P. falciparum malaria across diverse transmission settings in Africa. Longitudinal follow-up of patients in these trials supported previous findings that DP had a longer post-treatment prophylactic effect than AL, reducing the risk of reinfection and conferring additional health benefits to patients, particularly in areas with moderate to high malaria transmission.Recent multi-centre trials showed that dihydroartemisinin-piperaquine (DP) was as efficacious and safe as artemether-lumefantrine (AL) for treatment of young children with uncomplicated We developed a Markov model to assess the cost-effectiveness of DP versus AL for first-line treatment of uncomplicated malaria in young children from the provider perspective, taking into consideration the post-treatment prophylactic effects of the drugs as reported by a recent multi-centre trial in Africa and using the maximum manufacturer drug prices for artemisinin-based combination therapies set by the Global Fund in 2013. We estimated the price per course of treatment threshold above which DP would cease to be a cost-saving alternative to AL as a first-line antimalarial drug.First-line treatment with DP compared to AL averted 0.03 DALYs (95% CI: 0.006–0.07) and 0.001 deaths (95% CI: 0.00–0.002) and saved $0.96 (95% CI: 0.33–2.46) per child over one year. The results of the threshold analysis showed that DP remained cost-saving over AL for any DP cost below $1.23 per course of treatment.P. falciparum malaria in young children. A paediatric dispersible formulation of DP is under development and should facilitate a targeted deployment of this antimalarial drug. The use of DP as first-line antimalarial drug in paediatric malaria patients in moderate to high transmission areas of Africa merits serious consideration by health policymakers.DP is superior to AL from both the clinical and economic perspectives for treatment of uncomplicated  Plasmodium falciparum malaria is critical in preventing the progression of acute infections to severe disease and reducing the risk of further morbidity, disability and premature mortality from the disease P. falciparum malaria worldwide Despite a rapid scaling up of malaria control efforts and recent reports of decreasing transmission intensities in African countries, malaria remains an important cause of morbidity and mortality, particularly in young children Recent multi-centre trials showed that dihydroartemisinin-piperaquine (DP), a newer fixed-dose co-formulated ACT, was as efficacious and safe as AL for treatment of young children with uncomplicated malaria across diverse transmission settings in Africa Significant financial resources have been put into scaling up the access to ACTs through global subsidies on manufacturing prices of drugs Cost-effectiveness analysis was performed using a Markov model  that defined a set of mutually exclusive health states, simulating the progression of malarial disease and the risk of recurrent malaria in children younger than five years of age, receiving DP or AL for first-line treatment of uncomplicated malaria . The MarThe Markov model had 11 health states: State 1: Healthy; State 2: Uncomplicated malaria; State 3: Severe malaria; States 4–10: Post-treatment from week one to week seven; and State 11: Dead . The posPublished estimates were used for the probability of developing severe malaria following oral antimalarial treatment Children entered the Markov model in the “healthy” state. They were subjected to a hazard rate for unscomplicated malarial disease estimated from the trial data and flowed through the subsequent health states in weekly cycles. We assumed that all children with uncomplicated malaria would be treated promptly with either DP or AL, and all children with severe malaria would receive inpatient care. Children, who were treated orally with antimalarial drugs, would either recover and enter the “post-treatment” states, or develop severe malaria. Depending on the antimalarial treatment choice, children in the post-treatment states were subjected to a weekly hazard rate for recurrent malaria during the first seven weeks following treatment . ChildreHealth outcomes were measured in terms of disability adjusted life years (DALYs) averted. DALYs combine years of life lost because of premature mortality with years of life lived with disability. We used an average life expectancy of 57.25 years for children aged 1–4 years on the basis of the life tables for African men and women for the WHO sub-region with high child and high adult mortality We considered the costs of oral antimalarial drugs for treating uncomplicated cases and the costs of inpatient care for severe cases over one year , but excWe used published estimates of inpatient care costs at primary level hospitals from a recent Kenyan costing study To assess the uncertainty in key model parameters and the robustness of the results to model assumptions, we performed a probabilistic sensitivity analysis using a Monte Carlo simulation technique with 10,000 iterations. For each iteration, a value for each input variable was selected randomly from its distribution given in First-line treatment with DP was the economically dominant treatment strategy over AL with a 90% probability , resultiP. falciparum malaria that considered the post-treatment prophylactic effects of antimalarial drugs – an important secondary benefit of antimalarial treatment, particularly in areas with moderate to high malaria transmission in Africa. DP was both clinically superior and less costly compared to AL for first-line treatment of uncomplicated malaria in young children. It proved to be the economically dominant treatment strategy in such transmission settings. The threshold analysis showed that first-line treatment with DP would remain cost saving over AL for any DP cost below $1.23 per course of treatment.This is the first economic analysis of first-line treatment options for uncomplicated P. falciparum malaria per child when treated with DP and AL, respectively. A recent study predicted an annual incidence rate of 1.7 (95% CI: 1.4–3.9) cases of uncomplicated malaria per child for this age group living in such transmission settings in Africa Our analysis is based on the primary data from a cohort of children who participated in a multi-centre trial on the efficacy of ACTs conducted in areas with moderate to high malaria transmission in Africa We assumed full access to treatment with AL and DP and same level of patient compliance to both therapies to evaluate the costs and benefits conferred by the post-treatment prophylactic effects of the drugs. While both drugs are administered over three days, DP has a simple, once daily dosing regimen whereas AL should be given twice daily and ideally with a fatty meal. Therefore, DP has the potential to improve patient compliance and treatment effectiveness and hence overall management of pediatric patients. However, such an advantage of treatment with DP could not be evaluated in a clinical trial with an observed drug intake A limitation of our analysis is the lack of data on the long-term post-treatment prophylactic effect of DP compared to AL. Our model incorporated data from a drug efficacy trial with limited (63 days) longitudinal follow-up of patients Maximum manufacturer prices are negotiated by the Global Fund for several ACTs, including AL and DP. However, these prices do not necessarily reflect the first-line buyer prices; co-payment amounts go up to 98% of manufacturer sales prices for some African countries P. falciparum endemicity will affect the efficacy and the post-treatment prophylactic effect of these drugs. Decision makers should contextualise costs and assess patient compliance, pattern of clinical malaria, and other key parameters in their own settings to arrive at more locally representative results.It should be mentioned that a change in national malaria treatment policies may require substantial resources P. falciparum (and P.vivax) malaria P. falciparum malaria in children compared to the conventional treatment. From a patient's perspective, the differentiation between recrudescence and re-infection is insignificant. PCR-correction discounts the post-treatment prophylactic effect of a drug by excluding new infections that occur during the follow-up period. Large-scale clinical trials in African settings have consistently shown that DP was more efficacious than AL using PCR-uncorrected cure rates P. falciparum malaria in children in areas with moderate to high transmission.There is limited evidence on the cost-effectiveness of ACTs for treatment of uncomplicated malaria in children. A recent study from Papua New Guinea compared the cost-effectiveness of three different ACTs against the conventional treatment with chloroquine plus SP for treatment of A main concern with DP and other long-acting antimalarial drugs is that residual drug levels play a critical role in the emergence and spread of drug resistance P. falciparum malaria in young children. A paediatric dispersible formulation of DP suitable for use by children between the ages of six months and five years is under development There is an urgent need to improve the management of malaria in African children. Decision makers who aim to provide optimal treatment strategies to the populations at risk need to consider various aspects of antimalarial drugs – including safety, tolerability, dosing schedule, level of drug resistance, post-treatment prophylactic effect, and cost – and malaria endemicity. Our study demonstrates the superiority of DP over AL from the clinical and economic perspectives for treatment of uncomplicated


We develop a method to interpret our experimental results in the context of game theory, and show that the game wild-type and mutant bacteria play in an unstructured environment changes markedly over time, and eventually obeys a prisoner’s dilemma leading to cheater dominance. In contrast, when wild-type and mutant E. coli co-inhabit a spatially-structured habitat, cooperators and cheaters coexist at intermediate frequencies. Our findings show that even in microhabitats lacking patchiness or spatial heterogeneities in resource availability, surface growth allows cells to form multi-cellular aggregates, yielding a self-structured community in which cooperators persist.Cooperative organisms are ubiquitous in nature, despite their vulnerability to exploitation by cheaters. Although numerous theoretical studies suggest that spatial structure is critical for cooperation to persist, the spatial ecology of microbial cooperation remains largely unexplored experimentally. By tracking the community dynamics of cooperating ( Escherichia coli in various habitats that differ in spatial structure and interpret our results in a game-theoretic framework.Communities of cooperative organisms sharing limited resources or contributing to a common good are ubiquitous in nature, yet are vulnerable to disruption by free riders E. coli bacteria collectively restrain growth to avoid population collapse when resources become scarce before all resources are depleted is a form of altruistic cooperation, and one can think of the entry into stationary phase as a social contract: a collective rule of density-dependent growth regulation E. coli, this social contract is implemented through the switching of sigma factors. Sigma factors enable RNA polymerases to address distinct parts of the genome to adequately cope with changing environments rpoS gene encodes for the starvation/stationary-phase sigma-factor E. coli populations rpoS819 mutant used in this study, is caused by an attenuated functioning of rpoS WT bacteria invest energy in enhancing stress resistance and maintain a constant population size in which birth and death rates are balanced. By squandering resources and delaying stationary phase, GASP mutants defect on the social contract in which WT cooperate.In Saccharomyces cerevisiaeMyxococcus xanthusE. colirpoS gene of E. coli, can be interpreted in a game-theoretic framework rpoS WT) meet cooperators, both inhibit growth and get rewarded (receiving payoff rpoS819) get cheated upon as their savings get consumed prematurely, hence cooperators receive the sucker’s payoff Game theory can be applied when different types employ different strategies in social interactions rpoS WT cooperators and rpoS819 GASP mutants in various habitats that vary in spatial structure. We first quantitatively describe the community dynamics in a simple well-mixed ecological scenario and develop a method to interpret the WT-GASP interaction in a game-theoretic framework. In order to explore the influence of spatial structure on cooperator-cheater competition we performed competition experiments in microfabricated habitats. Time-lapse fluorescence microscopy allows us to characterize WT-GASP community dynamics in habitats that are structured at the micrometer scale and address whether cooperators can persist in the presence of cheaters.Here, we study the community dynamics of rpoS819 GASP mutants behave as cheaters and defect on the social contract of density-dependent regulation, by determining the amount of culturable cells in well-mixed cultures  starting from various WT/GASP ratios after one and four days of growth. We began by validating that E. coli  and predictions about the community dynamics can be made Note that the fraction of cooperators If we envision a lonely cheater in a majority of cooperators, i.e. Because we are only interested in the limits of This frequency-dependent fitness landscape rpoS WT E. coli and rpoS819 GASP mutants for 8 days in continuously shaken flasks. To gain insight into WT-GASP community dynamics in an unstructured environment, we used flow cytometry to monitor the relative abundance of neutrally labelled rpoS function not only influences stationary-phase physiology, but also affects the expression of genes involved in several metabolic functions, chemotaxis, motility and several other functions during earlier phases of growth During the first 24 hours of co-culture, the relative abundance of the cheater strain decreases for cultures starting from GASP fractions During the first day of WT-GASP co-culture, the environment changes dramatically as resource levels decrease and metabolic products build up. Consequently, the GASP mutant starts to defect on the social contract: it delays entry into stationary phase and sustains growth at the expense of WT cells. The temptation to defect thus gradually increases and after 24 hours it becomes bigger than the reward for cooperation  in a spatially structured but otherwise homogeneous environment, we performed competition experiments . Measuring occupancy rather than fluorescence intensity is less sensitive to differences in brightness of the two fluorescent proteins (GFPmut2 and mRFP) and the differential influence of environmental factors such as medium conditioning and population density on those proteins. To negate any remaining bias towards one of the reporters, half of the experiments were performed with GFP-labelled WT and RFP-labelled GASP strains, whereas the other half of the experiments were performed with a WT-GASP pair in which the fluorescent reporters were swapped – yielding equivalent results . This analysis, which is not affected by random migration events, shows how patches with a similar GASP fraction change in population composition over time and enables us to estimate payoffs at the patch level see . Figure within a single patch. This raises the question whether the patchiness of the microhabitats is a prerequisite for WT-GASP coexistence: does the imposed patchiness induce a population structure that facilitates coexistence? In order to investigate this, we performed experiments in microhabitats . Our results however, reveal that extrinsically imposed spatial heterogeneities are not required for coexistence of WT and GASP cells. Even in a homogeneous microfabricated habitat, where patchiness is not inherent in the spatial structure, a spatially heterogeneous community of coexisting WT and GASP cells arises. It is likely that surface growth and attachment is important for the formation of multi-cellular aggregates and thus facilitates self-structuring of the WT-GASP community. Hence, surface growth and attachment may have a key role in supporting coexistence of WT and GASP E. coli.WT and GASP Experimental studies addressing the influence of spatial structure on cooperation often introduce structure by imposing propagation and dispersal regimes on unstructured subpopulations Understanding how cooperation may have emerged and is maintained presents a major challenge rpoS allele and are Escherichia coli W3110 lacYZ::GFPmut2 (labelled with green fluorescent protein) and W3110 lacYZ::mRFP (labelled with red fluorescent protein), respectively. JEK1032 (green GASP) and JEK1033 (red GASP) have the GASP phenotype and carry the rpoS819 allele (described in Ref. de novo mutations leading to the GASP phenotype from occurring.All strains used in this study have been described before in Ref. Pure cultures of JEK1036 (WT) and JEK 1033 (GASP) were grown overnight in LB medium containing 100 Pure cultures of JEK1036 (WT) and JEK 1033 (GASP) were grown in LB medium containing 100 Flow cytometry of competition experiments was performed using a FACScan flow cytometer (Becton Dickinson) controlled using FlowJo software. GFP was excited at 488 nm and emission was filtered using a 530/30 band-pass filter, RFP was excited at 561 nm and emission was filtered with a 590/20 band-pass filter. Overnight cultures of JEK1036 and JEK1033 were separately diluted 1/50 in fresh LB supplemented with 100 Microhabitats were fabricated in silicon following a previously published protocol Microhabitats were imaged at 10 minute intervals for up to 5.5 days using an inverted Olympus IX81 microscope equipped with a 10x (N.A.Images were processed in MatLab using a custom script. We noticed that the two fluorescent proteins that we used (GFP and RFP) do not have the same brightness, and, more importantly react in different ways to growth phase and environmental factors like conditioning of the medium and population density. As a result, fluorescent intensity is not a good proxy for measuring biomass. To circumvent the problems associated with intensity measurements, we converted all images to occupancy data which measures the area a strain occupies. Based on the pixel values that corresponds to the autofluorescence of the medium for a certain color channel (i.e. strain), time point, and position, a threshold pixel value is calculated above which a pixel  migration of bacteria. In order to only analyze the competitive component of population dynamics, we group patches at each time point according to their GASP fraction in 8 equally sized bins and analyze the trend of the GASP fraction in each group. For example, all patches time see  we can stime see . As can GFP and GASP with mRFP (strains JEK1036 and JEK1033), and half of the experiments with a strain pair in which the fluorescent markers swapped, i.e. red WT and green GASP (strains JEK1037 and JEK1032). We have performed this analysis for these two datasets separately, The surfaces obtained using this approach, shown in Figure S1Kymographs of all (n = 20) microhabitat experiments. Each pixel corresponds to the GASP fraction of a single habitat patch ((EPS)Click here for additional data file.Figure S2Game dynamics of well-mixed WT-GASP competition. (a) Surface plot of time versus GASP fraction versus b) Surface plot of time versus GASP fraction versus c) Time-evolution of (EPS)Click here for additional data file.Figure S3Binned GASP fraction versus time. Bin intervals are denoted on the y-axis and depicted by dashed lines. (a) Data from patches of all microhabitat experiments binned in 8 equally sized bins. The GASP fraction within each bin is nearly constant. (b) Data from the well-mixed experiments binned in 8 equally sized bins. The GASP fraction within bins changes over time.(EPS)Click here for additional data file.Figure S4Game dynamics of WT-GASP competition in microhabitats. (a) Surface plot of time versus GASP fraction versus b) Surface plot of time versus GASP fraction versus c) Time-evolution of d and e) Half the microhabitat experiments was performed with a strain pair in which WT was labeled with GFP and GASP with mRFP (strains JEK1036 and JEK1033), and half of the experiments with a strain pair in which the fluorescent markers swapped, i.e. red WT and green GASP (strains JEK1037 and JEK1032). We have performed the same analysis as in (c) for these two datasets separately, the results of this analysis for strains JEK1036 and JEK1033 are shown in (d) and for strains JEK1037 and JEK1032 in (e). Both datasets show similar dynamics as the pooled dataset (c), the (EPS)Click here for additional data file.


E. coli and triacetic acid lactone (TAL) in S. cerevisiae revealed that the identified interventions tend to cause less dramatic rearrangements of the flux distribution so as not to violate concentration bounds. In some cases the incorporation of kinetic information leads to the need for additional interventions as kinetic expressions render stoichiometry-only derived interventions infeasible by violating concentration bounds, whereas in other cases the kinetic expressions impart flux changes that favor the overproduction of the target product thereby requiring fewer direct interventions. A sensitivity analysis on metabolite concentrations shows that the required number of interventions can be significantly affected by changing the imposed bounds on metabolite concentrations. Furthermore, k-OptForce was capable of finding non-intuitive interventions aiming at alleviating the substrate-level inhibition of key enzymes in order to enhance the flux towards the product of interest, which cannot be captured by stoichiometry-alone analysis. This study paves the way for the integrated analysis of kinetic and stoichiometric models and enables elucidating system-wide metabolic interventions while capturing regulatory and kinetic effects.Computational strain design protocols aim at the system-wide identification of intervention strategies for the enhanced production of biochemicals in microorganisms. Existing approaches relying solely on stoichiometry and rudimentary constraint-based regulation overlook the effects of metabolite concentrations and substrate-level enzyme regulation while identifying metabolic interventions. In this paper, we introduce k-OptForce, which integrates the available kinetic descriptions of metabolic steps with stoichiometric models to sharpen the prediction of intervention strategies for improving the bio-production of a chemical of interest. It enables identification of a minimal set of interventions comprised of both enzymatic parameter changes (for reactions with available kinetics) and reaction flux changes (for reactions with only stoichiometric information). Application of k-OptForce to the overproduction of L-serine in  Computational strain design procedures aim at assisting metabolic engineering efforts by identifying metabolic interventions leading to the targeted overproduction of a desired chemical using network models of cellular metabolism. The effect of metabolite concentrations and substrate-level enzyme regulation cannot be captured with stoichiometry-only metabolic models and analysis methods. Here, we introduce k-OptForce, an optimization-based strain design framework incorporating the mechanistic details afforded by kinetic models, whenever available, into a genome-scale stoichiometric-based modeling formalism. The resulting optimization problems pose significant computational challenges due to the bilevel nature of the formulation and the nonconvex terms in the constraints. A tractable reformulation and solution procedure is introduced for solving the optimization problems. k-OptForce uses kinetic information to (re)apportion reaction fluxes in the network by identifying interventions comprised of both direct enzymatic parameter changes (for reactions with available kinetics) and reaction flux changes (for reactions with only stoichiometric information). Our results show that the introduction of kinetic expressions can significantly alter the identified interventions compared to those identified with stoichiometry-alone analysis. In particular, additional modifications are required in some cases to avoid the violation of metabolite concentration bounds, while in other cases, the kinetic constraints yield metabolic flux distributions that favor the overproduction of the desired product thereby requiring fewer direct interventions. Bio-production is emerging as a competitive strategy for the production of a wide range of chemicals ranging from biofuels, precursor chemicals and bioactive molecules  is typically solved to obtain the time-dependent variation in metabolite concentrations and reaction fluxes. Different forms of mechanistic expressions have been used extensively such as Michaelis-Menten or Hill Kinetic expressions The introduction of genome-scale models of metabolism Stoichiometry-based strain design algorithms are often structured as bilevel mixed integer linear optimization problems (MILP) et alet alet alE. coli model (comprised of 76 equations) to constrain an FBA simulation. Similarly, Cotton et alThe need to integrate the mechanistic detail (whenever available) of kinetic expressions with the genome-scale scope of stoichiometric models has been recognized early by the community. Dynamic Flux Balance Analysis (dFBA) E. coliS. cerevisiaeE. coli, and triacetic acid lactone (TAL) in S. cerevisiae. For the former, k-OptForce identified key regulatory bottlenecks in upper and lower glycolysis that must be overcome to redirect more flux towards L-serine, which regular OptForce fails to pinpoint. In addition, k-OptForce removed interventions identified by regular OptForce that resulted in kinetically infeasible flux re-distributions. Application of the k-OptForce for the microbial overproduction of TAL in S. cerevisiae revealed the impact of additional kinetic constraints in alleviating a severe worst-case simulation of regular OptForce, resulting in a higher prediction of TAL yield from fewer interventions as compared to regular OptForce predictions. The introduction of kinetic expressions in strain design can significantly affect the identified interventions in sometimes non-intuitive ways. In some cases additional modifications are needed to substitute interventions that cause enzyme saturation or concentration bound violations. The mechanism of action of these modifications is often subtle by alleviating substrate inhibition or draining away cofactors from competing pathways. In other cases, kinetic expressions shape flux distributions so as to favor the overproduction of the desired product requiring fewer direct interventions.Here, we introduce k-OptForce, which extends the previously developed OptForce procedure kinJ = {j|j = 1,2,…,kinN} and reactions with only stoichiometric information stoicJ = {j|j = 1,2,…,stoicN}  nonlinear ordinary differential equations (ODEs) denoting the non-steady-state balance for each metabolite. For a square system of ODEs integration yields steady-state metabolite concentrations and fluxes. The allowable metabolic phenotype  of the remaining portion of the network stoicJ is inferred so as to be consistent with the predicted fluxes of the reactions in kinJ.k-OptForce aims at utilizing the available kinetic information within a larger genome-scale stoichiometric model to more accurately characterize all possible metabolic phenotypes of the reference and engineered strains. The procedure builds upon the OptForce procedure oic} see . MetabolThe k-OptForce procedure is composed of the following steps:kinJ. The phenotypic space of the reference strain is then identified by iteratively maximizing and minimizing the flux of each reaction in stoicJ while keeping the fluxes of reactions in kinJ fixed at their steady-state values, and restricting the flux of any other reaction for which any experimental data is available at their experimentally determined values or ranges .The flux ranges of the strain consistent with a desired overproduction target are similarly constructed by successively maximizing and minimizing the flux of network reactions subject to network stoichiometry, overproduction target, while also incorporating the kinetic expressions for reactions in UMUST), down-regulated (LMUST), or be knocked out X) manipulations selected from within the MUST sets whose direct manipulation  ensures production of the desired chemical beyond a desired target even under the worst-case scenario where fluxes are re-apportioned to drain flux away from the target product.L,kiny and U,kiny associated with the LMUST and UMUST sets of reactions in kinJ respectively, are used to control the effect of engineering modifications to the corresponding L,kiny = 1 then U,kiny = 1 then stoicJ. A separate set of binary variables L,stoicy and U,stoicy identify interventions in stoicJ required to guarantee a non-zero yield of the target chemical consistent with the flux distribution in kinJ. It is important to note that the metabolic fluxes in kinJ remain unaffected by the worst-case simulation of the reactions in stoicJ in the inner problem.The worst-case scenario is mathematically described by extending the bilevel optimization problem used for original OptForce jv. The fitness function of the inner problem is imposed setting the objectives of the primal and the dual equal to each other. It is to be noted here that the dualization of the flux variable for each reaction in kinJ introduces a bilinear term to the single-level formulation. This can be avoided by imposing the Karush-Kuhn-Tucker (KKT) conditions of complementary slackness between the primal constraints and their dual variables. This leads to the introduction of a binary variable for each constraint in the primal problem which is generally more tractable than the original bilinear constraints (see Supplementary This bilevel formulation is converted into a single level mixed integer nonlinear problem (MINLP) using the conditions of strong duality. We construct the dual of the inner problem , and add all the dual constraints, along with those of the primal, to the outer optimization problem. Since all the nonlinear kinetic expressions are present in the outer problem, the inner  problem is linear in the reaction fluxes kinJ and subsequently within stoicJ  as in regular OptForce kinJ and stoicJ. It is to be noted that while the illustrated formulation only targets changes in kinJ the same analysis could be applied for the modulation of other kinetic parameters  in the model.Due to the nonconvex nature of the kinetic expressions and the large number of binary variables the resulting MINLP equivalent representation of the bilevel optimization problem may become computationally intractable. For these cases, we exploit the natural hierarchy of the model by first selecting interventions within n target . Keepingmaxv of one of the reactions has hit its upper bound, and, (5) analyze all alternate intervention strategies to identify common metabolites and/or enzymes that are being targeted.Once the FORCE set of interventions are identified , it is important to manually curate the results to understand the underlying metabolic reason behind each intervention. This is necessary since k-OptForce makes use of not just stoichiometry which imposes straightforward connections between reactants and products but also kinetics that introduce complex nonlinear couplings often between distal reactions through metabolite pools. While it is not possible to put forth an invariant set of rules for all applications, the following checks can be useful in general: (1) check whether any metabolite participating in affected reactions is hitting lower or upper concentration bounds, (2) identify if a common metabolite is fixing the branching ratio of fluxes in two pathways, (3) resolve whether a metabolite is limiting the flux of a reaction through substrate-level inhibition, (4) confirm if the flux in a pathway has been restricted because the E. coli and S. cerevisiae, respectively. The goal was to assess how the addition of kinetic information into stoichiometric models changes the list of identified interventions and more importantly what are the underlying reasons for the new interventions.We contrasted k-OptForce with the original OptForce Escherichia coli and Corynbacterium consists of a three-step pathway branching out of the glycolytic intermediate 3-phosphoglycerate (3pg). 3pg is converted to 3-phosphohydroxypyruvate (3php) by phosphoglycerate dehydrogenase , and phosphoserine transaminase  catalyzes the conversion of 3-phosphohydroxypyruvate (3php) to L-phosphoserine (pser-L) using L-glutamate as the amino acid donor. In the last step, phosphoserine phosphatase  catalyzes the final conversion to L-serine  flux ranges were identified by finding the maximum flux variability in the entire network while keeping the fluxes in kinJ fixed at the steady-state values obtained by solving the system of ODEs for the kinetic model . The L-serine overproducing network flux ranges were calculated for a target of 90% maximum theoretical yield . The minimum biomass production was kept at 10% of its maximum achievable. The maximum enzyme activity maxv of reactions in kinJ was allowed to vary from zero to two-fold up-regulation of its reference activity . Also, the concentration of metabolites in kinI was allowed to vary within a two-fold range from their steady-state concentrations in the reference strain.The reference strain  in oxidative pentose phosphate (PP) pathway consistent with the imposed L-serine overproduction ranged from 0 to 136 mmol gDW−1hr−1 when constrained by only stoichiometry and from 0 to 62 mmol gDW−1hr−1 when imposing also kinetic information. This range reduction is due to the limitations of the maximum enzyme activity as well as metabolic concentrations of glucose-6-phosphate (g6p) regulating G6PDH flux. The restrictions implied by kinetics also propagate throughout the stoichiometric part of the network leading to an on average ten percent range contraction for reactions in stoicJ. For example, the fluxes ranges in 2-oxogluterate dehydrogenase (AKGDH) and succinate dehydrogenase (SUCD) in the TCA cycle decreased 7.4% and 6%, respectively as a direct consequence of the flux range reduction for pyruvate dehydrogenase (PDH) in kinJ which supplies acetyl-CoA (accoa) to TCA cycle. As a result of the tighter flux ranges in the overproducing network (and characterization of base strain) many more reactions are identified that must depart from their original ranges  compared to regular OptForce both in UMUST (38 vs. 3) and LMUST (293 vs. 108) sets. For example, up-regulation of glucose-6-phosphate isomerase (PGI) in upper glycolysis supplies more flux towards 3pg and L-serine production. The flux range for PGI in the overproducing phenotype (−36–100 mmol gDW−1hr−1) was wide enough to overlap with its reference flux value (35 mmol gDW−1hr−1) suggesting PGI up-regulation is not necessary for L-serine overproduction. In contrast, using k-OptForce we find that the flux range of PGI in the overproducing phenotype is restricted to 38–98 mmol gDW−1hr−1 which does not include the reference value of 35 mmol gDW−1hr−1. This implies that it is impossible to produce L-serine at 90% theoretical yield without directly (or indirectly) increasing the flux through PGI which becomes a member of UMUST. We also observe a significant increase in the number of reactions in LMUST. This is because the kinetic expressions in kinJ fix the branching ratios for fluxes emanating from metabolites in kinI. As a result, many reactions in stoicJ involving metabolites participating in reactions from kinJ appear as down-regulations. For example, k-OptForce identifies that the acetyl-CoA carboxylase (ACCOAC) flux, which branches away from pyr and accoa towards membrane lipid metabolism must decrease  as it goes from (9–527) mmol gDW−1hr−1 in the reference strain to (2.1–2.3) mmol gDW−1hr−1 in the overproducing strain.et al−1 hr−1) and pyruvate dehydrogenase (PDH) (93 mmol gDW−1 hr−1) reactions. The high concentrations of 6pgc and pep both prevent the up-regulation of upper glycolysis and the down-regulation of lower glycolysis. To alleviate this, k-OptForce suggests removal of PDH coupled with an at least three-fold down-regulation in the activity of either transaldolase (TALA) or transketolase (TKT1) reactions in PP pathway. Removal of PDH also allows the concentration of pep to be reduced from 2.8 to 1.43 mM thus alleviating its inhibitory effect on PFK. Likewise, down-regulation of the PP pathway activity reduces the flux in GND lowering the concentration of 6pgc (from 0.8 to 0.4 mM). Removal of substrate inhibition on PFK and PGI by pep and 6pgc leads to increased flux towards L-serine. The original OptForce procedure cannot identify such interventions, as substrate inhibition is not captured through stoichiometric modeling.in vivo−1 hr−1 and the PP flux is down-regulated further (from 22 to 20 mmol gDW−1 hr−1) to provide the extra flux for L-serine (results not shown here). As a result, k-OptForce suggests down-regulation of GHMT by at-least 3 folds from its reference flux instead of its reversal. All other interventions remain un-altered.The inhibitory effect of 6pgc and pep cannot be completely removed due to their prescribed lower limits in concentration (0.4 mM and 1.43 mM respectively). Moreover, the upper limits on concentration of metabolites involved in upper glycolysis put an upper bound on the amount of flux that can be carried by upper glycolysis. Therefore, additional interventions are needed to meet the L-serine target yield by modulating pathways away from glycolysis. k-OptForce suggests the reversal of glycine hydroxymethyltransferase (GHMT) thus converting glycine to L-serine see . In cont−1 hr−1) of either citrate synthase (CS) or succinate dehydrogenase (SUCD) reactions to reduce the TCA cycle activity which arrests ATP production in the network. This prevents the conversion of L-serine to acetaldehyde whose activity requires five units of ATP per unit of flux. The original OptForce achieves the same goal by simply down-regulating the transport of oxygen and up-regulating the transport of ammonium into the cell. These interventions were not chosen by k-OptForce as they lead to flux values that are inconsistent with the kinetic expressions in kinJ.The remaining interventions suggested by k-OptForce aim at preventing the drain of metabolic flux from L-serine. Removal of L-serine deaminase (SERD_L) prevents the conversion of L-serine to pyruvate. This is followed by an at least six-fold down-regulation .Consistent with k-OptForce predictions, metabolic engineering studies on In addition to suggesting intervention strategies consistent with the kinetic constraints in the network, k-OptForce also pinpoints which ones and how original OptForce interventions violate network kinetics. For example, the original OptForce framework suggested the reversal of lower glycolytic reactions that converge to 3pg. This is accomplished by removing PGI and either GND, TKT1 or TALA in PP pathway to reroute the metabolic flux toward 3pg and pyruvate through E-D pathway by using 2-dehydro-3-deoxy-phosphogluconate aldolase (EDA). Reversal of enolase (ENO) and phosphoglycerate mutase (PGM) in lower glycolysis converts pyruvate to 3pg. k-OptForce finds that this redirection is not feasible. Reversible reactions PGM and ENO rely on the relative concentrations of its reactants and products to inform their directionality. Their reversal, to the extent suggested by the original OptForce procedure requires the respective metabolic levels of 2-phosphoglycerate (2pg) and pep to increase to 1.162 and 6.05 mM respectively, beyond the imposed upper limits of (0.856 and 4.726 mM respectively). Therefore, k-OptForce provides both a check on stoichiometry-only derived interventions and more importantly quantifies the impact of flux redirections on metabolite concentrations and required enzyme levels.E. coli kinetic model by Chassagnole et al−1hr−1 to 70 mmol gDW−1hr−1), as the glycolytic pathway was inhibited by increased 6pgc concentration. As a result, down-regulation of GND was suggested as an additional intervention to reduce the increased PP activity and route more flux from glycolysis towards L-serine. In all other cases metabolite concentrations and fluxes in the kinetic model were minimally affected by perturbations in enzyme activity.Previous reports E. coli and S. cerevisiae has been explored E. coli nor S. cerevisiae can natively synthesize TAL, production routes for TAL rely on the heterologous expression of non-native enzymes such as 2-pyrone synthase (2-PS) (found in Gerbera hybrida) fasBS. cerevisiae of only up to 6% of the theoretical maximum (with a titer of 1.8 g/l) in glucose medium. S. cerevisiae. We used the iAZ900 model S. cerevisiae as the stoichiometric network of metabolism. The kinetic expressions for reactions in central metabolism were imported from the kinetic model of central metabolism of S. cerevisiae described by van Eunen et alet alS. cerevisiae central metabolism from Gombert et al4-Hydroxy-6-methyl-2-pyrone, commonly known as TAL, is a precursor for the production of phloroglucinol maxv from their wild-type values. Contrary to the previous example, here the kinetic expressions do not restrict further the flux ranges as the ABC metric (see Methods) for all kinJ and stoicJ fluxes is zero. As a result, we find no difference in the UMUST (19 reactions) and LMUST (61 reactions) sets for kinetic and original OptForce. This is due to the relatively few fluxes with kinetic expressions and the already fairly tight flux ranges achieved by stoichiometry-alone. For example, the PGI flux varies within the narrow range between 91 and 97 mmol gDW−1hr−1 for a 90% yield for TAL even when no kinetic expressions are used. This is because the imposed high production target for TAL largely fixes the flow in glycolysis. As a consequence of negligible contraction in flux ranges due to the kinetic constraints, no difference in predicted MUST sets by regular and k-OptForce is observed.As in the first example, we allowed for up to two-fold changes in the metabolite concentrations and kinJ. −1 hr−1). This eliminates all flux through G6PDH and PP (from 19 to 0 mmol gDW−1 hr−1) to maintain the steady-state metabolite balance of g6p. Without the benefit of any kinetics, the original OptForce suggests the removal of G6PDH reaction as a requirement for down-regulating the PP pathway. k-OptForce also requires that the concentrations of glyceraldehyde 3-phosphate (g3p) and 3pg be elevated by 6% and 31%, respectively from their reference states to up-regulate the lower glycolysis flux from 168 to 192 mmol gDW−1 hr−1. This re-direction in glycolysis prevents the loss of metabolic flux towards glycerol synthesis. Instead, the original OptForce procedure suggests the removal of glycerol-3-phosphatase (G3PT) in the glycerol synthesis pathway to serve the same purpose and channel the flux towards lower glycolysis and TAL.−1 hr−1) in the flux of aldehyde dehydrogenase (ALDD) which converts acetaldehyde towards acetate. The major routes of acetaldehyde production in S. cerevisiae are either from direct decarboxylation of pyruvate through pyruvate decarboxylase (PYRDC) or through the alternate threonine synthesis pathway followed by the cleavage of threonine by threonine aldolase (THRD) to acetaldehyde and glycine  resulting in a decrease in nadph levels. The original OptForce does not arrive at this intervention as the kinetic control on the fluxes of PYRDC and ALCD is not captured. It instead suggests the removal of cytosolic isocitrate dehydrogenase (ICDHy) to reduce the nadph production, and thus arrests fatty acid synthesis.The fatty acid synthase is in direct competition to TAL production. It uses the same precursors as TAL  to form medium chain fatty acids. Activity of fatty acid synthase requires the cofactor nadph for the reductive steps in the pathway. Not surprisingly, both kinetic and original OptForce identify strategies to lower the availability of nadph. k-OptForce achieves this by suggesting a 20 fold up-regulation . However, when a kinetic model is integrated with a stoichiometric genome-scale model, reactions in stoicJ that involve metabolites present in kinI add in effect additional degrees of freedom to the square-system of equations thus decoupling metabolite concentrations from enzyme activities. As a result metabolite concentrations can change in such a way that fluxes are altered without requiring any enzymatic interventions as observed in the TAL overproduction example. In the absence of kinetic expressions for all reactions associated with the metabolites in kinI a number of degrees of freedom remain for the metabolite concentrations. To avoid drastic concentration changes in response to the overproduction goal we explored penalizing deviations of metabolite concentrations from their reference steady-state values using a weight penalty factor ε. This posture in essence imposes a homeostasis term in the optimization objective function. The outer objective function of the bilevel formulation for identifying FORCE sets is thus modified as follows:kinI , the objective function for the first step is modified as follows:kinJ, scaled by the total number of reactions in kinJ identified in MUST sets (kinMUST). No other changes are made in the formulation of the algorithms. We tested this modified formulation on TAL production in S. cerevisiae.The steady-state balances of metabolites in a kinetic model kinJ for the overproduction of TAL in S. cerevisiae. We varied ε from 0.1  to 0.9  on the identified interventions. Up to a ε value of 0.6, the penalty is not high enough to require direct interventions instead of concentration changes. For a ε value of 0.7 k-OptForce identified up-regulation of enolase (ENO) while maintaining the average deviation in concentration to 0.1053. Note that without the use of the penalty term the concentrations for 3pg and 2pg have to be elevated by 31% and 27% respectively, from their reference levels to redirect more flux through the lower glycolytic reactions of glyceraldehyde-3-phosphate dehydrogenase (PGM) and ENO. For ε = 0.8 the metabolite concentrations remain even closer to their reference levels (average deviation is 0.0754) thereby requiring the up-regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) in addition to ENO. For ε equal to or greater than 0.9 at least 6 additional enzymatic interventions in kinJ are necessary to increase glycolytic flux while concentrations remain very close to their reference values. By increasing the value of the penalty ε enables drawing trade-offs between allowable concentration changes and minimality of needed interventions for an overproduction goal while also providing a prioritization strategy for implementing engineering interventions.E. coli in S. cerevisiae that directly converts pyruvate to acetyl-CoA. Note that S. cerevisiae has a pyruvate dehydrogenase activity in mitochondria but not in cytosol. The PDH complex in E. coli uses nad as the cofactor, however, an nadp-dependent PDH enzyme  in yeast follows a long and tightly regulated path involving the intermediate production of acetaldehyde and acetate E. coli proposed in Chassagnole et al.The kinetic expression for PDH was extracted from the kinetic model of k-OptForce integrates kinetic relations (whenever available) with stoichiometry based models to identify genetic perturbations that are consistent with enzyme expressions and metabolite concentrations. The resulting optimization problems pose significant computational challenges due to the bilevel nature of the formulation and the nonconvex terms in the objective function and constraints. We introduced tractable solution workflows for recasting the problems as equivalent single-level mixed-integer nonlinear optimization problems (MINLP) solved using the global optimization solver BARON to optimality. A hierarchical decomposition approach is also introduced for first identifying interventions within the kinetic part of the model followed by the interventions in the stoichiometry-only part of the model. As with other computational algorithms that make use of kinetics the results can be dependent upon the kinetic model structure and parameterization.Computational results show that the introduction of kinetic expressions in strain design can significantly affect the identified interventions in sometimes non-intuitive ways. In some cases additional modifications are needed to substitute interventions that cause enzyme saturation or concentration bound violations. The mechanism of action of these modifications is often subtle by alleviating substrate inhibition or draining away cofactors from competing pathways. In other cases, kinetic expressions shape flux distributions so as to favor the overproduction of the desired product requiring fewer direct interventions. Uncertainties in both the accuracy of the kinetic models and allowable concentration ranges imply that predicted interventions need to be carefully scrutinized to pinpoint the reasons for their inclusion. An important finding in this study was that concentration ranges have a very significant effect on the identified interventions. By penalizing departures of concentrations from the reference strain values substantial re-arrangements in the predicted interventions are observed. Each one of these changes can be analyzed and the underlying reason for its inclusion can be identified.E. coli provides an example of how k-OptForce can be used to both identify interventions and trace the reason(s) for the exclusion of others. k-OptForce revealed that inhibition of upper glycolysis by pep and 6pgc must be alleviated to route more flux towards L-serine. This is achieved through removal of PDH and down-regulation of TALA or TKT1 respectively. Flux analysis on single gene mutant strains of E. coli show that deletion of either tala or tktA increases the flux through PGI lpdA mutant encoding the PDH enzyme in E. colikinJ. Alternatively, since lpdA also encodes for the activity of ICDHy and the glycine cleavage system (GLYCL), its removal could be have a combined effect on down-regulating the flux in upper glycolysis, which is not captured by the kinetic model. k-OptForce, however, correctly predicts that PDH removal down-regulates lower glycolysis which is observed in the lpdA mutant strain cra), determines the directionality of both upper  and lower glycolysis  A key contribution of kinetic descriptions is that they can attribute performance bottlenecks to specific concentration bounds and/or enzymatic parameter ranges bottlenecks revealing avenues for model improvement and strain optimization. The case study for L-serine overproduction in S. cerevisiaeald6 (which encodes for the ALDD enzyme) increases flux towards acetyl-CoA. However, a fraction of the flux from pyruvate to acetaldehyde was routed through threonine degradation without the requirement of any additional interventions. This direct intervention-free flux redistribution may be an artifact of the kinetic model and may require direct manipulations to engineer. Metabolome studies on single-gene mutant analysis in E. coliet alkinI that do not have a kinetic expression (i.e. not part of kinJ). This would restore the square-system of equations in kinJ and recouple all metabolite concentrations with enzyme activities.k-OptForce may require fewer direct interventions if the kinetic expressions shape fluxes so as to favor the desired overproduction product as observed for the production of TAL where up-regulation of ALDD was suggested to redirect flux from pyruvate to acetyl-CoA. This is consistent with an experimental study for isoprenoid overproduction in E. coli metabolism by Lee et alThe k-OptForce procedure is versatile enough to incorporate additional omics information, whenever available, to further improve prediction fidelity. For example, MFA data for reactions can be included as additional constraints to further tighten flux ranges. k-OptForce can also capture other types of metabolic regulation and select from a wider palette of direct interventions  such as the dynamic hybrid model of Text S1E. coli and S. cerevisiae, supplementary figures for steady-state flux distribution and metabolite concentrations for the kinetic models, and flux ranges for the reference and overproducing phenotypes for L-serine and TAL production.Optimization formulation and solution procedure of k-OptForce, kinetic models of central metabolism for (PDF)Click here for additional data file.


Antibodies against tau protein indicate an interaction between the immune system and the neurocytoskeleton and therefore may reflect axonal injury in multiple sclerosis (MS).The levels and avidities of anti-tau IgG antibodies were measured using ELISA in paired cerebrospinal fluid (CSF) and serum samples obtained from 49 MS patients and 47 controls. Anti-tau antibodies were significantly elevated intrathecally (p<0.0001) in the MS group. The CSF anti-tau antibody levels were lower in MS patients receiving therapy than those without treatment (p<0.05). The avidities of anti-tau antibodies were higher in the CSF than in the serum . Anti-tau avidities in the CSF were elevated in MS patients in comparison with controls (p<0.05), but not in serum.MS patients have higher levels of intrathecal anti-tau antibodies. Anti-tau antibodies have different avidities in different compartments with the highest values in the CSF of MS patients. Demyelination and axonal pathology are the main underlying processes in multiple sclerosis (MS) The targeted axonal antigens for anti-neurocytoskeletal antibody responses in MS have already been investigated. Several studies reported the presence of antibodies against axonal antigens in the serum and CSF of patients having a variety of neurological diseases In this study we were interested to see if there was a similar pattern of anti-neurocytoskeletal response against tau protein, a low-molecular-weight microtubule associated protein abundantly present in the central nervous system. It is primarly expressed in a body of the cell and then it is localized in axons We also investigated the biological activity of anti-tau antibodies in more details. It is known that the antibodies are heterogeneous relative to types, classes, and avidities. Since antibody levels may not be the only feature for characterization, we also assessed avidities of anti-tau antibodies in the serum and CSF of MS patients. We hypothesized that the levels and the avidities of anti-tau antibodies would be increased in MS patients compared to neurological controls. We also wanted to determine if anti-tau antibodies and their avidities could provide some insight regarding therapeutic effects. Furthermore, we studied the relationship between levels of anti-tau antibodies and their avidities in serum and in CSF separately and also between these fluids.All subjects gave written informed consents regarding study participation. The Ethics Committee of the Third Faculty of Medicine, Charles University, Prague approved the study.Paired CSF and serum samples were obtained from 49 MS patients and 47 patients with other neurological diseases. The clinical data about patients and the type of therapy for MS patients are presented in The diagnosis and the course of MS, evaluated at the time of lumbar puncture (LP), were determined using established criteria Twenty-three patients had not received any therapy prior to their lumbar puncture, twenty-six patients had been treated with immunosuppressive (IS) drugs (steroids or azathioprine or both) either alone or in combination with immunomodulators (IM) (interferon-beta or glatiramer acetate).Controls were recruited from neurological patients with heterogeneous diseases. This group consisted of individuals without obvious neuro-axonal disease , i.e. who were suspected to have neurological disease and thus received lumbar puncture but finally turned out not to be affected. It also comprised subjects mainly with polyneuropathy and cranial neuropathy. Twelve patients had aseptic neuroinfection. Except for the patients with neuroinfection we assumed the rest of the controls as normal in terms of antibody status. It is difficult or almost impossible due to ethical reasons to obtain cerebrospinal fluid samples from healthy people, people without neurological disease and with other inflammatory non-neurological diseases.Specimens were stored at −20°C until analyzed.CSF and serum IgG anti-tau antibodies were analyzed using newly developed ELISA techniques based on the ELISA method described by Silber et. al. Tau protein is a highly conserved protein and the amino acid sequences of bovine and human tau are highly homologous For the avidity determination, an extra step using a reducing agent (urea) was added; this step facilitated disruption of antigen-antibody binding. Usually the 6 M or 8 M urea solution is used in avidity experiments 2O2)  for 30 minutes at room temperature. The enzymatic reaction was stopped by adding 2 mol/L H2SO4. The absorbance of each well was read at 450 nm, with a reference filter of 620 nm, using a microplate reader .The procedure for estimating anti-neurocytoskeletal antibodies to tau proteins is as follows. Individual 96-well microplates  were coated with 50 µL of bovine tau protein (2.5 µg/ml) in bicarbonate coating buffer (pH = 9.6). The microplates were then allowed to incubate overnight at 4°C. Blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (pH = 7.2) was performed for 1 hour at room temperature. After washing, 50 µL of sera, diluted 1∶400 in 1% BSA in PBS or undiluted CSF, was added to the wells in quadruplicate and incubated for 2 hours at 37°C. The plates were then washed three times. One hundred microliters, of 8 mol/L urea solution, was added to half of the wells and 100 µl of PBS was added to the other half of the wells. After a 10 min incubation, at 37°C, the wells were washed with wash buffer. In the next step horseradish peroxidase-conjugated goat anti-human IgG , diluted 1∶5000 in bovine serum solution in PBS, was added to all appropriate wells and the plates were incubated for 90 minutes at 37°C. After incubation the plates were washed. Color was developed by adding substrate .Avidity results were expressed as an avidity index. The index represents the percentage of residual auto-antibodies bound in the wells, in the presence of urea solution, to the total auto-antibody bound in the absence of urea. It was calculated by dividing absorbance (or arbitrary units) values obtained from the avidity ELISA assay (with urea addition) by absorbance (or arbitrary units) values obtained using the standard ELISA method (without urea) and multiplying by 100. There was a strong correlation between these two ways of calculations . In this study we used absorbation for the calculation of avidity.Intrathecal synthesis of anti-tau IgG antibodies was estimated as an anti-tau antibody index. The following formula was used: [(CSF anti-tau/serum anti-tau)/].The concentrations of albumin and total IgG in serum and cerebrospinal fluid were assayed using immunonephelometry.Since data were not normally distributed and the number of patients in MS subgroups was low, differences among all groups were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test. The Wilcoxon pair test was used for statistical analysis of avidity differences in the serum and CSF. The Spearman's coefficient was used for correlation analyses. The significance level for all tests was p<0.05. The Bonferroni correction was applied for multiple comparisons. Statistical analysis was performed using Statistica 8 software .The basic clinical and laboratory variables of the MS patients and the control group are shown in The serum and CSF anti-tau IgG antibodies levels tended to be higher in MS patients (as a group) than in controls, but the elevation did not reach statistical significance. Intrathecal synthesis of anti-tau antibodies was significantly elevated in the MS group compared with controls (p<0.0001) . The levIn a more detailed analysis of the MS subgroups, based on clinical course, we observed similar serum and CSF anti-tau antibody levels in various individual clinical forms of MS as well as in controls. Intrathecal synthesis of anti-tau antibodies was significantly higher in all MS clinical forms than in the control group , but a large overlap among different groups was observed. Analysis of PPMS was not performed because of small group size (3 patients only) .Serum anti-tau avidities did not vary between total MS patients and controls. However, further analysis among the MS forms showed that patients in RR-rMS and SPMS subgroups had significantly higher serum anti-tau avidities compared with RR-aMS  . The serOn the other hand, CSF anti-tau avidities were significantly elevated in MS patients compared with controls (p<0.05). There was also a significant difference between the RR-rMS subgroup and the control group (p<0.05) .The avidities of anti-tau antibodies were significantly higher in the CSF in comparison with those in the serum in both the MS group and the control group .Additionally, we compared the anti-tau antibody levels and their avidities in the treated patients and untreated ones. Patients were divided into two subgroups – patients untreated prior to their LP and those treated with immunosuppressive therapy alone or in combination with immunomodulators at the time of their LP. The treated patients had significantly higher the EDSS score in comparison with the untreated group (p<0.0001).Serum anti-tau antibodies were suppressed but without statistical significance in the subgroup of MS patients receiving therapy. With regard to the CSF, MS therapy resulted in a significant decrease in CSF anti-tau antibodies. Untreated MS patients had significantly higher levels of CSF anti-tau antibodies than patients receiving therapy (p<0.05) or the control group (p<0.01) . On the There were no differences in serum anti-tau avidities between either of the MS subgroups and the controls. CSF anti-tau avidity was significantly higher in untreated MS patients than controls (p<0.005) while it did not differ between treated MS patients and untreated ones or controls .A highly significant correlation between CSF and serum anti-tau IgG antibodies was observed in the MS group as well as in the control group . There were three outliners in the MS group. We performed the same analysis without them and we received similar results.Specific CSF anti-tau IgG significantly correlated with total CSF IgG  in the MS groups. A similar relationship in serum was also found for specific anti-tau IgG and total IgG  .Similarly to anti-tau IgG antibodies, avidities in the serum of the MS group were related to those in the CSF, but with a weaker correlation . A relationship between total anti-tau antibodies and their avidities in the serum and CSF was not found in any group with the exception of an inverse correlation between total CSF anti-tau antibodies and their avidities in the controls .No relationship was found between anti-tau antibodies (levels or avidities) in the serum or CSF with regard to clinical variables (age or sex of the patients) in any group. However, intrathecal synthesis was inversely related to age in MS patients . Serum and CSF anti-tau levels and their avidities did not correlate with disease duration or patient disability, expressed as an EDSS in the MS group. No relationship between anti-tau antibodies and EDSS was observed in an additional analysis in subgroups of treated and untreated MS patients.This study demonstrates, for the first time, elevated intrathecal synthesis of anti-tau IgG antibodies in patients with multiple sclerosis compared to the patients with a range of disorders from normal to inflammatory. Therefore effects measured in this study might be even larger than proposed if we took healthy individuals as controls into account. This is another indirect indication of simultaneous axonal and immune processes which were observed in our previous studies as well as studies by other researchers Levels of neurocytoskeletal tau protein are increased during the early stages of MS and in those with higher intrathecal IgG synthesis The absence of a difference between the anti-tau CSF antibodies in the MS and the control groups may be explained by a therapeutic impact on results. Significantly elevated levels in untreated MS patients were masked and offset by lower levels in treated MS patients.CSF anti-tau levels were related to serum levels. Unfortunately, serum anti-tau antibodies cannot be regarded as a biomarker of axonal damage since there is no difference between MS and control groups. Similarly as Rosenmann et al. Antibodies differ in avidities as well as levels. Antibodies in infectious and some autoimmune diseases mature from initially having low-avidity antibodies to having high-avidity antibodies later A surprisingly, serum anti-tau antibodies with higher avidity were present in MS remission patients, unlike those experiencing a relapse. We hypothesize that the anti-tau antibodies with higher avidity form immune complexes more easily, with appropriate antigens, and are missed during detection; immune complexes participate in a variety of pathological actions such as complement activation. Therefore, it is difficult to evaluate possible pathogenic or protective effects of high-avidity anti-tau antibodies in MS from our results.Glucocorticoids, interferon-beta or glatiramer acetate alter B-cell functions. B-cells participate in MS processes at multiple levels In contrast to our previous studies on antibodies against light and medium subunits of neurofilaments Various studies addressed the role of autoantibodies in pathogenesis of some diseases The strength of our study relates to (i) a relatively large sample size of well characterized and balanced subjects in two main groups, (ii) simultaneous measurements of levels as well as avidities of specific anti-tau antibodies both in serum and CSF using newly developed ELISA methods and (iii) study of relationship between therapy and anti-tau antibodies status. Limitations include a small number of MS patients in subgroups other than that in the RRMS subgroup, partly due to ethical reasons (SPMS) or rare occurrence in general (PPMS).


A better understanding of changes in HIV-1 population genetics with combination antiretroviral therapy (cART) is critical for designing eradication strategies. We therefore analyzed HIV-1 genetic variation and divergence in patients' plasma before cART, during suppression on cART, and after viral rebound. Single-genome sequences of plasma HIV-1 RNA were obtained from HIV-1 infected patients prior to cART (N = 14), during suppression on cART (N = 14) and/or after viral rebound following interruption of cART (N = 5). Intra-patient population diversity was measured by average pairwise difference (APD). Population structure was assessed by phylogenetic analyses and a test for panmixia. Measurements of intra-population diversity revealed no significant loss of overall genetic variation in patients treated for up to 15 years with cART. A test for panmixia, however, showed significant changes in population structure in 2/10 patients after short-term cART (<1 year) and in 7/10 patients after long-term cART (1–15 years). The changes consisted of diverse sets of viral variants prior to cART shifting to populations containing one or more genetically uniform subpopulations during cART. Despite these significant changes in population structure, rebound virus after long-term cART had little divergence from pretherapy virus, implicating long-lived cells infected before cART as the source for rebound virus. The appearance of genetically uniform virus populations and the lack of divergence after prolonged cART and cART interruption provide strong evidence that HIV-1 persists in long-lived cells infected before cART was initiated, that some of these infected cells may be capable of proliferation, and that on-going cycles of viral replication are not evident. Anti-HIV compounds are highly effective for preventing the onset of AIDS but they do not cure infected individuals. Very low levels of virus remain detectable in the blood of most patients despite antiviral treatment and levels surge if treatment is stopped. It is crucial to understand why current treatments are not equipped to cure HIV infection so that new therapies addressing these shortcomings can be developed. By characterizing genetic sequences of HIV in patients before and during antiviral treatment, we found that the low levels of virus detected in the blood of treated patients did not result from newly infected cells but originated from cells, or the daughters of cells, that were already infected when treatment was initiated. This finding demonstrates that HIV present in blood after prolonged antiviral treatment is derived from cells infected prior to treatment which likely expanded over time through cell division. Such long lived, infected cells are likely the critical target for developing strategies to cure HIV infection. The consequences of broad HIV-1 genetic diversity include the presence of viral variants containing mutations that escape immune responses or confer resistance to individual antiretroviral agents. The use of antiretroviral agents in combination results in potent suppression of HIV-1 replication and reverses immune deficiency, at least in part. Despite the ability of cART to inhibit HIV-1 replication, treatment does not eradicate infection and plasma viremia persists at low levels in the majority of patients The HIV-1 lifecycle includes rapid and error prone nucleic acid replication that results in large and genetically diverse virus populations et al. described a third phase consisting of long-lived, perhaps latently-infected, cells with a half-life of 6–44 months as well as a fourth phase having a slope not significantly different from zero et al., McMahon et al., and Gandhi et al. showed no decrease in the level of persistent viremia in patients on long term suppressive therapy before, during, or after intensification with an additional antiretroviral suggesting the absence of ongoing new rounds of replication during suppressive cART et al. investigated plasma viral sequences after long-term cART and found that HIV-1 populations often contain sets of identical sequences, referred to as “predominant plasma clones,” suggesting that viral subpopulations are lost over the course of treatment et al. found an increasing frequency of identical sequences in blood cells during cART suggesting proliferation of infected cells et al. showed that homogeneous populations rebound after cART interruption The dynamics of HIV-1 decay after initiating cART can be divided into four phases et al. suggested that phylogenetic clustering of sequences obtained from different cellular compartments after long-term cART demonstrated cross-infection between reservoirs, consistent with full cycles of replication as a source of persistent viremia In contrast to these findings, other studies have indicated that low-level virus replication may occur in specific anatomical compartments despite suppression of plasma HIV-1 RNA by cART To investigate further the effect of cART on virus replication, we examined HIV-1 populations in patients prior to cART, during each phase of viral decay including long-term cART (fourth phase), and during viral rebound after interruption of cART. By investigating the genetics of HIV-1 in all phases of viral decay and comparing on-therapy populations to pre-therapy virus we were able to directly assess HIV-1 replication and molecular evolution during long-term suppressive cART. We found that both short and long lived cellular compartments were seeded with the same diverse virus populations and that new viral populations rarely emerged after up to 15 years of cART.Participants were enrolled in prospective studies aimed at determining the role of antiretroviral therapy on HIV-1 infection  or on HIV-1 population genetics in infected individuals (00-I-0110) conducted at the NIH Clinical Center in Bethesda MD All participants in this study were enrolled in clinical protocols  approved by the NIAID Institutional Review Board (FWA00005897) administered at the NIH Clinical Center in Bethesda, Maryland. Individuals underwent an informed consent process and provided written consent for participation.gag-pro-pol amplified from plasma HIV-1 RNA was performed as previously described http://www.megasoftware.net) and an in-house program −3 as described by the original report to account for the high number of comparisons between sequences and nucleotide sites −3 for assigning a significant change in intra-patient HIV populations was derived statistically taking into consideration that every nucleotide position is compared in every two possible sets of sequences. This approach results in more than 1012 comparisons between populations of only 10 sequences. The test was derived from a geographic population structure test proposed by Hudson et al.p-value for the probability that the randomized populations' structures are the same between sets of sequences. Neighbor-joining phylogenetic analyses were performed using MEGA5 http://www.HIV-1.lanl.gov). Tests for molecular evolution were done with BEAST http://beast.bio.ed.ac.uk) using the HYK+G model with a relaxed clock, uncorrelated log normal and constant size, followed by estimating the root to tip distances with TreeStat1.2 (www.tree.bio.ed.ac.uk/software/treestat). Linear regression was used to determine the slopes for the root-to-tip analyses.HIV-1 RNA levels were determined using bDNA Versant version 3.0  as previously described To investigate if genetic bottlenecks occurred after initiating cART, we evaluated changes in the number of heterozygous sites over time To investigate the effect of cART on plasma HIV-1 diversity, we assessed HIV-1 genetics by single-genome sequencing of plasma HIV-1 RNA in individuals undergoing cART. Plasma samples were obtained prior to and following introduction of cART; and, for some patients, after planned patient-initiated treatment interruptions. Single-genome sequences were obtained at time points throughout the study period, and population genetics parameters were measured. Genetic diversity was measured by APD of virus populations in patients' plasma prior to treatment, during each phase of viral decay, and during viral rebound , 2. GrouMost patients (13 of 14) showed no significant difference in APD of HIV-1 populations during any phase of viral decay, after long-term therapy, or after viral rebound, compared to pre-therapy virus populations , 2. ThisAlthough HIV-1 populations revealed no significant change in APD with cART, genetic bottlenecks may occur in large, diverse populations without producing a detectable change in the overall diversity. During a bottleneck, low frequency alleles, which do not contribute substantially to overall diversity or to phylogenetic signal, are lost To specifically investigate whether prolonged HIV-1 suppression resulted in changes in amino acid sequences, we investigated nonsynonymous changes alone in patients from groups 1 and 2 for which there were more than 7 sequences at time points with <50 copies/ml (N = 8) . We founp-value for the probability that the populations are the same −3 were considered to indicate significantly different populations, taking into account the large numbers of comparisons. Divergence of HIV-1 populations during cART could result either from on-going cycles of replication leading to the emergence of new variants or as a consequence of shifts in the viral variants present in the plasma during suppression, indicating a dynamic reservoir. To investigate the possibility of population shift (divergence) during cART, we used a test for panmixia to detect changes in the population structure during therapy compared to pretherapy virus. The panmixia test compares populations of single-genome sequences obtained from longitudinal samples and provides a To further determine if the population shifts detected in the plasma of some patients during and after long-term cART were the result of on-going cycles of virus replication or were due to a shift in the population of cells that express virus particles during therapy, we performed phylogenetic analyses and tests for molecular evolution. Such tests can detect with high sensitivity the emergence of new viral variants indicative of full cycles of replication during cART. We used neighbor-joining trees to first evaluate the direct relationship of the sequences obtained prior to, during, and after therapy and we subsequently used tests for molecular evolution and calculations of root-to-tip distances to detect the emergence of new virus populations during cART.Phylogenetic trees of virus sequences from 6 patients on long-term cART are shown in The genetics of rebound viremia are shown in two patients from group 3  in http://beast.bio.ed.ac.uk) to determine if the plasma virus populations present during therapy were newly emergent variants or were pre-existing. The molecular evolution test was performed by measuring the distances from the root of the tree (rooted on consensus B) to the tip of each branch (−5±4.5×10−5 nt/day) with no significant differences between groups , showing that the variants present during the second and third phases of decay and after prolonged therapy were not more distant from the root of the tree than variants present prior to initiating therapy. In a few cases the sequences were actually slightly closer to the root (consensus B) resulting in a negative slope. By contrast, the slopes in untreated elite controllers with similar levels of viremia have significantly positive slopes (median = 15 nt/day) (p = 0.009) when measured over similar intervals Neighbor-joining analyses allowed us to visualize the plasma virus populations present during cART compared to those in pretherapy, but cannot be used to determine if the variants present during treatment are newly emergent resulting from full cycles of replication or if they are merely the expression of variants from cells infected prior to treatment. For this purpose, we applied a test for molecular evolution using Bayesian analysis as implemented in BEAST  of persistent viremia during suppressive antiretroviral therapy remains uncertain, and there have been a number of studies to investigate whether repeated full cycles of virus replication occur during adherence to cART or if low-level viremia present in the plasma of successfully treated patients is the result of viral expression from long-lived cells infected prior to treatment. Population genetics and phylogenetic approaches represent powerful techniques to detect genetic change in temporally spaced samples, but in the setting of relatively high genetic diversity it is often difficult to determine whether observed change represents molecular evolution from ongoing replication or a shift in the population of reservoir cells producing virus. One way to resolve this issue is to compare HIV-1 populations prior to and following initiation of cART and to compare temporal changes in viral populations in treated patients to untreated elite controllers with similar levels of viremia and duration of control. In this study, we investigated HIV-1 First, we investigated virus populations in samples collected within the first 6 months of initiating cART and compared these populations to the viral sequences obtained from pretherapy samples. We found no change in the diversity, divergence, or phylogenetic structure in populations obtained before and after 6 months of ART, and no evidence of any genetic bottleneck in 8/9 patients on study. These results indicate that both short- and long-lived cellular compartments are seeded with the same viruses and that these compartments are sufficiently large to support highly diverse populations of HIV-1 genomes. The sustained diversity of HIV-1 populations over months of suppressive therapy without a genetic bottleneck or loss of low frequency alleles also implies that pre-existing low-level drug resistance mutations are not likely to be lost during antiretroviral therapy.To investigate the genetics of persistent HIV-1 during long-term cART, we also sequenced plasma virus populations during 4–15 years of suppressive therapy; and again, compared these populations to those obtained from pretherapy samples. In contrast to earlier samples on cART, we found clusters of identical sequences in plasma samples collected after long-term treatment. However, using phylogenetic tests for molecular evolution , we found no evidence for the appearance of new variants during long-term cART and the clusters fit within the phylogenies of virus populations present before therapy (with the exception of one patient). The presence of identical sequences during ART suggests that virus particles are being produced by an HIV-1 infected clonal cell population, such as stem cell-like CD4+ memory T-cells or other proliferating cell types. These conclusions are consistent with previous studies that indicate that persistent plasma viremia during cART is derived from viral expression in long-lived cells Given that long term therapy reduces the average level of viremia from about 30,000 to about 1–3 copies of RNA per ml on average The conclusion that cART effectively and completely halts HIV-1 replication in those infected cells that are responsible for viremia is consistent with prior studies by us and others showing that low levels of viremia on therapy are independent of the therapeutic regimen used and they cannot be further suppressed by additional drugs Table S1Description of patient samples analyzed.(DOCX)Click here for additional data file.Table S2Patient HLA types and shifts in CTL Mutations during cART.(DOCX)Click here for additional data file.


Pediatric bipolar disorder (PBD) is a severely debilitating illness, which is characterized by episodes of mania and depression separated by periods of remission. Previous fMRI studies investigating PBD were mainly task-related. However, little is known about the abnormalities in PBD, especially during resting state. Resting state brain activity measured by fMRI might help to explore neurobiological biomarkers of the disorder. Methods: Regional homogeneity (ReHo) was examined with resting-state fMRI (RS-fMRI) on 15 patients with PBD in manic state, with 15 age-and sex-matched healthy youth subjects as controls. Results: Compared with the healthy controls, the patients with PBD showed altered ReHo in the cortical and subcortical structures. The ReHo measurement of the PBD group was negatively correlated with the score of Young Mania Rating Scale (YMRS) in the superior frontal gyrus. Positive correlations between the ReHo measurement and the score of YMRS were found in the hippocampus and the anterior cingulate cortex in the PBD group. Conclusions: Altered regional brain activity is present in patients with PBD during manic state. This study presents new evidence for abnormal ventral-affective and dorsal-cognitive circuits in PBD during resting state and may add fresh insights into the pathophysiological mechanisms underlying PBD. Pediatric bipolar disorder (PBD) is a severely debilitating illness, which is characterized by episodes of mania and depression separated by periods of remission. Nearly 1% of adolescents and children have diagnosis of PBD So far, functional studies investigating PBD have mainly used emotional or cognitive process tasks. Using varied kinds of emotional tasks,findings in PBD exhibited either reduced or increased activity in the ventrolateral prefrontal cortex (VLPFC) Many studies have proposed structural alterations in the cortical To fully understand the neural pathophysiology of PBD, we need to investigate how the brain allocates most of its resources. However, task-dependent altered neural activity only represents no more than 5% of the total brain activity Regional homogeneity (ReHo), a newly developed method, reflects the temporal homogeneity of the regional blood oxygen level-dependent (BOLD) signal rather than its density, and serves as a complement for RSFC. Abnormal ReHo may be relevant to the changes of temporal aspects of neural activity in the regional area, and can be used to find abnormal activity in the whole brain According to recent reviews, Pavuluri and Wessa We hypothesized that abnormal activity in the ventral-affective and dorsal-cognitive neural systems could be found in PBD. Our aim was to find out whether the abnormal regional activity detected by RS-fMRI and analyzed by the ReHo method in PBD patients differed from that in healthy controls.Twelve to seventeen-year old subjects were enrolled at Mental Health Institute of The Second Xiangya Hospital, Key Laboratory of Psychiatry and Mental Health of Human Province, Central South University  and were scanned as soon as possible after initial contact (within 1 to 2 days). Healthy comparison participants (HC) were recruited through local middle school. The study was approved by the ethical committee of the Second Xiangya Hospital, and written informed consents were obtained from both the children and their parents. Recruitment included advertisements in physician offices and advertisements in local middle school.The PBD patients (n = 15) were outpatients from psychiatric clinic in the Second Xiangya Hospital. The inclusion criteria were: meeting DSM-IV-TR criteria for BD, including at least one episode meeting full DSM-IV-TR criteria for hypomania (≥4 days) or mania (≥7 days),and being manic at the time of screening For healthy controls (n = 15), the inclusion criteria were a negative history of psychiatric illness in the subject and his/her first-degree relatives. Exclusion criteria were IQ≤80; ongoing medical or neurological illness; pregnancy; or past/present psychiatric or substance disorder.All participants were evaluated by a board-certified child psychiatrist using the Affective Disorders and Schizophrenia Scale for School-Age Children-Present and Lifetime Version (K-SADS-PL) administered to parents and children separately All participants were completed the Wechsler Abbreviated Scale of intelligence as an overall measure of cognitive ability 2tests, respectively. Correlation between the YMRS score and activity of brain areas was assessed using Pearson’s correlations analysis.Statistical analysis was performed with SPSS11.5 software. Continuous data and categorical data were analyzed with t-tests and chi-square Functional images were obtained on a 3-Tesla scanner (Siemens Trio) using a standard whole-head coil. Functional images were acquired by using of single-shot gradient echo-echo imaging (GRE-EPI) sequence .Structural images were acquired by using a three-dimensional magnetization-prepared rapid gradient-echo (MPRAGE) sequence . During the scanning, all subjects were informed to relax, hold still, keep their eyes closed without falling asleep and think of nothing in particular.http://www.fil.ion.ucl.ac.uk/spm). The fMRI images were corrected for the acquisition delay between slices and for the head motion. The participants whose head motion exceeded 1.5 mm or rotation exceeded 1.5° during fMRI scanning were excluded. The fMRI images were normalized to a standard SPM8 echoplanar imaging template, resampling to 3×3×3 mm3. Data were temporal band-pass filtered (0.01–0.08 Hz) to reduce the effects of low-frequency drift and physiological high-frequency noise, and the linear trend was removed Spatial preprocessing of fMRI data was done using the SPM8 software (http://www.resting-fmri.sourceforge.net). ReHo maps were generated for each subject by calculating Kendall coefficient of concordance (KCC) of the time series of a given voxel and the nearest voxels (26 voxels) in a voxelwise analysis Regional homogeneity analysis was performed with software REST  rate correction) was first performed for both groups to show where the standardize KCC value was larger than 1 Finally, to study whether the ReHo correlates with the manic severity in the patients with PBD, a correlation analysis of ReHo versus YMRS score was performed in the patients with PBD at regions of interest . For correlation analysis, a threshold of P<0.05 was chosen as a threshold for statistical significance.The PBD and HC groups did not differ significantly in age, gender, full-scale IQ and handedness. The PBD sample consisted of 15 participants with type I BD; none had type II BD, although it was not excluded. Our PBD participants were manic by mood ratings . All PBD participants were receiving psychopharmacological treatments, including lithium (n = 6 [40%]), atypical antipsychotics (n = 11 [73.3%]), Valproate (n = 8[53.3%]), antidepressants (n = 2 [13.3%]), and benzodiazepines (n = 1[6%]). Two PBD participants had comorbid obsessive-compulsive disorder (OCD) and three PBD participants had psychiatric symptoms. Only one PBD participants (6%) had one first-degree relative with BD. .Compared with the healthy controls, the PBD patients showed significant increase of ReHo in the bilateral hippocampus, the right anterior cingulate cortex, the right parahippocampal gyrus, and the left caudate. Regions showed decreased ReHo included the bilateral precuneus, the bilateral precentral gyrus, the bilateral superior frontal gyrus, the bilateral superior parietal lobe, the right orbitofrontal cortex, and the right superior temporal gyrus. ; Table 2Analysis by regressing ReHo at each ROI  against the score of YMRS in PBD group revealed significant positive correlation in the bilateral hippocampus  and the ACC . Negative relationship between ReHo and the Score of YMRS was found in the bilateral superior frontal gyrus in the PBD group . .To our knowledge, this study is the first research that shows neural synchronization of local brain areas in PBD during resting state. The latest researches also showed that PBD was dysfunctional in ventral-affective and dorsal-cognitive circuits Studies of pediatric patients can provide important information about interactions between BD and brain development, and are less likely to be confounded by medication exposure, substance use, and the long-term effects of mood symptoms, compared with adult samples. Thus this study chose PBD as experimental subject.Specifically, ReHo was developed to characterize the local synchronization of spontaneous fMRI BOLD signals and has the dominant characteristics of simple operation and good inter-experiment consistency This result showed that the PBD manic patients had decreased ReHo in the orbitofrontal cortex (OFC). OFC is involved in the cognitive processing of decision-making This study also found decreased ReHo in the superior frontal gyrus (SFG) in pediatric manic patients compared with healthy controls. Reduced ReHo in SFG represents decreased neural coherence in this region. The SFG was involved in cognitive process, such as executive function and memory retrieval When PBD patients are compared with HC patients, decreased coherences were also observed in the precentral gyrus and the superior parietal lobe (SPL), which are located in the cognition process network In this study, decreased ReHo was found in the superior temporal gyrus (STG). The STG takes part in the process of emotions In this study, we found decreased ReHo in the precuneus. It has been proposed that the precuneus is associated with episodic memory retrieval ReHo was significantly increased in the dorsal anterior cingulate cortex (dACC) in PBD patients, which reflects the enhancement of the local synchronization of spontaneous neural activities in this region. Increased ReHo may reflect neural hyperactivity in a regional brain area The dACC is the dorsal part of the ACC (BA32), which is connected with the parietal cortex, the prefrontal cortex as a central hub for processing top-down and bottom-top controls ReHo of the hippocampus and the parahippocampal gyrus (PHG) were both increased in this study. Several studies exhibited increased hippocampal activation in BD and PBD patients in affective tasks involved in emotional faces We also found ReHo increase in the caudate in the PBD patients compared to the healthy controls. The caudate is involved in both emotional and cognitive regulation We found positive correlation of abnormal ReHo in the hippocampus and the ACC with the manic severity (YMRS Score) in the PBD group. We also showed negative correlation between abnormal ReHo in the STG and the manic severity in the PBD group. The patients who had worse symptoms may have severer dysfunction in the neural activity. Combined with more large-sample experiments in the future, this finding will make it possible to assess clinical symptom severity in PBD patients.The caudate was associated with the dorsal-cognitive circuits In this study, we found diffused activities in many brain regions, probably because that regional activity in children and adolescents were more diffused compared with adult Firstly, almost all the PBD participants were taking psychotropic medications. It would be unethical to withhold anti-manic, anti-depressant and other antipsychotic medications for research purposes alone; and brief discontinuations would be ineffective, given their longer half-lives. In past fMRI studies of PBD, either no significant Secondly, Although we excluded the prominent confounded factors of the comorbid ADHD and anxiety Thirdly, although the PBD and control subjects were matched in age and gender, it need an larger study with sufficiently statistical power to meaningfully explore the potential confounding effects of age, gender and treatment in the future research.Our study for the first time shows neural synchronization of local brain areas in PBD during resting state. In summary, we find diffused dysfunctional regions associated with the ventral-affective and dorsal-cognitive circuits. The abnormal findings obtained from resting state-ReHo method are likely to be used for illustrating pathophysiologic mechanism of PBD. Combined with more large-sample experiments and clinical indicator in the future, this method may help in assistant diagnosis for PBD.