were also able to predict from first principles an energy range of -2 to -4 kcal/mol per site that effectively maximizes ultrasensitive behavior. This prediction is consistent with experimental measurements in phosphorylation sites. Another strategy predicted by some of our models is to select a subset of the sites and activate them uniformly, while silencing other modification sites in the protein. This strategy is also observed experimentally in many multisite phosphorylation proteins. Cellular systems rely heavily on signal transduction and environmental sensing pathways to successfully respond to internal and external environmental signals and conditions. To regulate signal transduction cascades, mammalian cells use ligand binding or post-translational modifications (PTMs) such as protein phosphorylation, methylation or ubiquitination. Several forms of disease can arise when there are defects in signal transduction pathways, including cancer, diabetes, and heart disease.Many proteins regulated by ligands or PTMs are multisite proteins, that is, they have multiple sites on which they can be modified or where a ligand can bind. For example, activation of mitogen activated protein kinases requires phosphorylation on two sites , and theA common role for multisite modifications lies in the creation of switch-like, or ultrasensitive, dose response curves –9. TheseA classical 1965 model by Monod, Wyman and Changeux (MWC)  uses mulClassical mathematical models of allostery and cooperative ligand binding, such as the MWC model, were based on observations of cooperativity between symmetric subunits of oligomeric proteins, such as hemoglobin (a tetramer), threonine deaminase  and aspartate transcarbamylase (a hexamer) . Given ti is assigned an activation parameter ci, generalizing the parameter c in the original formulation of the MWC model. Small values of the parameter ci correspond to a strong ability for the i-th site to activate the protein. One can also associate to each site a corresponding conformational free energy contribution, that is, the difference in the Gibbs energy function associated to the site i, ΔGi = rt ln(ci). In other words, rt ln(ci) is the site-specific free energy contribution to tense-to-relaxed flipping from ligand binding at site i. Notice that the conformational free energy contribution ΔGi is negative number when the activation parameter ci is less than one, and becomes more negative as ci approaches 0. Also note that a large negative ΔGi  corresponds to a strong conformational free energy contribution, which will promote flipping to the active state. In contrast, if ci > 1, then ΔGi will be positive, meaning that the modification does not promote flipping to the relaxed/active state but instead makes it more likely that the target will stay in the tense state.In the current paper, we set out to explore multisite systems in which the modification of some sites may have a stronger effect on the induced conformational change than the modification of other sites. To do this, we generalize the classical MWC system and assign different parameters to different sites. We aim to determine what combinations of parameters lead to a high level of ultrasensitivity. Each site ci smaller) has a strong tendency to increase the ultrasensitivity of the system, as measured by its associated Hill coefficient H. This effect is not guaranteed as there are some exceptions, especially for low values of the number of sites n, but it holds in most circumstances and under several orders of magnitude for the parameters in the system.Our main results can be summarized as follows. First, making the conformational free energy contribution associated with ligand binding to a single site i more favorable  or tense (IT), where I ∈ {0, 1}n is a binary vector representing the modified-form of the target. In the case of protein activation models, relaxed and tense states correspond to different levels of activity. We assume that all modified-forms in the relaxed (R) conformation are active and have the same activity, whereas all modified-forms in the tense (T) conformation are inactive, and have the same (low) activity. Under MWC assumptions, the relaxed state has a higher affinity to the ligand than the tense state, this is assumed here for the states IR and IT. The unmodified state, n modified-forms are possible. The modification of site i on the target will result in modified-form J, where J = I ∪ {i}. In other words J is the modified-form consisting of adding one more modification at site i to the modified-form I. For example, in R and be reversibly modified to R or R and subsequently to R. A target in a relaxed state can also flip to the tense state in that form. For instance, R can flip to the T state. Similarly, the tense target in modified-form T can be reversibly modified to T. We call u the kinase concentration in the case of multisite phosphorylation and u denotes the ligand concentration in the case of ligand binding.In this section, we carry out a generalization of the MWC model to account for different activation parameters at distinct sites. See Enciso and Ryerson , where aαi is the microscopic association constant for ligand binding at site i. Note that αi is an affinity, i.e. not a dissociation constant but the inverse thereof. If instead of ligand binding the protein is modified by post-translational modification , αi represents the modification efficiency of site i, which, for example, will be determined by the relative suitabilty of the site to be phosphorylated by a given kinase and dephosphorylated by a given phosphatase. The parameter L is the equilibrium constant between L is typically assumed to be greater than 1, so that, in the absence of modification, the tense/inactive state is favored over the relaxed/active state. Indeed, L > 9 is required for the target to be less than 10% active in the absence of modification. This network has the property of detailed balance, as the product of the equilibrium constants around any closed cycle of states is 1; this is the same as saying that the net free energy change around any closed cycle of states is 0. For this reason each forward and reverse reaction pair is in equilibrium.The general system can be described by the chemical reaction network in LcI is the equilibrium constant between the relaxed (active) state RI and the tense (inactive) state TI. In this sense, one can think of ci as the contribution to this equilibrium constant made by each individual site i, and ΔGi as the free energy differential between active and inactive protein contributed by modification at site i.We use the notation n = 2 is listed in R is defined as the ratio between the statistical weight and the partition function Z. In the n = 2 case, Z = 1 + α1 + α2 + α1α2 + L + Lα1c1 + Lα2c2 + Lα1α2c1c2. n = 3 and the associated Z. For general n, The statistical weights for each state possible when u to represent the concentration of ligand or the concentration of modifying enzyme. If the MWC target molecule is a receptor that is regulated by ligand binding, then u is the concentration of ligand. If the MWC target molecule is instead regulated by post-translational modification, then u is the concentration/activity of the modifying enzyme. In the latter case, we assume that the modifying enzyme is in steady state with a corresponding demodifying enzyme (e.g. a kinase-phosphatase system), and that both enzymes are far from saturation. Under these assumptions, the dose response relating kinase concentration u to the fraction of the target sites that are modified is the same as the dose response relating ligand concentration to the fraction of the target sites that are bound. See . The parameter L ≥ 1 was fixed, and the parameters αi were chosen to be identical to each other, In this section we carry out a computational analysis of the dose response and its Hill coefficient, where the parameters EC90 and EC10 with a standard numerical solver. Here, we solved for u such that f − βf∞(c) = 0 for both β = 10% and 90%. With both EC10 and EC90, we can calculate H asH > 1 implies the dose response curve is ultrasensitive, while H = 1 implies there is no ultrasensitivity, and H < 1 shows negative ultrasensitivity. One can also think of H > 1 showing that the dose response has a good switch  logarithmically for i = 1, 2, ⋯, n. The proportion of simulations where H decreases on c1 is almost always one for n > 4. For n = 2 there are many parameter sets where that is not the case.In −4, 0.9] logarithmically. This vector of activation parameters has arithmetic mean c one can assign a second vector H for each case, we can see in ci (solid line), with parameter values αi = 1 and L = 1000, H decreases with increasing mean of c for n = 2, 3, 4, 6 and 8. Any variation among the ci does not significantly affect H when H depends on the variability among the ci as well as their mean. When αi is sampled from the range  the dependence of H on αi = 1 . For fixed n, as the total conformational free energy contribution increases, ultrasensitivity generally tends to increase, and after some threshold, it tends to level off. To increase ultrasensitivity at that point, a target/receptor cannot profitably utilize more conformational free energy contribution, but instead must evolve more sites. As a concrete example, let us consider an MWC molecule with two sites under selective pressure to increase its ultrasensitivity. Changes to the microscopic modification affinities/efficiencies  will either decrease ultrasensitivity , or at best leave ultrasensitivity unaltered  , logarithmically. This v has an arithmetic mean H for each case, we can see in vi (solid line), ki = 1 and d = 1000, H can increase or decrease depending on the mean of v for n = 2, 3, 4 and 8. Here, we can see that any variation between the vi may affect H (asterisks).To determine the effect the variability between parameters CV(v) vs H. Here we see that CV(v) has some effect on H, regardless of n. This is particularly interesting since, contrary to MWC, the variation between vi affects H. We also see that there are values of H increases and values where it decreases. How often is H increasing with increasing v1?In H increases with increasing v1 based on n and d. The proportion was found in a similar fashion as in the MWC system. Here, we logarithmically sampled vi ∈  and ki = 1.In H increasing on v. When H ≈ 1). We can also see that if d is slightly past the 50% of max activation, H can be maximized by increasing the free activation of energy v.The computational and analytical results described in the section below titled “Independent System Mathematical Analysis” suggest that that To summarize, in this section we show that (1) ultrasensitivity increases under specific parameter regimes and (2) may depend on the variability between the activation parameters.H is roughly independent of the variation of c. We will show that H is essentially a function of H to the extent that it changes the mean, f from In this section, we provide a mathematical analysis of the generalized MWC system showing that C2 function f such that For notation purposes, let We will use this to show thatH essentially does not vary if the mean of c is preserved, as illustrated in This formula demonstrates in particular that Proposition 1Proof: For simplicity, assume ST = 1 and assume for now that u and n-th power, M > 0 such that |ω(x) − ω(y)| ≤ M|x − y| for all x, y > 0. Then,Taking the Then Thus,H is unaffected by increasing or decreasing its value, as explained in the sections above. Also, the above analysis is carried out for u in a neighborhood of u does not vary significantly.One can assume that Proposition 2Proof: Similar to Proposition 1,n-th power, Taking the Proposition 3Proof: The first-order Taylor approximation of H around From Proposition 1, n = 2, H increases when In this section, the following theorem and proposition provide mathematical analysis for the independent system showing that for Theorem 1Suppose thatf > 0 is a saturatingC2function defined for allu, z > 0, such thatfu > 0. If the functionis strictly increasing on u, then H(z) is increasing for every parameter z > 0.Proof: Let p(z) and q(z) represent the EC10 and EC90 values of the dose response respectively, that isf∞(z) is the maximum value of f. Differentiating both sides, it follows thatThat isRecall f(p(z), z)/f∞(z), 0.90 = f(q(z), z)/f∞(z). Overall, we have thatThe last inequality follows since 0.10 = σ is an increasing function of u, then Thus, if σ increase on u? Here, we provide a derivation for the associated σ function for the Independent dose response when n = 2. More specifically, we now apply the theorem to the independent model with z = v1.Given the dose response in , does σ 1 and v2 ifis increasing on vystem in , for n =Proof: We use the above result and show that when σ is increasing on u.f as given by  from  from . For theThen,σ = τ1τ2. Note that τ2 is a strictly increasing function on u since Let τ1 is also strictly increasing on u if and only if τ1 is strictly increasing if and only if C3k < C4. This is equivalent toIn the following text, we show that τ1 is an increasing function on u making σ the product of two increasing functions and thus, σ is increasing on u.As long as Theorem 2Suppose n = 2. If Q01 > 1/2 and Q10 > 1/2, then H is increasing as a function ofv1andv2.Proof: If Q01 > 1/2 and Q10 > 1/2, it follows that Then,d2 < v1v2 and so H is increasing as a function of v1 and v2.Thus, v1 and/or v2 are then increased so that d, the singly modified forms now have more than half the conformational free energy contribution of the doubly modified forms, and the Hill number increases. The Hill number will continue to increase until the two singly modified forms, collectively, contribute exactly the same conformational free energy contribution as the doubly modified form. At this point, the system is in the ‘+ regime’, where modification of one, the other, or both sites lead to the same level of activation.The point where v1 or v2 reduces the Hill number by pushing the system away from ‘both or none’ and closer to ‘linear’.On the other hand, if In a protein with multiple ligand binding sites, the individual sites can differ from each other in two ways: in their microscopic ligand binding affinity, and in the energetic contribution they make, once bound or modified, to functional outcomes such as a ligand-induced conformational change in the bound protein. Likewise, for a protein that is post-translationally modified on multiple sites, the individual sites may have different modification efficiencies, and may also, independently, make differential contributions to downstream functional consequences once modified. For example, in the case of phosphorylation, the amino acid sequence around the target phosphoacceptor residue can substantially influence the efficiency of phosphorylation by the relevant kinase, as well as the efficiency of dephosphorylation by cellular phosphatases . Such tuPreviously, Enciso and Ryerson  asked thHere we examined how differential energetic contributions of the sites might affect the performance objective of ultrasensitivity. We considered a simple model in which binding/modification promotes a conformational change that flips the modified molecule from an inactive to an active state; this example is readily extended to other known consequences of ligand binding or post-translational modification. We generalized the classic allosteric MWC model to allow for differences in the energetic contributions for any number of distinct sites. We also considered an independent modification model that does not assume allostery or cooperativity among sites. For the generalized MWC system, we found that ultrasensitivity generally increased when the energetic contribution  of one or more of the sites was strengthened. Here, ‘strengthened’ means that the conformational free energy contribution became more negative; this results in the corresponding activation parameter c becoming smaller. Furthermore, we found that there was no benefit derived from balancing the conformational free energies, nor any penalty for unbalancing them. Our results have implications for understanding the potential trajectories that can be pursued by a protein under selective pressure to increase the ultrasensitivity of its response to modification.ci of individual sites has a strong tendency to increase the Hill coefficient, this result is analogous to work by Rubin and Changeux  with L = 1000 and n = 2, (b) n = 4, ci = 0.01 for i ≥ 3, similarly with (c) n = 8. These figures are the same data points from Heat maps for H when (TIF)Click here for additional data file.S3 FigH and the arithmetic mean of c where ci are independently and logarithmically chosen from , L = 1000, and αi are independently and logarithmically chosen from  for n = 2, 3, 4, 8. (b) H from (a) with the coefficient of variation (CV) along the x-axis. (c) Scatter plot for H when increasing total conformational free energy with ci ∈ , L = 30 and n = 2, 3, 4, 8. (d) Scatter plot for H for when L = 10, 000. (e) Proportion of 10000 parameter sets in which H decreased when a ci is marginally increased for different values L and n. (f) H values for key scenarios. When a target molecule has 3 sites, H = 2.40 when L = 1000, and ci = 0.01. Adding a site with c4 = 1 will yield the same H. However, if c4 = 0.01, H = 3.08.(a) Scatter plot for (TIF)Click here for additional data file.S4 Figci ∈ , L = 1000 and n = 2, 3, 4, 8 and 10000 points. Ultrasensitivity is measured with (a) a non-linear regression fit to the Hill function H is the Hill number labeled HFit and (b) a generalized Levitzki derivation for ultrasensitivity , L = 1000 and n = 2, 3, 4, 8 and a maintenance cost of (a) Mc = 2, (b) Mc = 4  (TIF)Click here for additional data file.S1 TableL and n. Parameters Ultrasensitivity as measured by the Goldbeter-Koshland formula described in (TIF)Click here for additional data file.


The antigenicity of the hemagglutinin esterase (HE) glycoprotein of influenza C virus is known to be stable; however, information about residues related to antigenic changes has not yet been fully acquired. Using selection with anti-HE monoclonal antibodies, we previously obtained some escape mutants and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1. To confirm whether the residues identified as the neutralizing epitope possibly relate to the antigenic drift, we analyzed the growth kinetics of these mutants. The results showed that some viruses with mutations in antigenic site A-1 were able to replicate to titers comparable to that of the wild-type, while others showed reduced titers. The mutants possessing substitutions in the A-2 or A-3 site replicated as efficiently as the wild-type virus. Although the mutant containing a deletion at positions 192 to 195 in the Y-1 site showed lower titers than the wild-type virus, it was confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. Then, we revealed that antigenic changes due to substitutions in the A-1, A-3, and/or Y-1 site had occurred in nature in Japan for the past 30 years. These results suggest that some residues  in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability. Influenza C virus was first isolated in 1947 (C/Taylor/1233/1947) from the throat washings of a man living in New York City who had a mild headache and backache and coryza ,6 or in Information about the antigenic characterization of influenza C virus is limited ,8, becauOrthomyxoviridae family of enveloped and segmented negative-sense RNA viruses, together with the influenza A, B, and D viruses. The HE protomer, which is encoded from the fourth segment of the viral genome, is composed of two subunits, HE1 (432 amino acids) and HE2 (209 amino acids) [Influenza C virus is a member of the o acids) . The HE o acids) ,23,24. Ro acids)  determinRecently, we mapped the neutralizing epitopes of influenza C virus onto the three-dimensional (3D) structure of the HE glycoprotein by using escape mutants selected by anti-HE monoclonal antibodies (MAbs) and identified four antigenic sites, namely, A-1, A-2, A-3, and Y-1 . The A-1The residues identified as the neutralizing epitope in these antigenic sites may possibly be associated with the antigenic drift of influenza C virus. To resolve the problem regarding the functional constraints on variation in the antigenic region, we examined the growth kinetics of these escape mutants and found some residues that affect the antigenicity while retaining replicative fitness. Then, we reviewed antigenic mutations that occurred in our surveillance work of the past 30 years and investigated the possibility of the occurrence of antigenic drift in nature.The escape mutants and their parental viruses were obtained from our previous study . Ten mutNine anti-HE MAbs  characterized in our previous reports ,27 were Sequencing of seven gene segments  for C/Yamagata/11/88 and C/Yamagata/29/2004 was performed as previously described [Amino acid positions were plotted on the 3D structure of HE molecules using the PyMOL Molecular Graphics System, version 1.8.6.1 . The crystal structure of HE was obtained from the Protein Data Bank (PDB ID: 1FLC).Madin-Darby canine kidney (MDCK) cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001 in the presence of 20 μg/mL TPCK-trypsin at 34 °C. The culture medium supernatants were harvested every 24 h from 1 to 6 days post-infection (dpi). Viral titers were determined by plaque assays with MDCK cells as described previously . BrieflyWe previously reported that some escape mutants of C/Ann Arbor/1/50 (C/Taylor lineage) and C/Yamagata/15/2004 (C/Yamagata lineage) have reduced HI activity against the MAbs used for selection . To furtWe tested the antigenic cross-reactivity of the mutants with chicken antisera raised against viruses from the C/Taylor and C/Yamagata lineages. Although antigenic differences between mutants of C/Ann Arbor/1/50 and mutants of C/Yamagata/15/2004 were detected using polyclonal antisera, all mutants reacted with both antisera. Among them, we found that a mutant carrying the substitutions E193K or D269K had a two- or four-fold decrease in cross-reactivity, compared to the wild-type C/Ann Arbor/1/50, and the Δ198 deletion mutant virus had a four- or eight-fold decrease in cross-reactivity, compared to the wild-type C/Yamagata/15/2004.To examine the replicative fitness of the viruses possessing mutations in the antigenic sites , we compSome viruses with mutations in antigenic site A-1, including the N173I, S192L, and K235R mutations, were able to replicate to titers comparable to that of the wild-type C/Ann Arbor/1/50 A–C, whilThe escape mutants possessing the R68W or A351V mutation in the A-2 site replicated as efficiently as the wild-type C/Ann Arbor/1/50 D. For thFor the Y-1 site, the Δ198 deletion mutant virus replicated as efficiently as the wild-type C/Yamagata/15/2004, while the Δ192–195 deletion mutant showed lower titers from 2 to 6 dpi F. NevertSince 1988, we generated 509 HI test results for the antigenic analysis of influenza C virus isolates in Japan ,18,19,31HI titers and amino acid sequence alignment of the reference strains of six HE lineages are shown in The locations of the amino acid changes that occurred in natural isolates are shown in As shown in In Japan, the isolation of C/Sao Paulo lineage strains has increased since 2006, and antigenic variants possessing the K190N mutation (Y-1 site), which failed to react with MAbs YA3 and YA5, have increased since 2014 and accounted for 32% of total strains by 2018. Among them, two strains isolated in 2016 showed a 100-fold higher reactivity to J9 due to the further mutation at position 125 (D125N) (A-1 site) [Since the escape mutant virus with the D269N mutation (A-1 site) replicated to lower titers than the wild-type C/Ann Arbor/1/50 C, we exaEvolutionary tree analyses have revealed that six antigenic lineages of influenza C virus diverged by approximately 1980 ,34. AlthWe revealed that two variable regions, residues 165 to 172 and residues 190 to 195, exist in the A-1 site of the HE molecule, suggesting that six different antigenic lineages were generated by the stepwise accumulation of mutations that occurred in these regions . The A-1In the A-1 site, the two escape mutants possessing the E193K or D269N mutation have affected their replicative fitness. Although amino acid differences at each position were not found between lineages , a straiFor the A-2 site, the MAb K16, which recognizes near the 9-O-acetylesterase active site , had litIn the A-3 site, escape mutants possessing the L164P or K198E mutation replicated as efficiently as the wild-type virus. Although variation at residue 164 was not observed among the natural isolates, the K198E substitution was found in viruses belonging to the C/Aichi, C/Yamagata, and C/Kanagawa lineages . Some stIt is well known that the glycosylation of HA on influenza A viruses interferes with antibody access ,38. HoweFor the Y-1 site, we examined the growth kinetics of the deletion mutant at positions 192 to 195, which failed to react with MAb U9 (A-1 site) and MAbs YA3 and YA5 (Y-1 site), and confirmed that this region in the 190-loop on the top side of the HE protein is not essential for viral propagation. This region is also variable among the lineages; therefore, further accumulation of mutations occurring in this region would contribute to antigenic changes within the lineage, such as the C/Sao Paulo lineage [Finally, although antigenic drift may be a rare or nonoccurring event for the influenza C virus, our long-term surveillance work indicated that the replacement of the dominant antigenic lineage periodically occurs . We suggWe revealed that some residues  in the A-1 site, residue 198 in the A-3 site, and residue 190 in the Y-1 site are likely to mediate antigenic drift while maintaining replicative ability, and some residues  in the A-1 site and residue 164 in the A-3 site are unlikely to be involved in antigenic drift. These findings may facilitate future surveillance of influenza C virus.


Agaricus bisporus ) pizza. He denied any drug intake, hymenoptera stings or other possible triggers, and no identifiable cofactors were present. Since then he avoided all types of mushrooms, however an accidental contact occurred with mushroom sauce that resulted in angioedema of the lip within minutes. The allergy workup included measurements of total IgE and specific IgE to mushroom, and skin prick test to aeroallergens sources, possible food allergen sources and mushroom extract, a prick to prick test with raw and cooked A. bisporus , in addition to a SDS-PAGE and immunoblotting assay. The study revealed a specific IgE to mushroom of 0.76kUA/L positive skin prick test to mushroom extract, and prick to prick test positive to white and brown A. bisporus (raw and cooked). The immunoblotting identified two IgE binding proteins with 10kDa and 27kDa. We report a case of A. bisporus anaphylaxis probably due to primary mushroom sensitization. We detected two IgE-reactive proteins with 10kDa and 27kDa as possible culprit allergens.A 33-year-old male with house dust mite allergic rhinitis and asthma reported an episode of facial and lip angioedema, dyspnea, cough and dysphagia at the age of 25, minutes after eating a mushroom (  Anaphylaxis is a serious allergic reaction with a rapid onset and potentially fatal outcome.In adults the main anaphylaxis triggers are food, insect stings and drugs. In a suspected food allergy, a clinical history with a food diary is extremely important to identify unusual food allergens.Concerning food allergens, the main identified triggers are peanut, fish, shellfish, tree nut and fresh fruits, especially in pollen allergic patients; however we must be aware that the implicated allergens can change with different eating patterns.Species in the Fungi kingdom can cause different types of allergic symptoms. Allergens from mold spores are mainly associated with airborne respiratory allergies. Mushroom species can be implicated in contact dermatitis and also cause digestive symptoms, accounting for 1% of mushroom allergy.Agaricus bisporus is the largest cultivated mushroom, accounting for 38% of the world production. Only few cases of allergy due to ingestion of A. bisporus have been described. The majority of cases reporting mushroom allergy due to ingestion refer to the species Boletus edulis , Boletus badius , Lentinus edulus and Tricholoma matsutake.A. bisporus in a patient without any other sensitization to molds, in which ingestion appears to be the primary route of sensitization.We report a case of anaphylaxis to Dermatophagoides pteronyssinus and Dermatophagoides farinae .A 33-year-old man with allergic rhinitis and asthma under treatment with inhaled and nasal corticosteroids, and with a known allergy to He experienced an anaphylactic reaction at the age of 25 years, with facial and lip angioedema, dyspnea, cough and dysphagia minutes after ingestion of a mushroom pizza. No other suspected triggers were present, such as drug intake or stings. He also denied physical exercise or alcohol intake in the hours before the episode. He stopped eating mushrooms and any food containing mushrooms.The patient reported two posterior episodes of lip angioedema, without any other symptoms after accidental contact with mushroom sauce.D. pteronyssinus , D. farina e, Lepidoglyphus destructor , cat and dog dander) molds  and mushroom were carried out.Measurements of serum total IgE  and specific IgE  to airborne allergenic sources ( D. pteronyssinus (100HEP/mL); D. farinae (100HEP/mL); L. destructor (10HEP/mL); pollens from Olea europaea (30HEP/mL), Corylus avellana (30HEP/mL), and Platanus occidentalis (30HEP/mL); cat and dog dander (30HEP/mL); A. fumigatus (150ug protein/mL); A. alternata (30HEP/mL); Cladosporium herbarum (150ug protein/mL); Candida albicans (125ug protein/mL); pollen from grass mixture (30HEP/mL); cow's milk , egg ; wheat flour ; tomato (10HEP/mL) and mushroom (10HEP/mL). Histamine was used as positive control (10mg/mL).We also performed skin prick tests with commercial extracts  of A. bisporus was also carried out.Besides skin testing with commercially available extracts, a prick to prick test with raw and cooked A. bisporus .A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting assay was performed to assess the molecular mass of the IgE-binding proteins from A/L), D. peteronyssinus (30.90kUA/L) and L. destructor (2.33kUA/L).Total IgE was 240kU/L and specific IgE was positive to mushroom (0.76kUD. pteronyssinus (10mm), D. farinae (8mm), L. destructor (7mm).Skin prick tests were positive to extracts from mushroom (7mm), A. bisporus , and raw (14mm) and cooked (11mm) brown A. bisporus ( Prick to prick test was positive to raw (9mm) and cooked (11mm) white isporus   .A. bisporus extract revealed two main IgE binding bands of approximately 10kDa and 27kDa ( The immunoblotting assay with d 27kDa   .(-)In the literature there are a few cases of mushroom food allergy, most of them occurred in China, where mushrooms are included in the diet and the last known report was published in 2013.A. bisporus allergy in a patient with asthma and rhinitis, an uncommon case highlighting the importance of being aware of unsuspected food allergens.We report a case of anaphylaxis due to A. alternata , who had one episode of exercise-induced anaphylaxis after ingestion of A. bisporus ; Dauby et al. described a woman with allergic rhinitis, allergic to Hormodendrum cladosporioides , A. alternata , Fusarium vasinfectum , Helminthosporium interseminatum and to Epicoccum nigrum , with an oral allergy syndrome to raw A. bisporus, and in both case reports the authors believed the hypersensitivity reaction was attributable to cross reactivity between mold and mushroom allergens.According to skin tests and serum measurements, the patient was allergic to house dust mites and mushroom. The skin tests were negative to molds and all other suspected food allergen sources. Without mold sensitization, this patient probably presents a primary sensitization to mushroom by ingestion, and not a cross reactivity with molds or mushroom spores. The cases reported in the literature associated mushroom food allergy to cross reactivity: Carrapatoso et al. described a case of a young man with allergic rhinitis allergic to et al . were able to identify mannitol, the major carbohydrate component in Fungi , as one of the possible allergens of A. bisporus in a woman who had anaphylactic episodes, after ingestion of A. bisporus and pomegranate. Dauby et al . identified thermolabile A. bisporus proteins with molecular weight of 43kDa to 67kDa, which seemed to cross react with aeroallergens from mold, and were involved in a case similar to oral allergy syndrome.Two thermostable IgE-reactive proteins with approximately 10kDa and 26kDa were detected, although protein class was not established, and these two allergens were unknown until our case report. In other case reports other mushroom allergens were identified. Hegde After the diagnosis of mushroom allergy, the patient was advised to eliminate mushroom and mushroom-containing products from his diet. He was prescribed an adrenaline autoinjector  to use, if necessary.A. bisporus . The first episode described by the patient fulfills clinical criteria for anaphylaxis, which is rather uncommon presentation. The immunoblotting assay revealed two IgE-reactive proteins with approximately 10kDa and 27kDa.There are few described cases of mushroom food allergy, and the majority are due to cross-reactivity between molds and food allergens. The scarce data related to primary sensitization to edible mushrooms and food allergy became our diagnostic approach difficult, but make our results even more important, since our patient has a probable primary sensitization to Although a food oral challenge was contraindicated, the test results and clinical history were essential to make diagnosis. The importance of considering less common allergenic sources as possible culprits is challenging, but it is of utmost relevance in the field of food allergy. A anafilaxia é uma reação alérgica grave, com início rápido e desfecho potencialmente fatal.Em adultos, os principais desencadeadores de reações anafiláticas são alimentos, picadas de insetos e fármacos.Em casos suspeitos de alergia alimentar, a história clínica, combinada a um diário alimentar, é extremamente importante para a identificação de alérgenos alimentares menos comuns.Em relação aos alérgenos alimentares, os principais desencadeadores identificados são amendoim, peixe, crustáceos, frutos secos e frutas frescas, principalmente em pacientes alérgicos a pólen; no entanto, temos que estar cientes de que os alérgenos implicados podem mudar conforme os diferentes padrões alimentares.Espécies do reino Fungi podem causar diferentes tipos de sintomas alérgicos. Alérgenos dos esporos de bolores são predominantemente associados a alergias respiratórias. Espécies de cogumelos podem estar implicadas em dermatite de contato e causar também sintomas digestivos, que correspondem a 1% das alergias a cogumelos.Agaricus bisporus é a espécie mais cultivada de cogumelos e representa 38% da produção mundial.Foram descritos apenas raros casos de alergia por ingestão de A. bisporus . A maioria dos casos relatados de alergia após ingestão de cogumelos referem-se às espécies Boletus edulis, Boletus badius, Lentinus edulus e Tricholoma matsutake.A. bisporus num paciente sem qualquer outra sensibilização a fungos, em que a ingestão parece ser a via de sensibilização primária.Relatamos aqui um caso de anafilaxia por ingestão de Dermatophagoides pteronyssinus e Dermatophagoides farinae .Indivíduo do sexo masculino, 33 anos, com asma e rinite alérgica, em tratamento com corticosteroides inalatórios e nasais e com alergia conhecida a Apresentou reação anafilática aos 25 anos, com angioedema facial e labial, dispneia, tosse e disfagia alguns minutos após ingestão de pizza de cogumelo. Não havia qualquer outro desencadeador presente, como fármacos ou picadas de insetos. O paciente também negou ter realizado exercício físico ou consumido álcool nas horas que antecederam o episódio. Desde então, o doente evita cogumelos ou qualquer alimento contendo cogumelos.O paciente relatou dois episódios posteriores de angioedema labial, sem nenhum outro sintoma, após contato acidental com molho de cogumelo.Dermatophagoides pteronyssinus , D. farinae , Lepidoglyphus destructor , faneras de gatos e cães), bolores  e cogumelos.Foram realizados doseamentos séricos de IgE total  e IgE específico  para aeroalérgenos ( D. pteronyssinus (100HEP/mL); D. farinae (100HEP/mL); L. destructor (10HEP/mL); pólen de Olea europaea (30HEP/mL), Corylus avellana (30HEP/mL) e Platanus occidentalis (30HEP/mL); faneras de gatos e cães (30HEP/mL); A. fumigatus (150ug proteína/mL); A. alternata (30HEP/mL); Cladosporium herbarum (150ug de proteína/mL); Candida albicans (125ug de proteína/mL); pólen de gramíneas (30HEP/mL); leite de vaca (1.600ug de proteína/mL); ovos (1.400ug de proteína/mL); farinha de trigo (1.900ug de proteína/mL); tomate (10HEP/mL) e cogumelos (10HEP/mL). A histamina foi usada como controle positivo (10mg/mL).Realizamos também testes cutâneos com extratos comerciais  de prick to prick com A. bisporus cru e cozido.Além dos testes cutâneos com extratos disponíveis comercialmente, foi realizado teste immunoblotting por eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE) para avaliar a massa molecular das proteínas de ligação ao IgE do A. bisporus .Foi efetuado A/L), Dermatophagoides peteronyssinus  e L. destructor .A IgE total foi de 240kU/L e a IgE específica foi positiva para cogumelos , D. farinae (8mm) e L. destructor (7 mm).Os testes cutâneos foram positivos para extrato de cogumelos (7mm), prick to prick foi positivo para A. bisporus branco cru (9mm) e cozido (11mm) e A. bisporus castanho cru (14mm) e cozido (11mm) ( O teste  (11mm)   .immunoblotting com extrato de A. bisporus revelou duas bandas principais de ligação com IgE, de aproximadamente 10kDa e 27kDa. ( O teste de  27kDa.   .(-)Na literatura, há poucos casos de alergia alimentar a cogumelos, os quais são relatados principalmente na China, onde os cogumelos fazem parte da dieta. O último relato conhecido foi publicado em 2013.A. bisporus num paciente com asma e rinite. Trata-se de um caso incomum, que destaca a importância de estarmos alertas para alérgenos alimentares incomuns.Relatamos aqui um caso de anafilaxia por alergia a A. alternata , que apresentou episódio de anafilaxia induzida por exercício após ingestão de A. bisporus ; Dauby et al. descreveram uma paciente do sexo feminino com rinite alérgica e alergia a Hormodendrum cladosporioides , A. alternata , Fusarium vasinfectum , Helminthosporium interseminatum e Epicoccum nigrum , com síndrome alérgica oral a A. bisporus cru – em ambos os relatos de caso, os autores associaram a reação de hiperssensibilidade à reatividade cruzada entre alérgenos de bolores e cogumelos.De acordo com testes cutâneos e dosagens séricas, o paciente era alérgico a ácaros e cogumelos. Os testes cutâneos foram negativos para fungos e para todos os outros alérgenos alimentares considerados suspeitos. Sem sensibilização a fungos, esse paciente provavelmente apresentava sensibilização primária a cogumelos por ingestão e não reatividade cruzada com esporos de bolores ou outros fungos. Os casos relatados na literatura associam alergia alimentar a cogumelos com reatividade cruzada: Carrapatoso et al. descreveram o caso de um jovem do sexo masculino com rinite alérgica desencadeada por Fungi , como um dos possíveis alérgenos do A. bisporus numa paciente do sexo feminino, com episódios de anafilaxia após ingestão de A. bisporus e romã.Dauby et al. identificaram proteínas termolábeis de A. bisporus com peso molecular de 43kDa a 67kDa, com suposta reatividade cruzada com aeroalérgenos de fungos e relacionadas a um caso semelhante a uma síndrome alérgica oral.Duas proteínas IgE-reativas termoestáveis de aproximadamente 10kDa e 26kDa foram detectadas, embora a classe das proteínas não tenha sido estabelecida, e esses dois alérgenos fossem desconhecidos até a data do nosso relato de caso. Em outros relatos de casos, alérgenos de cogumelos diferentes foram identificados. Hegde et al. identificaram o manitol, principal componente carboidrato dos Após o diagnóstico de alergia a cogumelos, o paciente foi instruído a eliminar da dieta cogumelos e produtos que os continham. Foi prescrito um autoinjetor de adrenalina  para ser usado, se necessário.A. bisporus . O primeiro episódio descrito pelo paciente atende aos critérios clínicos de anafilaxia, uma apresentação bastante incomum. O teste de immunoblotting revelou duas proteínas IgE-reativas, de aproximadamente 10kDa e 27kDa.Há poucos casos descritos de alergia alimentar a cogumelos, e a maioria é explicada pela reatividade cruzada entre fungos e alérgenos alimentares. Os dados escassos relativos à sensibilização primária a cogumelos comestíveis e à alergia alimentar dificultam nossa abordagem diagnóstica, mas tornam nossos resultados ainda mais importantes, já que nosso paciente apresenta provável sensibilização primária a Apesar da contraindicação do teste de provocação oral, os resultados dos outros testes e a história clínica foram essenciais para que se estabelecesse o diagnóstico. A importância de se considerarem fontes alergénicas menos comuns como possíveis responsáveis traz desafios, mas é essencialmente relevante no campo das alergias alimentares.