Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design. The 50th anniversary of Monod-Changeux-Jacob seminal paper “Allosteric proteins and cellular control systems” became the hallmark of a new wave in the allostery studies and the turning point in our vision of allostery and its implications in protein engineering and drug design. Recent experimental and theoretical works clearly show relevance of allosteric phenomenon to drug design, unraveling advantages of allosteric drugs in comparison to traditional orthosteric compounds. Remarkable simplicity of allosteric effectors and, at the same time, their potentially high specificity is one of the most important traits. The non conserved nature of allosteric ligands is a basis for avoiding drug resistance, and existence of latent regulatory sites make them attractive drug targets. The model presented in this work provides a theoretical framework for the quantification of the causality and energetics of allosteric regulation, which is a prerequisite for design of effector molecules with required characteristics. The synthesis between the thermodynamics of allostery and the intrinsic atomic nature of proteins and their interactions with the allosteric effectors accomplished in this work is a small initial step in the long endeavor towards future allosteric drugs. Activity modulation through ligand binding in sites other than the catalytic ones is a formidable way of performing biological function in allosteric proteins. Consideration of the experimental activity curves  togetherFrauenfelder’s concept of the energy landscape with multiple conformational states –9 compleThe current vision of allostery is based on the understanding that intrinsic protein dynamics is modulated by the ligand binding which affects the structure’s function-related degrees of freedom. Hence, the coupling between the ligand binding and protein dynamics was shown to be instrumental in detecting the allosteric and catalytic sites. The strength of this link can be expressed via binding leverage  that meaIn this work we present a new method for quantifying the configurational work exerted in different parts of the protein as a result of the ligand binding to the allosteric site—the allosteric free energy. Causal effects of the binding is modeled in a statistical mechanical framework by estimating the per residue free energy difference between the ligand bound and ligand free proteins. An allosteric potential based on normal modes is introduced on a “per residue approximation” for evaluating the energetics of structural changes induced by the overall protein dynamics of the protein in the local environment of a residue. Specifically, starting from the protein’s set of low frequency normal modes, the allosteric free energy of the system is obtained by estimating and comparing the partition functions that describe the conformational ensembles of the ligand free and ligand bound states at the single residue level. Thus, this model allows one to analyze a change in the allosteric free energy of a residue caused by the binding of a single ligand or by the sequential binding of several effector molecules.e.g. Aspartate carbamoyltransferase ATCase. A different mode of regulation was found, for example, in NAD-dependent Malic Enzyme (NADME) where binding at the allosteric site induces overall stabilization of the structure, including the functional sites. The inhibition of activity in this protein is supported by the increased dynamics in the catalytic site caused by the binding of ATP effector. The model also successfully discriminates between the negative and positive cooperativity in allosteric regulation, where Catabolite Activator Protein (CAP) and Dihydroxyacetone Kinase (DAK) are examples of the former and Phosphofructokinase (PFK) is an example of the latter. Finally, redistribution of the configurational work in D-3-phosphoglycerate dehydrogenase (PGDH) is strongly influenced by the ring-like shape of this protein, which is an example of the role of topology in allosteric communication.We used here a set of proteins collected on the basis of thorough experimental data with well documented presence of the allosteric mechanisms in the regulation of their functions. We found several ways of regulation, dependent on the structures, oligomerization states, and functions of corresponding proteins. The increase of configurational work at the functional site is an archetypal mode of allosteric communication for hetero-oligomers, Ri and the active Ra states with the Gibbs free energy gain/loss ΔG(Ri → Ra) = −RTln[Ra]/[Ri], where [Ra]/[Ri] is the equilibrium constant for the transition Ri → Ra, R is the ideal gas constant, and T is the temperature. In the event of binding of an allosteric ligand the equilibrium between active and inactive states is shifted, that is the bound states ARi and ARa yield the following free energy difference ΔG(ARi → ARa) = −RTln[ARa]/[ARi]. The global stabilizing/destabilizing effect of the active state caused by an allosteric effector is thus given by the free energy difference ΔΔG(Ra → ARa) = −RTlnα, where α = [ARa][Ri]/[ARi][Ra] reflects the population shift in the active state.A structure-based model of protein allostery should in general address two related issues: (i) providing a quantitative description of the causal effect of allosteric ligand binding on the protein’s structural dynamics and (ii) evaluating the communication between functional and regulatory sites and the modulation of the protein’s functional activity. Thermodynamical description of the protein states, the effects of the ligand binding, and allosteric cooperativity are globally characterized by the free energy changes of the protein. If, for example, thermodynamics of the two state model of allostery is considered, the ligand free protein populates the inactive A bound—A and one functional site F, is a structure-based model that should link the ligand binding at the site A to the energetics of the site F. It is important to emphasize that this free energy is precisely the difference between the work exerted in the catalytic site of the ligand free and ligand bound protein states. We express the free energy gain/loss i belonging to a site F such asnF is the number of residues in the site F. In order to estimate a relative strength of the allosteric effects, the average effect caused by the allosteric binding on all protein residues should be also evaluated:n ligands bound to the protein Protein conformational states are the key determinants in the phenomenological thermodynamic model of allostery . On the Δgi(P→AP)with nP i upon binding an effector molecule to an allosteric site. The method we propose for estimating the allosteric free energy Cα harmonic model constructed on the basis of the available structures. The ligand bound system A. In other words, the stabilization of the allosteric binding site A indirectly models the presence of the actual bound molecule, affecting local dynamics of the protein. Considering all the above, the main task of a structure-based model is the evaluation of the free energy gain/loss for any arbitrary residue allosteric potential, which is the second component of our method. The potential is used for evaluating the energetics that reflects structural differences induced by the different dynamics in the ligand free and ligand bound systems at the single residue level. The per residue allosteric potential used here is:εμ, i are characteristics of the ligand free and ligand bound systems ))Δgi = -3.35 kcal/mol and 1rac: ΔgATP(0 → 6×ATP) = -3.27 kcal/mol) induces an overall freezing of the regulatory monomers R − mer (3d7s: ΔgR − mer(0 → 6×ATP) = -2.02 kcal/mol and 1rac: ΔgR − mer(0 → 6×ATP) = -1.61 kcal/mol, red colored surfaces of the outer dimers, gPAL(0 → 6×ATP) = 1.98 kcal/mol and 1rac: ΔgPAL(0 → 6×ATP) = 1.94 kcal/mol, blue surfaces of the two internal trimers, C − mer (3d7s: ΔgC − mer(0 → 6×ATP) = 1.47 kcal/mol). At the same time, preliminary binding of the inhibitor (CTP in 1rac) practically eliminates a difference between the work exerted at the catalytic sites and the average work exerted in the whole catalytic unit (1rac: ΔgC − mer(0 → 6×ATP) = 1.83 kcal/mol, see also gPAL(0 → 6×ATP) = 1.88 kcal/mol and 1d09: ΔgPAL(0 → 6×ATP) = 1.28 kcal/mol, see gC − mer(0 → 6×ATP) = 1.73 kcal/mol and 1d09: ΔgC − mer(0 → 6×ATP) = 1.18 kcal/mol). These observations show that corresponding structures (7ati and 1d09) are already in the activated state, while binding to the ATP/CTP site in the inactive state (apo form 3d7s) induces a transition from the inactive to active states.Aspartate carbamoyltransferase (ATCase)  is one oAnthranilate Synthase (AnthS) is a hetero-tetrameric enzyme catalyzing reaction that produces anthranilate (BEZ), pyruvate (PYR), and glutamate from two substrates—chorismate and L-glutamine (GLU). Substrates of this enzyme bind to two distinct sites, BEZ, PYR, and GLU , mutuallWith about 3000 residues the bovine glutamate dehydrogenase (BGDH) is the largest complex analyzed in our protein list see .gADP(0 → 6×ADP) = −1.51 kcal/mol) of the configurational work at the activator site ADP induces a small overall stabilization of the protein (Δgnr71(0 → 6×ADP) = -0.16 kcal/mol)—see gGLU(0 → 6×ADP) = -0.88 kcal), whereas NDP site yields lightly increased configurational work (ΔgNDP(0 → 6×ADP) = 0.11 kcal). If the inhibitor (GTP) is bound, the active state is destabilized Δgnr71(0 → 6×GTP) = 0.61 kcal, while the NDP and GLU substrate sites are destabilized less than the units containing them (ghwz1(0 → 6×ADP) = −0.41 kcal/mol) along with quite pronounced freezing of the catalytic sites. These observations are in agreement with the role of activator as a stabilizer of the active apo form (in 1nr7 and 1nqt structures). This homo-hexamer protein catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. It is up-regulated by ADP and down-regulated by GTP. The coenzyme NADP+ that binds in the NAD site is an antagonist. Catalytic sites (where substrates GLU and NDP bind) are located very close to each other in the catalytic cleft. ADP is found in the active and inactive conformations. It supposedly favors transition between two states by facilitating the opening of the catalytic cleft . GTP bining them . Free engB − mer(0 → 1×cAMP) = -0.13 kcal/mol), along with a significant destabilization (ΔgF − mer(0 → 1×cAMP) = 0.24 kcal/mol) of the free monomer, as well as destabilization (ΔgDNA(0 → 1×cAMP) = 0.88 kcal/mol) of the DNA-binding regions. If the cAMP sites are occupied in both monomers the overall conformational work of the whole protein is further reduced Δgwc22(0 → 2×cAMP) = -0.30 kcal/mol, reflecting the loss of structural flexibility in qualitative agreement with the experimental observations. The conformational work at the DNA-binding regions (ΔgDNA(0 → 2×cAMP) = 0.85 kcal/mol) is similar to the value observed for the structure with the only one allosteric site ligated. The difference ΔΔgwc22 = Δgwc22(0 → 2×cAMP) − Δgwc22(0 → 1×cAMP) = −0.54 kcal/mol is consistent with the known negative cooperativity of CAP upon cAMP multiple binding. In the other structures with cAMP bound (1run) and both cAMP and DNA bound (1o3q) a qualitatively similar picture is observed. Analysis of these two forms shows that binding of both cAMPs relaxes the DNA binding site (an increase in flexibility) and facilitates DNA binding. If DNA is already bound, cAMPs binding provides further stabilization of the DNA binding site. In Catabolite Activator Protein (CAP), dimeric transcription factor, is a very well studied example of DNA-binding activity modulation by means of what is commonly understood as dynamically driven  allostery . AllostegPHE(0 → 4×PHE) = −2.33 kcal/mol) in the active form induces an increase of configurational work at the PGA functional site (ΔgPGA(0 → 4×PHE) = 0.33 kcal/mol), which is higher than the overall increase of the mean configurational work Δggg11(0 → 4×PHE) = 0.25 kcal/mol (see 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS) is a key enzyme involved in the biosynthesis of aromatic amino acids such as phenylalanine, tyrosine, and tryptophan. The amino acid products work as allosteric effectors that provide a feedback regulation and inhibit the catalytic activity. We study two DAHPS forms: inactive (PDB ID: 1kfl) and active (PDB ID: 1gg1). The former is crystallized in presence of the allosteric inhibitor-product L-phenylalanine  and substrate phosphoenolpyruvate (active site PGA); the latter—in the presence of the substrate only. The over-stabilization of the PHE site (Δ/mol see .gB − mer(0 → 1×(ADP + ARG)) = −0.72 kcal/mol), increasing the configurational work in the free monomer (ΔgF − mer(0 → 1×(ADP + ARG)) = 0.74 kcal/mol). As a result, the free energy loss by the ligated allosteric site monomer is compensated by the work exerted at the active site monomer. When both active sites are ligated the overall configurational work of the whole protein is greatly reduced (Δgju53(0 → 2×(ADP + ARG)) = −0.05 kcal/mol). The difference, ΔΔgju53(1×(ADP + ARG)→2×(ADP + ARG)) = Δgju53(0 → 2×(ADP + ARG)) − Δgju53(0 → 1×(ADP + ARG)) = −0.79 kcal/mol, reflects a typical phenomenology of the negative cooperativity, and it is consistent with the inhibition mechanisms proposed elsewhere [The dihydroxyacetone kinase (DAK) is a protein kinase that catalyzes the phosphoryl transfer between phosphagen and ADP necessary for the energy exchange in cell metabolism. The enzymatic activity in DAK is homotropically regulated by its substrate that works as a negatively cooperative inhibitor . There ilsewhere . The resgG16(0 → 6×16G) = -0.04 kcal/mol). At the same time, we detected an overall destabilization of the whole complex (Δgcd51(0 → 6×16G) = 0.31 kcal/mol) and the functional sites (ΔgAGP(0 → 6×16G) = 0.37 kcal/mol). The destabilization effect Δgi(0 → 6×16G) is illustrated in Hexaoligomeric glucose-6-phosphate dehydrogenase (G6PD) catalyzes a regulatory step of N-acetylglucosamine catabolism, producing fructose 6-phosphate and ammonia via isomerisation and deamination of the glucosamine 6-phosphate . Activitgefk1(4×FUM) = −0.14 kcal/mol), as well as on the active site NAD (gefk1(4×ATP) = −0.25 kcal/mol), but a mild increase in configurational work at the NAD active site (ΔgNAD(4×ATP) = 0.11 kcal/mol). One can presume, therefore, that the allosteric activation by the fumarate is coupled with a stabilization of the active site, thus providing a proper binding of a substrate. On the other hand, the work increasing at the active site caused by the ATP binding might partially induce inhibition as it would facilitate the substrate release. Similar qualitative results were obtained in the model based on the inactive form  enzyme (NADME) is a homo-tetrameric oxidoreductase that catalyzes decarboxylation of L-malate to pyruvate and the concomitant reduction of the cofactor NAD+ or NADP+. ATP works as a competitive inhibitor of the NAD active site, and it can also bind at the tetrameric interface (site ATP). Fumarate binds at the dimeric interface (site FUM), working as an allosteric activator . We conssite NAD . Interesgxtu1(0 → 4×CTP) = -0.53 kcal/mol), as well as an increase in the configurational work at the functional sites. An increase in the configurational work at the active sites, along with unchanged overall protein stability in the other form  is a homo-tetramer that catalyzes the transformation of 5-phosphate-alpha-1-diphosphate and uracil into uridine 5’-monophosphate (site U5P) and diphosphate. The complex is allosterically inhibited by CTP (site CTP). It has been suggested that CTP binding and consequent structural changes of the complex stabilize the flexible loop in a closed conformation . Four CTgPLP(0 → 1×SAM) = 0.35 kcal/mol). Restraining both SAM sites causes significant stabilization of the dimer (Δge5x1(0 → 2×SAM) = −0.24 kcal/mol), and, at the same time, it increases configurational work at the PLP sites (ΔgPLP(0 → 2×SAM) = 0.52 kcal/mol, gPLP(1×SAM → 2×SAM) = 0.17 kcal/mol) was also observed.Threonine synthase (ThrS) is a homo-dimeric enzyme that is in charge of the final steps of the threonine synthesis (TRS), using the pyridoxal-L-phosphate PLP substrate. It has been shown that allosteric activation caused by the S-adenosylmethionine (SAM) binding triggers a large reorganization of the substrate site in one monomer that induces a conformation change of PLP to an active conformation . Restraigpsd1(0 → 8×SER) = 0.03 kcal/mol), while the substrate sites, in particular NAD (ΔgNAD(0 → 8×SER) = 0.38 kcal/mol), yield some increase of the configurational work. As shown in The D-3-phosphoglycerate dehydrogenase (PGDH) is a ring-shaped tetrameric enzyme that catalyzes formation of 3-phosphohydroxypyruvate from 3-phospho-D-glycerate with NAD as a cofactor. PGDH is allosterically regulated by serine as a cooperative inhibitor. Binding of the pair of serines to dimer interfaces inhibits PGDH, affecting mainly the rate of catalytic reaction . We analgpfk3(0 → 4×PEP/ADPa) = 0.16 kcal/mol), while the F6P sites yield an increase of the mean configurational work (ΔgF6P(0 → 4×PEP/ADPa) = 0.71 kcal/mol) along with mild stabilization of the functional ADPf binding sites (ΔgADPf(0 → 4×PEP/ADPa) = −0.11 kcal/mol). Interestingly, having the only substrate ADPf binding sites ligated not only causes an overall destabilization of the protein (Δgpfk3(0 → 4×ADPf) = 0.50 kcal/mol), but it also results in a significant increase of configurational work at the F6P site (ΔgF6P(0 → 4×ADPf) = 1.74 kcal/mol). Binding at both PEP/ADPa and ADPf sites also leads to a major increase of configurational work at the F6P site (ΔgF6P(0 → 4×(PEP/ADPa + ADPf)) = 2.22 kcal/mol). gADPf(0 → 4×PEP/ADPa) = −0.11 kcal/mol). At the same time, it can work as a modulator increasing configurational work in the functional sites upon binding the ADP to both PEP/ADPa and functional ADPf sites (ΔΔgF6P(4×PEP/ADPa → 4×(PEP/ADPa + ADPf)) = 1.51 kcal/mol). Modulating activity of the PEP/ADPa site agrees with an early detected positive cooperativity in binding of two substrates (F6P and ADP) in presence of the PEP [Phosphofructokinase (PFK) is an important regulatory enzyme of glycolysis and is one of the best studied allosteric proteins . PFK’s s the PEP . PFK’s rThese are two small monomeric proteins for which we analyzed the apo forms and structures with bound inhibitors . Our data also indicated positive cooperativity in PFK. In all cases of activation except BGDH, the activation mode is coupled with increased dynamics at the catalytic site. A combination of the activation with negative cooperativity in CAP and DAK and with positive cooperativity in ThrS was detected. An increase in catalytic site dynamics in BGDH, DAHPS, SSUPRT, PKA, and PTP1B provides an inhibition mode. In AnthS and PFK inhibition is based on stabilization of the catalytic sites (decreased dynamics). We found that either increase or decrease in dynamics of the catalytic site can be associated with activation or inhibition. For example, increase of dynamics can result in activation favoring the binding of substrate, or contribute to inhibition supporting release of the substrate. At the same time, a decrease in dynamics can stabilize the active state of the catalytic site, or can prevent binding of the substrate (inhibition). As a result, any change in a catalytic site’s dynamics is indicative of the regulation, albeit the mode of regulation should be further explored. Another important aspect that should be studied in the future is the issue of agonists/antagonists that bind to the same allosteric site. For example, opposite modes of the regulation were detected, depending on the bound allosteric ligand in ATCase and PFK. Current and future work in this direction includes development of the atomic resolution model that can determine agonistic/antagonistic nature of individual residues and their combinations within the same binding site. Merged with state-of-the-art experimental techniques, e.g. disulfide trapping [There are different scenarios of allosteric regulation in 14 proteins analyzed in this work. Both activation and inhibition modes are observed for BGDH and PFK; only activation modes for ATCase, CAP, DAK, G6PD, NADME, and ThrS; and only inhibition modes for AnthS, DAHPS, SSUPTR, PGDH, PKA, and PTP1B. In CAP and DAK activation modes are coupled with negative cooperativity; in ThrS—with positive cooperativity; and in PFK there are both modes of regulation and positive cooperativity. Since there is no experimental data on free energy changes associated with allosteric signaling at a single residue level, the allosteric free energy obtained in our model can be only qualitatively compared with experimental observations. Computationally obtained free energy changes allow one to conclude on the modulation of dynamics in the catalytic sites under regulation. Positive and negative values of Δtrapping , this aptrapping .The rise in interest in allosteric processes and, as a consequence, turning the focus to the design of allosteric drugs , 44, 22 Cα harmonic model), since the lack of atomic detail affects the quality of the allosteric effects’ estimation. An indirect modeling of the binding effect can be considered as both a limitation and an advantage of the model. First, it does not take into account the structure of the ligands or the actual set of interactions in the binding sites. On the other hand, the indirect way of mimicking the ligand binding is a generic framework that can be applied to different types of proteins without any preliminary knowledge of the allosteric sites. While the model could benefit from empirical parametrization on the basis of experimental observations, it provides an energy estimate of the allosteric signaling.The structure-based statistical mechanical model of allostery introduced here consists of three key components: (i) it models the effector binding via local perturbation/stabilization of the protein harmonic model; (ii) per residue allosteric potential estimates structural changes due to the effect of protein dynamics on the residue environment; (iii) statistical mechanical ensemble treatment provides a quantification of the allosteric free energy obtained from the per-residue partition functions in the ligand free and bound states. The model is applied to a large set of allosteric proteins previously analyzed in the context of allosteric site prediction . The lisground statemodel, as the reference structure used in a harmonic model of a protein is the same in the ligand free and ligand bound systems. The free energy difference between the ligand free and ligand bound states is chiefly determined by the contribution of the configurational entropy. Allosteric communication detected by the model is indicative of the signaling between the regulatory and functional sites in both directions. Therefore, perturbation of the functional site can point to the potential locations of relevant the allosteric ones. As a result, the perturbation of the functional sites would allow one to determine the allosteric sites that modulate activity of corresponding catalytic sites, to identify specifics of the allosteric communication at the atomic level resolution, and to discriminate between the agonism and antagonism of allosteric effectors.To conclude, this approach should be regarded as a Cα harmonic model. Despite this radical simplification, harmonic models are surprisingly powerful in describing the large-amplitude protein motions based on the consideration of the low-frequency normal modes [Cα atoms isr is the 3N-dimensional vector of coordinates of the Cα atoms, r0 is a vector of Cα positions of the reference structure, dij is the Euclidian distance between the Cα atoms i and j, kij is the distance-dependent force constant decaying as n ligands bound to sites s1, …, sn. We define the latter by adding a binding site’s stiffening harmonic term to the energy function of the free system, that iss and α > 0 is the stiffening factor. The effect of ligand binding is indirectly modeled as a stabilization effect on the atoms that belong to the binding site of interest. Thus, the factor α gives the extent to which these indirect interactions affect the atoms of the binding site. To ensure a pronounced allosteric effect, a stiffening factor α = 100 was chosen based on the analysis of the allosteric free energy profiles obtained for the list of proteins (see K(0) = ∂2E(0)/∂ri∂rj and Kn)( = ∂2En). In ordeins see . From thperturbation-based harmonic models have been employed in previous works [The additional harmonic terms in us works –55 with i of the protein. In order to do that, we introduce an allosteric potential of residue i determined by the normal mode μ as:c = 1 kcal/mol/Å2, cd = 11Å a distance cutoff, Cα distances in the reference structure R, and Cα distances as a result of the normal mode μ. Thus, the potential evaluates the total elastic work that is exerted on the residue i as a result of the deforming action of a normal mode μ on its neighboring residues j. The idea behind the allosteric potential per residue i and mode μ is to compare the different deforming effects on a residue i induced by the normal mode of the free system  undergo different displacements upon a normal mode μ, changing the inter-residue distances In the previous section the sets of normal modes were obtained for the ligand free and bound protein states. Here, the normal modes are used as a basis for defining an allosteric potential in the context of a statistical mechanical framework. We are interested in evaluating the effect of the ligand binding on an arbitrary chosen residue i due to mode eμ is i and σμ is a gaussian coefficient. Thus, the allosteric potential in n ligated binding sites only)σμ and residue intensity parametersi isσ =  is a set of gaussian coefficients that can be regarded as a configurational state of a residue i.The displacement of the residue G = 1/2kBT∑μ lnλμ + const, with λμ the eigenvalues of the Hessian matrix K of the model (ωμ the frequency associated to the mode μ). Thus, the free energy difference between the state with n bound ligands m ligands bound m < n. The relations in Eqs λμ(0)and the )(λμ(m))2where ani in the ligand free and n-ligated states of the protein, respectively. The set of coefficients σ =  in σμ are gaussian variables with zero mean and variance 1/εμ, i εμ,i(0)and the )εμ,i(m)2with theThe obtained free energies in the eqs  and 17)17) clearThe method introduced in this work is applied to the list of allosteric enzymes used in previous studies in the context of binding leverage and leverage coupling calculations , 21. MosCα effective harmonic model described in [The protein modeling as well as the normal mode calculations were carried out with the MMTK package . The Cα ribed in  was usedribed in . The MMTS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 TableThe table includes the complete data obtained for all available forms of proteins. For designation of columns see (PDF)Click here for additional data file.


Head-up tilt test is useful for exploring neurally mediated syncope. Adenosine is an ATP derivative implicated in cardiovascular disturbances that occur during head-up tilt test. The aim of the present study was to investigate the impact of hyperoxia on adenosine plasma level and on hemodynamic changes induced by head-up tilt testing.2 = 21%) and 1 session with administration of pure oxygen (FiO2 = 100%) in double-blind manner. Investigations included continuous monitoring of hemodynamic data and measurement of plasma adenosine levels.Seventeen healthy male volunteers (mean age 35 ± 11 years) were included in the study. The experiment consisted of 2 head-up tilt tests, 1 session with subjects breathing, through a mask, medical air  and during head-up tilt test (0.33 ± 0.09 μM) when compared with baseline (0.6 ± 0.1 μM). Adenosine plasma level was unchanged during medical air exposure at rest (0.6 ± 0.1 μM), and slightly decreased during orthostatic stress. In 2 volunteers, the head-up tilt test induced a loss of consciousness when breathing air. In these subjects, adenosine plasma level increased during orthostatic stress. In contrast, during hyperoxic exposure, the head-up tilt test did not induce presyncope or syncope. In these 2 volunteers, biological study demonstrated a decrease in adenosine plasma level at both baseline and during orthostatic stress for hyperoxic exposure compared with medical air.These results suggest that hyperoxia was able to increase blood pressure during head-up tilt test via a decrease in plasma adenosine concentration. Our results also suggest that adenosine receptor antagonists are worth trying in neurocardiogenic syncope. Indeed, strong adenosine release is associated with hemodynamic disturbances during HUT.4 Furthermore, there is a positive correlation between the increase in adenosine plasma level (APL) and the rapidity of onset of tilt-induced syncope.4 Lastly, an overexpression of adenosine A2A R has been reported in patients with unexplained syncope and a positive HUT.7Head-up tilt test (HUT) is a useful tool for exploring neurally mediated syncope.8 An increase in APL has been associated with bradycardia and loss of consciousness during experimental hypoxia induced by breath-holding.9 Altitude-induced hypoxia alters cardiovascular response to orthostatism,10 and experimental hypoxia favored vasovagal reaction via a vasodilation mechanism.11 Conversely, hyperoxia is associated with a decrease in extracellular adenosine level.12 Consequently, partial pressure of oxygen (PO2) impacts cardiovascular status, partly via the modulation of extracellular adenosine level. It was shown that hyperoxic chemoreflex sensitivity is impaired in patients with neurocardiogenic syncope,13 but the influence of hyperoxia on HUT had never been evaluated. The aim of this study was, therefore, to assess the effects of oxygen administration on both the hemodynamic status and the APL during HUT in healthy volunteers.Adenosine is released in the extracellular spaces during hypoxia or inflammatory process.According to previous knowledge, our hypothesis was that hyperoxic exposure increased blood pressure (BP) during HUT when compared with medical air exposure, via its vasoconstrictor effect and a decrease in APL.15 To exclude this potential bias, only men were included in the study.Seventeen male volunteers (mean age 35 ± 11 y) were investigated to assess the cardiovascular impact of hyperoxia during abrupt changes in position. In women, the fluctuations in hormones during the menstrual cycle lead to variations in central arterial compliance and sympathetic neural responses to orthostatic stress.The study was performed in a double-blind manner, and the protocol was approved by the local ethics committee . Informed consent was obtained from volunteers, and the study was carried out in accordance with the WHO code of ethics (Declaration of Helsinki).2 = 21%) and 1 session with administration of pure oxygen (FiO2 = 100%), in a random order  was recorded using an analog 3-lead ECG . BP was continuously monitored using a finger cuff . The arm was supported to maintain the transducer at the heart level. HR and BP data were the average of the recordings during the last 5 minutes of each period .4 The sample collection method allowed whole blood to mix quickly with the stop solution to prevent red blood cell uptake and degradation of adenosine.17 The stop solution was composed of dipyridamole (0.2 mM); ethylene diamine tetracetic acid disodium (Na 2 EDTA 4 mM); erythro-9-(2-hydroxy-3-nonyl adenine [EHNA 5 mM]); alpha, beta-methyleneadenosine 5’ diphosphate (AOPCP 79 mM); 2’ deoxycoformycine 10 μg/mL; and heparin sulfate 1 IU/mL. Adenosine plasma concentration measurement has been described.17 In brief, after collection, samples were put in ice, carried to the laboratory, quickly centrifuged, and deproteinized (perchloric acid 70% 1/10 V). Supernatants were analyzed by high-pressure liquid chromatography . The intra and interassay coefficients of variation ranged from 3% to 5%.Blood samples (3 mL) were collected through the catheter and processed as described previously using laboratory-prepared tubes containing 2 mL of cold-stop solution under vacuum.18 All data  recorded during baseline were subtracted from data recorded during rest, tilt, and recovery to produce delta values. Delta values were chosen because the sessions  were not performed the same day. Consequently, the hemodynamic data recorded at baseline could be slightly different. Linear mixed model (LMM) analyses were performed with R package “lme4.”19 The mixed model approach was chosen over generalized linear model  for the reason that it allows post hoc analysis and is adapted to crossed random effects—here, time and subject.20 Least-squares mean calculations and post hoc comparisons were performed with R package “lsmeans,”21 with Bonferroni adjustment for P value adapted for multiple comparisons.All the statistical analyses were performed with R statistical software.For BP, HR data, and plasmatic adenosine level, several LMMs were compared, and the most appropriate model (based on lowest Akaike Information Criterion and Bayesian Information Criterion) was the following: “gas type,” “protocol period,” and interaction between “gas type” and “protocol period” for fixed factors; random factors were “subject” and the interaction between “protocol period” and “subject.”Fifteen volunteers presented no clinical manifestations during the experiment, whereas 2 experienced presyncope or syncope during HUT.Head-up tilt test was well tolerated by 15 volunteers. Table Fadjusted  = 8.54, P = 0.0056). No difference was found for delta SBP between the protocol periods . No interaction between “gas type” and “protocol period ” was found for delta mean BP .Figure Fadjusted  = 1.73, P = 0.195). During protocol period , delta DBP tended to be higher during HUT than recovery  and rest .Delta diastolic blood pressure (DBP) was not significantly different during hyperoxia and medical air  during medical air exposure. During HUT, delta HR was higher  than during other periods , but no difference was found between recovery and rest . No interaction effect between “gas type” and “protocol period ” was found .Figure Fadjusted  = 107.16, P = 0.0000) was lower during hyperoxia than medical air exposure. Delta APL was lower  during HUT than in recovery  and rest period . A drop of delta APL was found under medical air exposure during HUT with restoration during recovery , but not under pure oxygen .Figure 22In 2 volunteers, presyncope or syncope appeared during HUT. In these two subjects, the timing of faint argues in favor of vasovagal symptoms rather than orthostatic hypotension.Volunteer 1 was a healthy student in sport sciences . Under the first test with hyperoxic exposure, the test was well tolerated until the duration initially planned (45 min). During the medical air session, after 8 minutes and 30 seconds in the upright position, the volunteer felt like passing out. A marked decrease was recorded in both HR (from 79 to 41 beats/min) and SBP (from 110 to 60 mm Hg). HUT was immediately terminated and the subject recovered spontaneously.Volunteer 2 was a well-trained breath-hold diver . He was first submitted to the test with medical air exposure. During this test, a syncope appeared 11 minutes after the passage in upright position. Simultaneously, a sudden drop was recorded in SBP (from 110 to 69 mm Hg) and in HR (from 90 to 56 beats/min). The volunteer experienced a second test under hyperoxia, 1 week later. To compare the APL at the moment of the syncope, the blood sample was collected 11 minutes after the passage in head-up position. No clinical or hemodynamic impairment was observed during this test. Interestingly, a third HUT was performed in this volunteer in the course of another experiment. It was positive with medical air breathing. Table The study presents for the first time the impact of hyperoxia on both hemodynamic data and APL in healthy subjects submitted to orthostatic stress. In our study, hyperoxic exposure improved hemodynamic status during HUT.Systolic BP was increased during hyperoxic session when compared with medical air session in the 15 volunteers with normal tolerance to HUT Figure . A drop 23 It was reported that the difference in the R-R intervals before and after oxygen inhalation (for 5 min), divided by the difference in the oxygen pressure, evaluated in the venous blood, is lower in neurocardiogenic patients than healthy subjects, suggesting that in these patients, chemoreflex sensitivity is impaired in hyperoxic conditions.13 This mechanism may participate in the improvement in orthostatic tolerance of HUT during oxygen exposure. Our results suggest that the improved orthostatic tolerance secondary to oxygen breathing can also be attributed to the vasoconstrictor effect of hyperoxia.25 The mechanisms explaining the vasoconstriction induced by hyperoxia remain unclear. Some studies have implicated a direct effect of O2 or reactive O2 species on vascular smooth muscle through an action on calcium channel.27 Other studies have reported an alteration in endothelial-derived vasoactive agents, such as nitric oxide, Prostaglandins, or endothelin-1.31 Here we found that hyperoxia was associated with a decrease in APL.Appropriate cardiovascular adaptation to the passage from supine to standing position includes increase in HR and vasoconstriction. In some subjects, the cardiovascular response is inappropriate and it is well-recognized that fainting and syncope, during a passive transition from supine to upright position, are secondary to a marked decrease in cerebral blood flow secondary to a paradoxical triggering of bradycardia and/or vasodilatation. These abnormal cardiovascular responses are the essential features of reflex syncope and particularly vasovagal syncope.4 may contribute to the cardiovascular adaptive response to orthostatism, probably via a quick adenosine uptake by blood cells.An increase in APL was recorded in upright position in the 2 volunteers with a positive HUT under medical air exposure Table . This de2. It has been shown that experimental hypoxemia leads to adenosine plasma release, favoring bradycardia and loss of consciousness.9 The main cardiovascular changes induced by an increase in APL included decreased HR through A1 receptor stimulation and vasodilatation via extraluminal A2A adenosine receptors. These alterations are able to counteract the appropriate cardiovascular adaptation to orthostatic stress in subjects with vasovagal reaction.The extracellular generation of adenosine by nucleotidases is facilitated by the reductions in extracellular pH and in POSeventeen volunteers were investigated in this study. Two of them experienced presyncope or syncope during HUT. To strengthen our results, a larger sample is needed. Also, further work should study the impact of hyperoxia on the tolerance to orthostatic stress and APL in subjects with vasovagal syncope.In our study, oxygen breathing led to a decrease in APL, when compared with medical air breathing. This decrease was particularly large in upright position Table , and couThe authors gratefully acknowledge the volunteers and Mrs Karine Ayme for technical support.


We consider interventions with hypothetical CTL-inducing vaccines in a range of epidemiologically plausible pandemic scenarios. We estimate the achievable reduction in the attack rate, and, by adopting a model linking epidemic progression to the emergence of IAV variants, the opportunity for antigenic drift. We demonstrate that CTL-inducing vaccines have limited utility for modifying population-level outcomes if influenza-specific T cells found widely in adults already suppress transmission and prove difficult to enhance. Administration of CTL-inducing vaccines that are efficacious in "influenza-experienced" and "influenza-naive" hosts can likely slow transmission sufficiently to mitigate a moderate IAV pandemic. However if neutralising cross-reactive antibody to an emerging IAV are common in influenza-experienced hosts, as for the swine-variant H3N2v, boosting CTL immunity may be ineffective at reducing population spread, indicating that CTL-inducing vaccines are best used against novel subtypes such as H7N9. Unless vaccines cannot readily suppress transmission from infected hosts with naive T cell pools, targeting influenza-naive hosts is preferable. Such strategies are of enhanced benefit if naive hosts are typically intensively mixing children and when a subset of experienced hosts have pre-existing neutralising cross-reactive antibody. We show that CTL-inducing vaccination campaigns may have greater power to suppress antigenic drift than previously suggested, and targeting adults may be the optimal strategy to achieve this when the vaccination campaign does not have the power to curtail the attack rate. Our results highlight the need to design interventions based on pre-existing cellular immunity and knowledge of the host determinants of vaccine efficacy, and provide a framework for assessing the performance requirements of high-impact CTL-inducing vaccines.Vaccines that trigger an influenza-specific cytotoxic T cell (CTL) response may aid pandemic control by limiting the  Producing the current generation of IAV vaccines—which reduce host susceptibility by inducing antibodies against viral surface proteins—requires antigenic characterisation of the IAV strain. Intensive global surveillance of seasonal strains enables predictions for the dominant IAV in the coming influenza season, facilitating the production of sub unit B cell vaccines with average efficacy of approximately 60 per cent in adults aged 18–65 , may reqPrime-challenge experiments in animal models demonstrate that CTLs induced by exposure to a heterologous strain reduce viral loads and disease severity , 10. WhiRecent cohort and challenge studies have established that influenza-specific T cells that cross-react with pandemic and seasonal IAV are present in most adults without relevant strain-specific antibodies , 21–23. et al. identified late effector cytokine secreting influenza-specific T cells as correlated with reduced severity of pH1N12009 infection  respectively denote the infectiousness of vaccinated naïve, unvaccinated experienced and vaccinated experienced hosts relative to unvaccinated naïve hosts. Variables are expressed in numbers of hosts unless otherwise specified. Our choice for Te and Ti yields a generation time between successive cases of 2.9 days, consistent with average estimates from pH1N12009 surveillance data [Te and Ti.Our model has 4 main classes of hosts, each occupying an nfection , 54). Wey period , 56), annce data –59, howefE of hosts are “influenza-experienced”; having had a previous infection with IAV that confers cross-reactive cellular, and potentially also cross-reactive humoral, immunity to the pandemic virus. The remaining hosts are denoted “influenza-naïve”. The strength of the cellular immunity amongst influenza-experienced hosts ϵ and the prevalence of the humoral immunity amongst influenza-experienced hosts are specified independently of fE (see below). With the simplifying assumption that lifetime risk of infection increases with age, our choice of fE = 0.8 roughly translates to assuming that hosts older than 15 have had an infection with IAV . Alternate values for fE are explored in a sensitivity analysis. The population size N is nominally chosen to be 1 million.We assume that a fraction mNN = mEE = mNE = mEN = 1) or weakly assortative mixing with mEE = 0.7, mNN = 1.4, mEN = mNE = 1. The latter choice mimics the increased contact rate between younger hosts and between adults and younger hosts seen in survey data [We explore results assuming homogeneous/random mixing , but consider alternative vaccine efficacy assumptions in experienced hosts between the two scenarios (see below). Such protection may reflect the distribution of effective cellular immunity if the influenza-specific T cells found in most adults [ϵ = 0.5).In the absence of firm knowledge of the immunological determinants of host response to a CTL-inducing vaccine, and the epidemiology associated with CTL-immunity, we explore a range of assumptions for prior immunity and vaccine action. In particular, we consider a scenario in which the population has no prior cellular or humoral immunity (A). Such an assumption is likely appropriate if the pandemic strain is of novel subtype and if influenza-specific T-cells commonly found in the population are alone insufficient to suppress transmission from infected hosts (perhaps because elevated levels of effector T cells are required ). We alst adults , 21–23 rS2 = (1 − fE)N and S3 = fEN (Scenarios A–C). Hosts with pre-pandemic sterilising immunity begin in the compartment R3 in our model, yielding initial conditions S2 = (1 − fE)N, S3 = fEN/2 and R3 = fEN/2 for scenario D. Variations on these scenarios are explored in the sensitivity analysis.Initially all states are empty except for ϵv. In scenario A, when there is no pre-pandemic immunity, the infectiousness of all vaccinated hosts is uniform. In scenario B we consider a ‘saturating’ vaccine that may not boost existing CTL responses ). Such vaccine action may be representative of the action of live influenza vaccines able to induce T-cell responses in children more readily than in adults [i.e.ϵN = ϵv, ϵE = ϵϵv). Viral vector vaccines that show promising results in adults may have such an action if they prove efficacious in children. Lipopeptide vaccines may also have the potential to boost CTL immunity in hosts with a range of pre-existing naïve and memory CTL pools, depending on their design [We denote the infectiousness of vaccinated naïve hosts relative to unvaccinated naïve hosts n adults . In scenr design . Table 1ϵv . This parameter can be considered to subsume both ‘uptake’ (vaccinated proportion) and ‘take’ (effectively immunised proportion) given the anticipation of only partial population coverage, resulting from HLA class restriction of CTL epitopes.For simplicity we assume that vaccine protection lasts for the duration of the simulation. We explore rapid vaccine distribution to the general population early in the pandemic (with baseline assumption that vaccines are distributed over 10 days) at a range of coverage levels, or pre-emptive targeted distribution of a stockpile allowing 50 per cent coverage. As vaccines inducing T cell responses are still in the design phase, we explore the full range of possible values for R0—indicating the average number of transmitted infections per infected host—expected for future pandemic outbreaks is uncertain. Inferences using mortality and morbidity data from the 1918 Spanish influenza pandemic suggest that R0 may be as high as 4–6 [The value of the basic reproduction number h as 4–6 . The meah as 4–6 . Estimath as 4–6 , and theh as 4–6 , also raReff = 2. At the time of emergence of a pandemic virus, the existing immune status of a given population is unknown, allowing estimation of Reff but not R0 from early observations of the epidemic growth rate [Reff = 2 prior to intervention, enabling us to consider the influence of underlying prior immunity on vaccine impact for epidemics that may otherwise appear the same. We explore more severe pandemic scenarios, and a scenario in which the final size of the epidemic, rather than the initial growth rate, is held fixed in the sensitivity analysis. Reff depends on the population susceptibility profile (including ϵ and fE) and is the maximum eigenvalue, M, of the matrix constructed by multiplying each row i of βij (Si(t). In scenario A the absence of any pre-pandemic immunity renders the basic and effective reproduction numbers equal (i.e.R0 = Reff). To fix Reff between scenarios the factor bf must vary. If there is pre-existing cellular or humoral immunity, R0>Reff and βf is higher than for a scenario without prior immunity. Assuming homogeneous mixing βf = Reff/(NTi) for scenario A, βf = 5Reff/(3NTi) for scenarios B & C, and βf = 5Reff/(2NTi) for scenario D at baseline (i.e. unimpeded by vaccination).We consider a pandemic virus that at pandemic onset and prior to vaccination spreads at a moderate rate wth rate . For thi of βij  by Si(tt → ∞∑iRi(t) or equivalently, The proportion of the population infected over the course of an outbreak is known as the ‘final epidemic size’ or ‘attack rate’ (despite not being a ‘rate’). We adopt the latter terminology here. We evaluate it numerically as limChanges in the pandemic virus may have been responsible for the increased severity of the second wave of the 1968–9 H3N2 pandemic . ChangesIi has the potential to transmit a new variant. We calculate the “drift opportunity” Pemg—the cumulative opportunity for drift variants to emerge over the course of the epidemic—by integrating over all infections as below:tend corresponds to the resolution of the epidemic. Note that we only report dimensionless values of Pemg that are normalised by the value for the same epidemic without intervention. Pemg > 1 is possible and indicates an epidemic in which drift opportunity under intervention was greater than than in the absence of intervention. The non-linear dependence on time in Pemg > 1 is motivated by results from theoretical models for antigenic drift suggesting that new variants may be more likely to circulate later after introduction of the initial strain [et al. [te is the time since the cumulative incidence reaches 1 (host) in our deterministic model.To estimate the opportunity for antigenic drift, we assume that each infectious host in each stratum l strain , 71. The [et al. . We havegi0, in i. Arinaminpathy et al. assume that the per infection rate generating new variants depends only on cluster age [i.e.gi0, = 1). However, the emergence and thus transmission of variants from a host may be more common when infections are prolonged and viral replication occurs in the presence of sterilising or cross-reactive antibody [gi0, = 1 (per infection rate of emergence of new variants independent of vaccination and pre-pandemic immune status), (ii) gi0, = ϵi , and (iii) the distribution g0,1 = g0,2 = g0,3 = ϵi, g0,4 = 0 . Here ϵi is the infectiousness of hosts in the ith stratum. It is also possible in principle that CTL responses may enhance selection of novel variants, in which case scenarios with gi0, > 1 may be justified. Such a possibility is not explored in this work.The factor ster age   is selected, we consider this ratio at the conclusion of the pandemic. The effective reproduction number of the emerging strain Reff,new is calculated assuming that it escapes all antibody-mediated immunity remnant after resolution of the initial pandemic wave. The ratio Reff,new/Reff naturally increases with the prevalence of immunity to the resident pandemic strain.Antigenic variants may have mutations that confer increased virulence/transmissibility and/or escape recognition of antibodies to the original pandemic virus and thus facilitate the process of antigenic drift. We focus on the selective advantage of the latter type of antigenic variant, assuming that the intrinsic transmissibility is unchanged from that of the original pandemic strain. The selective advantage of a new variant is largely governed by the ratio of effective reproduction numbers for the replacing and resident strains . To gaugOur predictions for the attack rate as a function of CTL-inducing vaccine coverage vary significantly with underlying population immunity and vaccine action for pandemic scenarios A–D with the same initial growth rate. The mitigating potential is very similar for homogeneous and assortative mixing scenarios when vaccines are distributed uniformly amongst the population see .ϵv = 0.25 relative to unvaccinated naïve hosts, can mitigate the pandemic with 65 per cent coverage. Attack rates are approximately halved to 40 per cent if coverage with such a vaccine is 50 per cent.In this subsection we focus our discussion on the homogeneous mixing case  but highReff. Note that this is only strictly true when mixing is homogeneous as the introduction of assortative mixing influences the value of R0 for fixed Reff, however the variation between scenarios A–C is very small for our choice of assortative mixing , predictions for vaccine impact are more promising. Indeed the mitigating impact of vaccination campaigns achievable in scenario A is recovered.For scenarios in which there is only prior cellular immunity to the pandemic strain (B & C) baseline attack rates are equal to that for scenario A for fixed xing see . In scenN. Baseline attack rates amongst the naïve subpopulation however are equivalent to those in scenarios A–C when mixing is random  is required to halt the pandemic than for scenarios A or C, as many vaccines are ‘wasted’ preventing transmission from hosts who are not susceptible . The impact of this targeted distribution when mixing is homogeneous is depicted in ϵv ≳ ϵ and the vaccine cannot reduce the potential infectiousness of experienced hosts. However, reduced attack rates when targeting naïve hosts are apparent even when the vaccine suppresses the transmission potential by the same factor in naïve and experienced hosts (scenarios C & D). Assuming Reff = 2 and a vaccine stockpile sufficient for 50 per cent population coverage, distribution of a vaccine that completely blocks transmission (ϵv = 0) won’t halt population spread unless ≳50 per cent of naïve hosts are vaccinated in scenarios B-D  shows a similar trend across scenarios B–D (ϵv < 1), vaccines.We explore the impact of distributing vaccines to varying proportions of naïve and experienced hosts assuming both homogeneous and assortative mixing for scenarios B–D . Adopting assortative mixing (as described earlier) instead of homogeneous mixing also increases the predicted benefit of reducing the coverage amongst influenza-experienced hosts for low to intermediate values of ϵv —as assumed in previously published work [ϵv = 0.75) for all scenarios, and remains approximately equal to that for an unimpeded pandemic in scenario B when ϵv ≳ 0.5. Assuming a boosting vaccine and no strain-specific humoral immunity (scenario C), 50 per cent coverage with a vaccine reducing the infectiousness of naïve hosts by ϵv = 0.25 reduces the drift opportunity to approximately 75 per cent of that for an unimpeded epidemic  than if this per infection rate is uniform . General population vaccination achieving 50 per cent coverage (i.e. exp coverage = 0.5) using a vaccine with ϵv = 0.25 can reduce drift opportunity to approximately 47 per cent of that for an unimpeded epidemic in this situation , then the drift opportunity is further reduced compared to the scenario where gi0, = ϵi for ϵv ≳ 0.2  if naïve hosts rather than experienced hosts are targeted. If ϵv ⪅ 0.25 the drift opportunity remains more efficiently suppressed by targeting naïve hosts to curtail the epidemic size. This trend holds for our assumed choice of assortative mixing (data not shown). Targeting experienced hosts has similar benefits for suppressing drift opportunity for moderate to high values of ϵv in scenario B, but has muted benefit in scenario D due to the smaller contribution of experienced hosts to the overall attack rate (data not shown). Note that for the final choice of gi0, presented  can completely suppress the emergence of new variants in experienced hosts. In practice a threshold vaccine efficacy may be required to generate such an effect.If CTL vaccination completely suppresses the per infection probability of emergence of variants from vaccinated experienced hosts , potentially triggering a second wave of severe infection unconstrained by the cross-reactive antibody that limited the initial wave. If a CTL-inducing vaccine offers truly heterologous protection that suppresses hosts potential to transmit all IAV strains equally, increasing vaccination coverage cannot erase the advantage afforded to antigenically novel strains that emerge in the presence of immunity-driven selective pressures unless they halt almost all transmission thereby rendering Pemg negligible . HoweveIn the above sections we have explored the sensitivity of expected vaccine impact—as measured by the attack rate and/or drift opportunity—on underlying population immunity, population mixing characteristics and vaccine action. Here we additionally consider the sensitivity of our results to the transmissibility of the pandemic virus, the period of vaccine distribution, and alternate profiles for pre-pandemic population immunity for the homogeneous mixing case.Reff), resulting in baseline epidemics with considerably smaller attack rates for scenario D due to the presence of sterilising antibodies reducing the size of the susceptible pool. If we instead assume a fixed baseline attack rate across scenarios , the additional coverage required to halt an epidemic in scenario D relative to scenarios A–C is even more pronounced than for the fixed initial Reff scenarios . This phenomenon is not observed for our nominal choice of Reff = 2 .Increasing vaccine coverage slows the rate of transmission, which while reducing the attack rate, also prolongs the epidemic. The higher probability of generating new variants later in an epidemic in  = 2 see . HoweverReff = 2, 4 in Reff) highlighting the value of rapid or pre-emptive vaccine distribution. As epidemics are resolved more rapidly when vaccination does not immediately reduce the effective reproduction number, the enhancement of drift opportunity with increased coverage seen for a severe scenario is diluted when vaccine distribution is prolonged (We have thus far assumed vaccines are distributed rapidly upon emergence of the pandemic (at a constant rate over 10 days) when exploring the impact of vaccine coverage. However the impact of vaccination campaigns on attack rates and epidemic duration is naturally less pronounced when vaccines are distributed over a longer-timescale. Focusing on scenario C, we show the effect of increasing the duration of vaccine distribution for rolonged .fE. Values of fE less than our baseline value of 0.8 may, for example, reflect population immunity if late effector cytokine secreting T cells found to correlate with reduced viral shedding [If population mixing is assumed to be homogeneous, we can decouple the concepts of youth and naïvety in our model, and freely vary the experienced proportion shedding  are rareshedding ).ϵv), attack rates for fixed initial Reff are independent of the prevalence (fE) and strength (ϵ) of suppressed infectiousness due to cellular immunity acquired from pre-pandemic seasonal IAV infection(s). If vaccine action is saturating, or doesn’t scale with pre-pandemic host CTL immunity in another fashion, characterising the landscape of underlying cellular immunity (determined by fE and ϵ in our model) is crucial for predicting the impact of a vaccine on the attack rate. ϵ, fE and ϵv; as ϵv increases the benefit of vaccination campaigns becomes increasingly dependent on having a low fraction of experienced hosts (low fE) and/or poor immunity amongst these hosts (high ϵ).For scenarios A and C  motivated by potential CTL-inducing vaccine candidates. Our analysis is novel in its aims to capture the current immunological understanding of cellular immunity in population-level IAV pandemic transmission models. Doing so enables us to highlight the uncertainties in predicting the impact of wide-spread use of CTL-inducing vaccines against a pandemic virus and provide a valuable reference for informing the performance requirements of high impact CTL-inducing vaccines.i.e. effectively assumed to be older) sub-populations, but not the strength and extent of cross-protective cellular immunity acting only on host infectiousness (i.e. large fE), effective (i.e. ϵ < 1), and difficult to boost with vaccines (i.e. moderate ϵv) as explored in scenario B, general population vaccination campaigns will be inefficient at controlling the spread of an emerging pandemic virus.We have focused on the pandemic attack rate and drift opportunity as proxies for the impact of an intervention with CTL-inducing vaccines. By fixing the initial pandemic growth rate but altering the underlying population immune profile, we have shown that if using a boosting vaccine , the anticipated attack rate of a pandemic outbreak depends strongly on the extent of sterilising cross-reactive antibody in influenza-experienced . Vaccinating influenza-naïve hosts most likely to transmit has the largest impact on the attack rate, even when there are no other differences in the immune status or mixing behaviour of naïve and experienced hosts. If influenza-naïve hosts are typically children, targeting such sub-populations is likely to be of enhanced benefit for controlling the overall outbreak when taking into account their increased mixing intensity . If a suReff .Reff. If the per infection probability of emergence of antigenic variants is uniform across the population, drift opportunity is difficult to suppress unless the pandemic can be mitigated. If there is little pre-existing CTL-mediated immunity or the vaccine is able to boost this further in most hosts (scenarios A and C), then extensive CTL-inducing vaccination campaigns may significantly decrease the attack rate and modestly reduce the opportunity for antigenic change. In such scenarios prolonged use of a CTL-inducing vaccine would likely modestly slow the rate of emergence of antigenically novel clusters [We have demonstrated that depending on the underlying population immunity and vaccine action, vaccination campaigns may have very different impact on the opportunity for the pandemic virus to undergo antigenic change over the pandemic season, for fixed initial growth rate clusters . Howevergi0,) scales with host infectiousness and/or the rapidity of viral clearance, the probability of antigenic replacement may decrease more rapidly with coverage than predicted by Arinaminpathy et al. [Uncertainties in the role of enhanced CTL responses in altering the within-host generation and selection of antigenic variants further cloud expectations for the long-term role of CTL-inducing vaccines on antigenic evolution. If the per infection probability of generating new variants . EpidemConsideration of the utility of multi-faceted public health responses that use CTL-inducing vaccines to buy time before a strain-specific vaccine can be distributed, and/or use of CTL-inducing vaccines in tangent with traditional anti-viral drugs, are also of interest. In future work we will explore strategies for the optimal use of CTL-inducing vaccines that take into account sociologically important differences in mixing behaviour for adults and children in addition to differences in immunological memory to IAVs.S1 FigReff = 2. Red lines indicate attack rates for naïve hosts, blue lines for experienced hosts, and black lines indicate population-average values.Predicted attack rate as a function of general population vaccination coverage for scenarios A–D (left to right) assuming (EPS)Click here for additional data file.S2 FigReff = 2 and random (left) or weakly assortative mixing (right) as described in the main text. As for the figures in the main text, solid lines indicate ϵv = 0.25, dashed lines ϵv = 0.5 and dotted lines ϵv = 0.75. Red lines indicate attack rates for naïve hosts, blue lines for experienced hosts, and black lines indicate population-average values. The grey lines show the results for scenario C with the same initial effective reproduction number and homogenous mixing.Predicted attack rate as a function of general population coverage for scenario D assuming (EPS)Click here for additional data file.S3 FigReff = 2 in scenarios B (left), C (middle) and D (right). The axis “exp coverage” indicates the fractional coverage in influenza-experienced hosts, with exp coverage = 0.5 corresponding to general population distribution of vaccines.Epidemic attack rates for targeted distribution of a vaccine stockpile enabling 50 per cent population coverage assuming weakly assortative mixing (as described in the main text) and (EPS)Click here for additional data file.S4 Figϵv and 50 per cent coverage) where influenza-experienced hosts are preferentially targeted  for scenarios B–D. Upper panels correspond to assumptions of homogeneous mixing and lower panels for weakly assortative mixing as described in the main text. Note that this figure is an alternate representation of the results in ϵv for each set of scenarios arises due to the benefit of redirecting an imperfect vaccine to the sub-population more likely to transmit infections.Absolute decrease in the attack rate (coded by the coloured legend) compared to a like scenario (with the same (EPS)Click here for additional data file.S5 FigReff) have been fixed at ∼ 48 per cent for all scenarios. In each panel the solid line indicates ϵv = 0.25, with dashed and dotted lines corresponding to ϵv = 0.5 and 0.75 respectively. Scenario A captures a population without pre-existing CTL mediated immunity to the pandemic strain that experiences uniform vaccine efficacy. In scenarios B and C, 80 per cent of the population have CTL-mediated immunity reducing infectiousness by 50 per cent, with vaccine action saturating and boosting respectively. Scenario D assumes that 50 per cent of those with naturally acquired CTL immunity also have cross-reactive protective antibody and vaccine action is boosting.Predicted attack rates for four different pandemic and vaccine action scenarios as a function of general population coverage with a CTL-inducing vaccine. Baseline attack rates Click here for additional data file.S6 FigUpper panels: Drift opportunity as a function of coverage for scenarios A–D with fixed baseline attack rate of ∼ 48 per cent across all scenarios  and gi0, = 1. Lower panels: The ratio of effective reproduction numbers of antigenically drifted and pandemic variants. In each panel the solid line indicates the relative infectiousness in vaccinated naïve and naïve hosts is ϵv = 0.25, with dashed and dotted lines ϵv = 0.5 and 0.75 respectively.(EPS)Click here for additional data file.S7 Figupper panels) and drift opportunity (lower panels) as a function of general population vaccine coverage for Scenario C with an initial Reff = 2 and vaccines distributed over 10 days , 90 days (middle) or 182 days (right). As for the figures in the main text, solid lines indicate ϵv = 0.25, dashed lines ϵv = 0.5 and dotted lines ϵv = 0.75.Predicted attack rate ((EPS)Click here for additional data file.S8 Figupper panels), drift opportunity (middle panels) and ratio of effective reproduction numbers of drifted and resident strains (lower panels) as a function of general population vaccine coverage for Scenario C with an initial Reff = 4 and vaccines distributed over 10 days (left panels), 90 days (middle panels) or 182 days (right panels). As for the figures in the main text, solid lines indicate ϵv = 0.25, dashed lines ϵv = 0.5 and dotted lines ϵv = 0.75. Note the differences in the scales of the y-axes when compared to Predicted attack rate ((EPS)Click here for additional data file.S9 FigUpper panels: Attack rate assuming 50 per cent coverage of a saturating vaccine in a homogeneously mixing population as a function of the fraction of influenza experienced hosts fE and suppression of infectiousness of unvaccinated experienced hosts ϵ. Lower panels: Attack rate assuming 75 per cent coverage of a saturating vaccine in a homogeneously mixing population as a function of the fraction of influenza-experienced hosts fE and suppression of infectiousness of unvaccinated experienced hosts ϵ. Parameters corresponding to scenario B are marked in each panel by a red dot.(EPS)Click here for additional data file.S10 FigfE = 0.8 (as for the main results section) a function of the strength of pre-pandemic cellular immunity ϵ and the proportion of influenza-experienced hosts with humoral immunity (“fraction of fE with Abs”). Results are shown for an unimpeded epidemic (left), assuming 50 per cent general population coverage with a boosting vaccine that suppresses the infectiousness of vaccinated naïve hosts by ϵv = 0.25 (middle) and as for the middle panel but assuming 75 per cent general population coverage (right). Parameter values corresponding to scenarios C and D are marked in each panel by red and blue dots respectively.Attack rate assuming (EPS)Click here for additional data file.


A recent report from the British Nuffield Council on Bioethics associated ‘emerging biotechnologies’ with a threefold challenge: 1) uncertainty about outcomes, 2) diverse public views on the values and implications attached to biotechnologies and 3) the possibility of creating radical changes regarding societal relations and practices. To address these challenges, leading international institutions stress the need for public involvement activities (PIAs). The objective of this study was to assess the state of PIA reports in the field of biomedical research.PIA reports were identified via a systematic literature search. Thematic text analysis was employed for data extraction.After filtering, 35 public consultation and 11 public participation studies were included in this review. Analysis and synthesis of all 46 PIA studies resulted in 6 distinguishable PIA objectives and 37 corresponding PIA methods. Reports of outcome translation and PIA evaluation were found in 9 and 10 studies respectively (20% and 22%). The paper presents qualitative details.The state of PIAs on biomedical research and innovation is characterized by a broad range of methods and awkward variation in the wording of objectives. Better comparability of PIAs might improve the translation of PIA findings into further policy development. PIA-specific reporting guidelines would help in this regard. The modest level of translation efforts is another pointer to the “deliberation to policy gap”. The results of this review could inform the design of new PIAs and future efforts to improve PIA comparability and outcome translation. New developments in biomedical research, such as biobank-based research and gene transfer methods, as well as biomedical innovations such as synthetic biology, regenerative medicine, neuroimplants and nanotechnology, particularly attract public attention. In 2009, for example, biobanking was included by the Time Magazine in a list of “10 Ideas Changing the World Right Now” Further, specific social, ethical and legal challenges, such as the communication and commercialization of research results, the balancing of individual rights against the collective good, and data protection, to name but a few, require careful consideration and governance. This may be relevant not only to the approval and application of new biomedical technologies, but also to the processes of the underlying research To address these challenges, leading international institutions stress the importance of public involvement in biomedical research and innovation one-way activity in which scientists provide the public with relevant information on a particular subject “to help them gain knowledge” For this paper, public information is understood as a Previous studies have assessed and discussed the state of PIAs in health policy These previous studies have revealed that A) the majority of PIAs are carried out in the USA, Canada and the UK The objective of this study was to assess a systematically-derived set of published PIAs in the field of biomedical research and innovation for A) their general characteristics ; B) their study aims; C) their use at the planning stage of frameworks or findings from previous PIAs; D) the types of PIA method used; E) the translation of PIA outcomes to policy and practice; and F) measures used for PIA evaluation.We conducted a systematic literature search in the bibliographic databases PubMed, PsychInfo and Scopus from June 14 to July 29, 2013 with an update after three months. We restricted the search to studies published between January 2000 and June 2013 to account for the (most) recent developments in the field of biomedical research, in any language. The search queries combined comprehensive, general search terms representing “public involvement” and “research and innovation” in the field of biotechnology . We chosWe used a thematic text analysis approach for data extraction All included studies were read in full and assessed independently by three researchers . According to thematic text analysis practice, all relevant text passages related to the core aspects were extracted. A code/keyword was assigned to each extracted text passage to allow for subsequent qualitative and quantitative analysis.A sample of five studies was assessed by three authors  to pilot-test the construct validity of the assessment matrix and the consistency of the extraction procedure. Results were compared and unclear or divergent coding was resolved after discussion with the fourth author (DS). The consistency of further extractions and codings was discussed in ongoing meetings and discussions between all four researchers.Based on international categorization schemes, we broadly distinguished the included PIAs either as a public consultation or a public participation/deliberation activity Of 3,791 publications identified via the comprehensive search, we finally included 46 PIA studies in our study . Four stOur studies were conducted in 15 different countries with 64% (n = 30) of studies being conducted in the USA (n = 18), Canada (n = 8) or Australia (n = 4). Those studies that provided a date  were carried out between 1999 and 2011 and were published between 2002 and 2013 in 28 different journals .Genetic research was mentioned 20 times as the topic of the PIA , and biobank research was mentioned 18 times . The other topics were biomedical research in general (n = 5), research with bloodspot samples (n = 3), synthetic biology (n = 1), nanotechnology (n = 1), and human cloning research (n = 1). 28% of studies (n = 13) mentioned more than one topic.Regarding the number of PIA participants, 35% of studies (n = 16) had fewer than 100 participants, 30% (n = 14) had 100–500 participants, and 35% (n = 16) had more than 500 participants.24% of studies (n = 11) did not specify a duration. Of those that did (n = 35), 67% (n = 31) took longer than a day and 9% (n = 4) took one day or less . 63% (n In the 46 PIAs we found 66 different (wordings for) objectives, such as “assess the public's perceptions”, “offer collective responses that should be understood as tailored policy input” or “determining the feasibility of conducting a citizens [sic] jury”. These different wordings were grouped under 6 distinguishable PIA objectives see .62% of objectives (n = 42) related to public consultation, i.e. to a broad understanding of ‘attitudes research’, for example, “to assess attitudes”, “to inquire perceptions [sic]” or “to query about preferences [sic]”.16% of objectives (n = 11) related to public participation/deliberation, for instance by offering “collective responses that should be understood as tailored policy input, rather than public opinion measurement”.3% of 68 objectives (n = 2) related to public information. Since our analysis did not include pure public information activities , these objectives were ‘only’ sub-objectives within two PIAs considered as public participation/deliberation see the  section.The remaining objectives related to a broad understanding of and contribution to PIA research, i.e. describing a PIA method (n = 2), investigating the impact of the PIA on participants (n = 3), and testing a PIA method (n = 6). Overall, 20% (n = 9) of all PIAs reported objectives related to PIA research (two PIAs reported more than one research objective).We rated 76% (n = 35) of the studies as public consultations, and 24% (n = 11) as public participations/deliberations. In total, we found 146 wordings for the methods applied in the 46 PIAs. However, these wordings often described similar methods and therefore were grouped according to the 6 PIA objectives see . Within ‘Public information’ contained many more methods (n = 36) than corresponding objectives (n = 2). This is due to the fact that several PIAs (n = 23) reported the use of informational material such as literature or introductory presentations without also explicitly stating the objective of providing information prior to consultation or participation/deliberation.93% (n = 43) of all studies referred to other PIA reports or PIA research. 42% (n = 18) of all studies explicitly used these references as the methodological framework for their own PIA or to validate their choice of PIA methods. Three studies did not refer to any methodology.Furthermore, no study reported the explicit or implicit use of public involvement guidelines that describe different PIA objectives and methods  reported some evaluation measures, which related to A) participants' satisfaction during participation (n = 6) or attitude changes (n = 2) and/or B) effectiveness and feasibility of the PIA method (n = 4); two PIAs reported both A) and B).One of the 35 public consultation studies (3%) and 8 of the 11 public participation/deliberation studies (73%) reported some sort of translational efforts to either disseminate study findings and recommendations or communicate them directly to decision-makers in policy and practice. Translational efforts included A) a final conference and (spoken) presentation of recommendations (n = 3); B) the writing and dissemination of a policy-oriented final report (n = 4); C) community guidance during and after the PIA (n = 3); D) dissemination via print and electronic media (n = 4); and E) dialogue-oriented meetings between researchers and politicians to discuss results (n = 2).This study assessed a systematically-derived sample of 46 peer-reviewed journal articles reporting on public involvement activities (PIAs) in the field of biomedical research and innovation. The 35 public consultation and 11 public deliberation/participation activities used a broad range of different wordings for PIA objectives and corresponding methods. Our categorization reduced this range to 6 different PIA objectives and 37 corresponding PIA methods. These findings confirm previous reviews of PIAs in other areas such as health policy, concluding that public involvement as a tool for governance and democratic decision-making is defined and practiced in many ways Regarding the clustered PIA objectives in particular, it emerged that a range of significant, classical objectives such as building public trust, empowering individuals to actively engage in political debate, and legitimizing policy were not explicitly stated as objectives in the respective PIA reports www.equator-network.org) might be a good partner for starting the development of such reporting standards.Though there is relatively established terminology for PIA categories and objectives While research into the quality of PIAs on biomedical issues is important to further strengthen the validity and practical relevance of the public involvement approach, it is currently not widespread. Only 10 (22%) of the identified PIAs reported on evaluation measures such as participants' satisfaction with their participation, changes in attitudes as a result of participation, and the effectiveness and feasibility of PIA methods.Only 9 (20%) of the 46 PIA studies reported on translation of their findings, despite the importance attributed to this aspect of PIAs Regarding the analysis of the aspect of translation, we stress the limitation that our analysis only assessed whether translation efforts were reported or not. It might be that translation of PIA findings is often practiced but seldom reported in scientific journals. This may be true especially for public consultation activities, which are not necessarily limited to merely gathering public opinion. However, if the findings of public consultation activities are in fact used to influence policy, future PIA reports could address this more explicitly. In order to improve insight into challenges for translation of consultation and participation/deliberation activities, further research could assess the importance policy and decision-makers actually place on PIA findings. In this sense, it could also be assessed whether there are sufficient ‘contact points’ in policy or practice for communicating PIA findings.Despite the fact that we employed narrow as well as general search terms for the broad field of biomedical research and innovation, most of the identified PIAs dealt with genetics or biobanking. The fact that biobanking and genetics  concern more of the public than other emerging biotechnologies such as gene therapy, nanotechnology, neuroimplants, tissue engineering and synthetic biology may partly explain this gap. With recent calls for more public involvement in the field of biomedical research, more PIAs on issues beyond genetics and biobanking are to be expected. The design of new PIAs on relevant issues in specific disciplines of biomedical research could be informed by the results of this review.We highlight that our categorizations and classifications of text passages from the included PIA reports involved interpretation. We addressed this challenge by having at least two researchers analyzing each text independently, discussing differences and finally agreeing classifications among all four authors. Also, PIAs conducted in disciplines other than biomedical research as well as PIAs published in formats other than peer-reviewed scientific journals may suggest different reporting practices, for instance regarding details on ‘direct’ objectives (to consult the public) and more indirect ones (to increase public trust).Further consideration of existing PIA guidelines and the development of PIA-specific reporting guidelines could improve the comparability of PIAs, streamline their reporting and thus enforce the still lacking (or underreported) translation of findings.Checklist S1PRISMA Checklist.(PDF)Click here for additional data file.Information S1Assessment matrix. The matrix used to analyze selected studies is divided according to the core aspects of Public Involvement Activities identified by a literature review.(PDF)Click here for additional data file.Information S2Overview of journals that published selected PIA reports. Sorted according to (1) the number of studies selected from the respective journal and (2) alphabetical order.(PDF)Click here for additional data file.Information S3Electronic search strategy.(PDF)Click here for additional data file.


The Na+-/K+ ATPase utilizes energy from ATP hydrolysis to extrude three Na+ cations and import two K+ cations into the cell. The minimum constellation for an active Na+-/K+ ATPase is one alpha (α) and one beta (β) subunit. Mammals express four α isoforms (α1−4), encoded by the ATP1A1-4 genes, respectively. The α1 isoform is ubiquitously expressed in the adult central nervous system (CNS) whereas α2 primarily is expressed in astrocytes and α3 in neurons. Na+ and K+ are the principal ions involved in action potential propagation during neuronal depolarization. The α1 and α3 Na+-/K+ ATPases are therefore prime candidates for restoring neuronal membrane potential after depolarization and for maintaining neuronal excitability. The α3 isoform has approximately four-fold lower Na+ affinity compared to α1 and is specifically required for rapid restoration of large transient increases in [Na+]i. Conditions associated with α3 deficiency are therefore likely aggravated by suprathreshold neuronal activity. The α3 isoform been suggested to support re-uptake of neurotransmitters. These processes are required for normal brain activity, and in fact autosomal dominant de novo mutations in ATP1A3 encoding the α3 isoform has been found to cause the three neurological diseases Rapid Onset Dystonia Parkinsonism (RDP), Alternating Hemiplegia of Childhood (AHC), and Cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS). All three diseases cause acute onset of neurological symptoms, but the predominant neurological manifestations differ with particularly early onset of hemiplegic/dystonic episodes and mental decline in AHC, ataxic encephalopathy and impairment of vision and hearing in CAPOS syndrome and late onset of dystonia/parkinsonism in RDP. Several mouse models have been generated to study the in vivo consequences of Atp1a3 modulation. The different mice show varying degrees of hyperactivity, gait problems, and learning disability as well as stress-induced seizures. With the advent of several Atp1a3-gene or chemically modified animal models that closely phenocopy many aspects of the human disorders, we will be able to reach a much better understanding of the etiology of RDP, AHC, and CAPOS syndrome.The transmembrane Na With each pump cycle, the Na+/K+ ATPases utilize the energy from hydrolysis of one adenosine triphosphate (ATP) to extrude three Na+ ions and import two K+ ions into the cell. The minimum constellation of an active pump consists of an alpha (α) and a beta (β) subunit  of which α1−3 are expressed in the CNS. While α1 is expressed ubiquitously and considered to maintain housekeeping cellular functions, the α2 isoform is expressed primarily in astrocytes and developing neurons and α3 isoform is restricted to neurons  and general loss of the Na+ electrochemical gradient. As the α3 isoform is detected in several basal ganglia neuronal subsets  and dopamine  use ion gradients for the active transport, generally by co-transport of Na+/K+-ATPase to maintain Na+ and K+ gradients will cause a decrease in Na+ and K+ currents (through voltage-dependent channels). This will lead to a decrease in the membrane potential, action potential and most likely a loss of neuronal excitability. This will further affect sodium-coupled co- and counter-transport. Moreover, it can also affect Na+/H+ and Na+/Ca2+ exchange, essential for cellular functions, and could lead to increased intracellular Ca2+ and acidification. Not surprisingly, mutations in the ATP1A3 gene have been associated with neurological disorders.Failure of the NaATP1A3 gene are associated with three related rare neurological disorders, Rapid-onset Dystonia-Parkinsonism (RDP)   constitute a group of organic compounds that show high binding affinity toward Na3 protein expression in the 1 expression but not of α2  showed no changes in protein expression levels of the α1, α2, and α3 isoforms relative to wild type, suggesting that the Myshkin mutation is a functional null allele of the Atp1a3 gene, which encodes a normally expressed, but inactive enzyme, as revealed by reduced total ATPase activity  regained approximately 80% of wild type total Na+/K+-ATPase activity and showed normal body size, thus supporting the theory that Na+/K+-ATPase activity loss correlates with symptoms  integrate inhibitory signals from GABAergic Purkinje neurons and excitatory glutamatergic mossy fibers and climbing fiber pathways and constitute the majority output fibers from the cerebellum. The DCN connects to premotor and motor nuclei in the basal ganglia Faull, . Abnorma+ channel density and short spike duration during which large amounts of Na+ ions enter the neurons, emphasizing the requirement for functional a Na+/K+-ATPase  injections of ouabain in high concentrations to be CNS stimulatory and convulsive  and demonstrated spontaneous recurrent seizures and neuronal excitability  are important for triggering several different forms of synaptic plasticity, including long term potentiation and long-term depression  showed relatively mild symptoms whereas the Myshkin and Mashl+∕− models carrying AHC mutations (with larger Na+/K+-ATPase reduction), recapitulated most of the key phenotypes. As a proof of concept, most symptoms of the Myshkin mouse were rescued by increasing the Na+/K+-ATPase activity. Future experiments will be required to establish if similar approaches can be translated into a possible treatment.Previous studies using CTS to study Na+/K+-ATPase activity by infusion of ouabain  in single cell populations or organs, using the Cre LoxP system  (PUMPKIN DNRF85).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.