We investigate whether individuals are more prone to act selfishly if they can passively allow for an outcome to be implemented (omission) rather than having to make an active choice (commission). In most settings, active and passive choice alternatives differ in terms of factors such as the presence of a suggested option, costs of taking an action, and awareness. We isolate the omission effect from confounding factors in three experiments, and find no evidence that the distinction between active and passive choices has an independent effect on the propensity to implement selfish outcomes. This suggests that increased selfishness through omission, as observed in various economic choice situations, is driven by other factors than a preference for selfish omissions. Are people more selfish if they can passively allow for a selfish allocation of resources, rather than actively having to implement it? This question speaks to a variety of different choice situations, as decisions about resource allocations often vary by whether they require active involvement or not. People may be more likely to walk by a charity solicitor on the street than to refuse to give to a solicitor who knocks on their door. An employer may passively allow for inflation to erode workers’ real wages even if she would not be willing to actively cut nominal wages. Citizens face governmental policies about redistributive matters that vary by the involvement they require, such as whether being an organ donor requires active registration or not, or whether declarations of taxable income require more or less active statements. In this paper, we conduct three experiments to test whether individuals exhibit preferences for selfish omissions over selfish commissions in economic decision-making.commission, if the decision maker implements an outcome by taking an action, whereas we define a choice as passive, or as an omission, if the decision maker allows for an outcome to be implemented by being inactive. A body of research in moral philosophy and psychology suggests that individuals favor harmful omissions over harmful acts. Subjects faced with hypothetical choice scenarios often judge harmful acts as morally worse than equally harmful omissions )Apart from the presence of a suggested option, commissions and omissions often differ in terms of the costs of taking an action and the decision maker’s awareness. In many choice situations remaining passive is less costly than taking an action. Making an active choice may require more costs in terms of time and effort, such as filling out an organ donation form. It may also be cognitively more costly for individuals that are unsure about what to choose to resolve this uncertainty under active choices. In some settings, passive choices might be the result of unawareness of being in a choice situation or a lack of enough time to make an active choice, rather than the expression of a preference. In such settings, self-serving commissions may be judged more harshly than self-serving omissions, merely due to differences in the revealed intent. For example, one motivation for legally distinguishing between the act of killing and the failure to help someone who is dying is that the former provides stronger evidence of harmful intentions. Our contribution is to provide a test that isolates the effect of active and passive choices from these other confounding factors . The main question we ask is whether there is an omission effect in the sense that individuals have a preference for implementing selfish options passively rather than actively.morally significant in and of itself. Accordingly, individuals that inherently care about behaving morally may reflect this distinction in their behavior. Such a moral distinction is not necessarily made consciously. Research by Cushman et al. . Thus, while the point estimate is very small and not nearly statistically significant, we can only rule out the existence of an effect larger than 17.1 percentage points.We also run an OLS regression, using an indicator for whether the participant chose the  default option as the dependent variable , no individual treatment effect is statistically significant. Since we do not find any order effects in the likelihood to choose the default, in the analyses below we pool all choices.We also test our hypotheses using data from all 14 allocation choices. Table C in Omission treatment. Model 1 restricts the sample to only the first choice, and verifies the result from the previous section . Model 2 pools all allocation choices with a selfish default, i.e. all allocation trade-offs where the default option is strictly payoff-dominant for the dictator. There is no significant omission effect in the likelihood to choose a selfish default even when pooling all choices , and the 95% confidence interval narrows from  in Model 1 to  in Model 2.By pooling all choices we can substantially increase the precision of our estimates, compared to when restricting the data to the first allocation choice. To test our first hypothesis, we look at all choices with a selfish default, i.e. all allocation trade-offs where the default option is strictly payoff-dominant for the dictator. Next, to see whether the omission effect varies across allocation trade-offs with different properties, we split our data into different subsets of choices. Model 3 restricts the analysis to allocations where the dictator’s payoff is at least as high as the recipient’s payoff. Model 4 only includes choices that trade-off selfishness and behindness, i.e. choices between one option which is weakly payoff-dominant for the dictator and another option giving the dictator less than the recipient. Model 5 is restricted to the choices where the selfish option is less efficient than the non-selfish option. Also for these subsets of choices, there is no significant omission effect. The 95% confidence intervals for the estimated coefficients are ,  and , respectively. Hence, even when substantially increasing the precision of the estimated omission effect by pooling all choices, we find no evidence that individuals are more likely to be selfish passively rather than actively. As in the previous section, we leave the analysis of the fair default to In χ2(1) = 1.17, p = 0.28), nor in the omission treatment  = 0.01, p = 0.92).To evaluate the size of the default effect in Experiment 1, we compare the share of selfish choices when the selfish allocation is the default to the share of selfish choices when the fair allocation is the default. In the first allocation choice, the share of  choices is not significantly larger when  is the default as compared to when  is the default, neither in the commission treatment  but not in the omission treatment . Using the sample restrictions from Models 3–5, there are no significant default effects, neither in the commission treatment nor in the omission treatment. Assuming that the default effects for selfish and fair defaults point in the same direction, this means that we find no strong evidence of default effects in Experiment 1.We estimate default effects for the pooled choices by running separate regressions for each treatment, using an indicator variable for selfish choice as the dependent variable, and an indicator variable for selfish default as the explanatory variable. Using the sample restriction from Model 2 in In Experiment 1, we find no evidence of an omission effect on prosocial behavior. However, nor do we find any strong evidence of a default effect. Thus, from Experiment 1, we can conclude that there is no evidence of an omission effect independent of the default effect. However, given that default and omission effects have been suggested to be based on similar mechanisms (see e.g. ), it canselfish allocation), giving $1.05 to the dictator and $0.05 to the recipient, and an allocation which is both fair and efficient , giving $0.70 each to the dictator and the recipient. Each subject is randomly assigned to one of three treatments: the commission treatment, the omission treatment or the no-default treatment. After the experiment, subjects are randomly matched and randomly assigned to either the role of the dictator or the role of the recipient and paid accordingly. As in Experiment 1, subjects have 40 seconds to make their choice, as indicated by a timer on the screen. To simplify the procedure, we do not include the slider task that was used in Experiment 1.The second experiment employs a one-shot dictator game with one binary allocation choice. The relative payoff between the two allocations resembles that used in the first choice in Experiment 1. Subjects choose between an allocation which is payoff-dominant for the dictator  = 2.01, p = 0.16). We include these two subjects in the analysis presented below, but our results are robust to excluding them. 40% of the participants are women and 75% are located in the US. 151 subjects participated in each treatment. No subject in the commission treatment and only 2 subjects (1.3% of subjects) in the no-default treatment failed to make a choice in the allotted time. These shares are not significantly different across the two treatments (χ2(1) = 17.29, p = 0.00). This corresponds to an increase in selfish choices of 70.4% through the introduction of a selfish default. Thus, as hypothesized, subjects are more prone to choose the selfish option when it is presented as the default.First, we test whether we succeeded in introducing a default effect into the decision. In the no-default treatment, 33.8% of the subjects choose the selfish option. Presenting the selfish option as the default in the commission treatment significantly increases the share of selfish choices by 23.8 percentage points (χ2(1) = 0.054, p = 0.816). Thus, even in a context where the default effect is strong and the mechanisms behind a potential omission effect are at work, we find no evidence that the possibility of choosing a selfish default passively rather than actively increases the share of selfish choices. The 95% confidence interval of the estimated omission effect is . Hence, assuming that the effect is positive, we can rule out the existence of an omission effect of 10 percentage points or larger.Second, we test whether an omission effect occurs in the presence of this default effect. The difference in the share of selfish choices in the commission treatment and the omission treatment is small with 1.3 percentage points and statistically insignificant (χ2(1) = 9.30, p = 0.00; omission vs. no-default: χ2(1) = 4.62, p = 0.03). Thus, a significant, but small, fraction of subjects in the omission treatment consciously use the option to be selfish by omission. However, since the share of selfish choices is the same across the omission and the commission treatments, choosing the selfish option passively only seems to serve as a substitute to being selfish by commission.Subjects in the omission treatment could implement the selfish default allocation either actively (by ticking the box) or passively (by letting the timer run down). Among the subjects that choose the selfish option in the omission treatment, 10.6% use the possibility to remain passive. This corresponds to 6% of all subjects in the omission treatment, which is a significantly larger share than the subjects failing to make an active choice in the no-default treatment and the commission treatment  whether the time was sufficient for making a choice on a scale from 1 (“strongly disagree”) to 7 (“strongly agree”), and (ii) how pressured they felt by the time constraint on a scale from 1  to 5 (“very time pressured”).χ2(1) = 1.00, p = 0.32). We include this subject in the analysis presented below, but our results are robust to excluding the subject.We recruited 448 subjects through a work task posted on Amazon Mechanical Turk in November 2016. Subjects received a fixed payment of $1.00 for participation on top of their earnings from the experiment, which were paid as bonuses. 45% of the participants are women and all participants are located in the US. No subject in the no-default treatment and only one subject (0.67%) in the commission treatment failed to make a choice within the allotted time. The share of subjects failing to make an active choice is not significantly different between these two treatments z = 2.57, p = 0.01). Importantly, 95.1% of subjects agree that time was sufficient. Similarly, subjects report significantly stronger feelings of time pressure in this experiment than in Experiment 2 , but 91.3% of subjects still report that they felt little or no time pressure. Thus, while the more restrictive time constraint significantly affected subjects’ perception of the available decision time, most subjects still agree that 20 seconds is sufficient time for making an informed choice without much time pressure.Compared to subjects in Experiment 2, subjects in this experiment agree less with the statement that they had sufficient time to make their choice. This different is significant but fairly small  = 3.13, p = 0.08). Thus, we find evidence of a default effect, albeit the size of this effect is 14 percentage points smaller than in Experiment 2 (p = 0.08 from a joint regression).χ2(1) = 0.17, p = 0.68). Thus, even when substantially reducing the time subjects need to wait when making a passive choice, we find no evidence that the possibility of choosing a selfish default passively rather than actively increases the share of selfish choices. The 95% confidence interval of the estimated omission effect is , ruling out effect sizes above 13.8 percentage points.The share of selfish choices in the omission treatment is only 4.4 percentage points larger than in the commission treatment. This difference is not statistically significant (χ2(1) = 4.58, p = 0.03; omission vs. no-default: χ2(1) = 7.12, p = 0.01). This suggests that, as in Experiment 2, a small but significant share of subjects in the omission treatment consciously use the option to be selfish by omission. However, again, the lack of difference in selfishness between the commission treatment and the omission treatment indicates that choosing the selfish option passively is merely a substitute to choosing the same option actively.As in Experiment 2, subjects in the omission treatment could implement the selfish default allocation either actively (by ticking the box) or passively (by letting the timer run down). Among the subjects who chose the selfish option in the omission treatment, 9.7% used the possibility to remain passive. In total, this corresponds to 4.7% of subjects in the omission treatment, which is a significantly larger share than the share of subjects remaining passive in the other treatments  and a setting with stronger default effects (Experiments 2 and 3). This indicates that prosocial preferences do not depend on the difference between commissions and omissions as attributes of the action taken by a decision maker. It has been suggested that a utility maximization framework in the presence of social norms may benefit from including actions as an argument of the utility function . OurOur result suggests that increased selfishness through omission, as observed in various settings, is not an omission effect in the sense that it expresses an individual’s preference for selfish omissions. Rather, observed behavioral differences between passive and active choices are likely to be driven by other factors than the difference between active and passive choices in and of itself. This leads to the question of which other situational factors could explain why passivity and selfish behavior often coincide. Below, we discuss the potential role of default and status quo effects, costs of taking an action, time constraints and awareness as potentially important confounding factors that commonly also differ across commissions and omissions in previous economics literature.First, the omission option is a passive default option. Default effects have been identified in a variety of consequential real-world decisions, such as whether to become an organ donor ,32, the Second, commission and omission options can differ substantially in terms of action costs. In our experimental setting action costs are reduced to a minimum, since making an active choice only requires clicking a button. An example of a field setting with minimal action costs is online purchases where choosing to add options, such as environmentally friendly delivery or a charity donation, only requires checking a box. However, in some real life situations, active choices are associated with considerable investments of effort and time. For instance, actively choosing to become an organ donor may require both time and effort, such as filling out a form and reflecting on the ethicality of one’s choice. Similarly, making an informed decision about a new retirement plan requires a careful evaluation of all alternatives. Thus, in some settings, an increased propensity to stay passive may simply reflect the costs associated with making an active choice.Third, it can be the case that passive choices, but not active choices, result from unawareness of the choice situation. In such settings, an increased willingness to be selfish by omission rather than commission can arise because agents are genuinely unaware of the possibility to commit a non-selfish act. Or, it can arise because they use this potential unawareness as an argument when justifying their choice to remain passive. For instance, individuals may be more likely to passively walk by a silent charity solicitor, allowing for potential unawareness, as compared to actively refraining from contributing if the solicitor explicitly asks for a contribution. Along these lines, several studies find that when given the possibility to opt-out of a situation in which a fairness norm would suggest a monetary contribution, some individuals choose this opt-out option –38. For Fourth, time constraints may be used by agents as a credible excuse for being selfish by omission. Dana et al.  conduct Investigating the specific role of each of the above-mentioned mechanisms is beyond the reach of this paper. While our second and third experiments indicate that the presence of a default plays a non-negligible role, an interesting direction for future research would be to more systematically address the importance of costs of taking an action, plausible unawareness and time constraints in the same setting. As suggested by an anonymous reviewer, one natural extension to the current study would be to first replicate previous findings of an omission effect and then remove each of these confounding factors one at a time.prosocial behavior in a context with economic outcomes. Whether omission effects exist for decisions that affect only the decision-maker, e.g. choices relating to risk or time preferences, is still an open empirical question. For example, consider the risky decision of whether to sell stocks at a loss or not. This decision may vary both in terms of the presence of a default  and whether the default is implemented passively or actively . Relatedly, Ritov and Baron [Furthermore, our experiment focuses specifically on omission effects in nd Baron  conduct Similarly, we cannot rule out the possibility of an omission effect in prosocial choices with non-economic outcomes. Gärtner  suggestsFinally, while we find no omission effect in choices, it is possible that there is nevertheless an omission effect in the moral judgment of others’ choices. Previous vignette studies in psychology –5 suggesS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S1 Data(XLS)Click here for additional data file.S2 Data(XLS)Click here for additional data file.S3 Data(XLS)Click here for additional data file.S1 Code(TXT)Click here for additional data file.S2 Code(TXT)Click here for additional data file.S3 Code(TXT)Click here for additional data file.


FDDG), garbanzo flour and corn grits were chosen as a source of high‐protein and high‐fiber extruded snacks. A four‐factor central composite rotatable design was adopted to study the effect of FDDG level, moisture content of blends, extrusion temperature, and screw speed on the apparent viscosity, mass flow rate or MFR, torque, and specific mechanical energy or SME during the extrusion process. With increase in the extrusion temperature from 100 to 140°C, apparent viscosity, specific mechanical energy, and torque value decreased. Increase in FDDG level resulted in increase in apparent viscosity, SME and torque. FDDG had no significant effect (p > .5) on mass flow rate. SME also increased with increase in the screw speed which could be due to the higher shear rates at higher screw speeds. Screw speed and moisture content had significant negative effect (p < .05) on the torque. The apparent viscosity of dough inside the extruder and the system parameters were affected by the processing conditions. This study will be useful for control of extrusion process of blends containing these ingredients for the development of high‐protein high‐fiber extruded snacks.A combination of different levels of distillers dried grains processed for food application ( Extrusion process is a combination of cooking, mixing and forming, resulting in good quality direct expanded product. It is widely used in industries owing to its characteristic high throughput and automatic control  and distillers dried solubles (DDS). Distillers dried grains with solubles (DDGS) is produced after mixing and drying these two co‐products Singh, . DDGS coCereal or grains are the primary source of most extruded snack foods because of their high expansion properties. However, they tend to be low in protein and essential nutrients. Usually, cereals lack lysine as the essential amino acid but have sufficient sulfur containing amino acids. Legumes on the other hand, are rich in lysine and deficient in sulfur containing amino acids. When combined together, the proteins of cereals and legumes complement one another to produce a protein of a better quality. Corn in different form has been widely used as raw material for extrusion. It is ideal for extrusion since it has a high starch content, which facilitates the expansion process. It is gluten‐free and contains protein, fiber, vitamins, and unsaturated fatty acids. Compared to wheat, oat, and rice, corn has higher phytochemical content such as phenolic compounds which have anticarcinogenic effects , low fat (6.48%), high available carbohydrate (50%) and crude fiber contents of 3.82%  during extrusion cooking.22.1Distiller's dried grains with solubles (DDGS) was obtained from Glacial Lakes Energy LLC, Watertown, SD. It was then washed, freeze dried, steam/pressure sterilized, oven toasted, and ground to make a wholesome food‐grade ingredient. The processing of DDGS for food application was done following method described by Rosentrater and Krishnan . The DDG2.2®, South Hackensack, NJ) having barrel inner diameter of 19.18 mm. A screw compression ratio of 1.5:1 was used in the experiments. A pictorial representation of the single screw extruder is shown in Figure Post conditioning, the blends were randomly extruded using single‐screw laboratory extruder (Brabender Intelli‐Torque Plasti‐Corders) at the screw surface (N/m2) and the shear rate  were calculated from the following equations: rcorr is the radius correction due to the screw's frustum geometryreff is the effective radius (m), Ω is the net torque exerted on the screw (N m), Ls is the screw length in the axial direction (m), ω is the angular velocity of the screw (rad/s), Css is an empirical correction factor for shear stress (which is 10321.5 for this study), γ˙s is the shear rate at the screw surface (1/s), rb is the inner barrel radius (m), and Csr is the empirical correction factor for shear rate (which is 3.48 for this study). The calibration value for this extruder has been calculated from the calculation reported elsewhere .The apparent viscosity of the dough in the extruder was calculated by approximating extruder behavior as that of a coaxial viscometer but corrected for the tapered screw geometry Figure  of the e2.3X1), temperature (X2), screw speed (X3), and moisture content (X4) each at five levels were taken as shown in Table Experiments were conducted using the central composite rotatable design which was developed using Design‐Expert 8.0.7.1 . Four numerical independent variables namely FDDG , specific mechanical energy (YSME), apparent viscosity of dough (YAV), and torque (YTor) were taken as the responses. Second‐order polynomial regression models were established for the dependent variables to fit experimental data for each response.yi is the predicted response; b0 is the interception coefficient; bi, bii, and bij are coefficients of the linear, quadratic, and interaction terms; and xi is the independent variables studied. The fitness of the model was evaluated and the interactions between the independent and dependent variables were identified using an analysis of variance (ANOVA) presented in Tables R2) and its statistical significance was determined by the F‐test. Three‐dimensional response surfaces were used to visualize the interactive effects of the independent variables.Mass flow rate (response.yi=b0+∑i=33.1R2) of 0.82. The second order model (Table p < .05), whereas lack‐of‐fit was not significant (p > .05). The selected model adequately represented the data for apparent viscosity. FDDG (x1) had significant (p < .05) positive linear effect, whereas temperature (x2) and screw speed (x3) had significant negative linear effects (p < .05) on apparent viscosity of the dough inside the extruder. Temperature also showed significant negative quadratic effect (p < .05) suggesting that viscosity decreased with excessive increase in temperature.The fitted model shown in Table el Table  for appap > .05) on apparent viscosity, the highest viscosity was observed at the lowest moisture content of the blend which was 14%. Response surface plots for apparent viscosity at different FDDG level, temperature and screw speed are shown in Figures The apparent viscosity varied between 1,654 and 4,397 Pas. Although in this study, moisture content did not have any significant effect (p < .05) on the apparent viscosity  in terms of coded variables is shown in Table p < .05), whereas lack of fit was not significant (p > .05). The response surface regression model on MFR yielded a good fit with a coefficient of determination (R2 = 0.81) for the extrudates. Regression analyses showed that MFR was significantly (p < .05) affected by linear effect of moisture content (x1) and quadratic effect of screw speed (x3). Interaction effects of temperature and screw speed (x2x3), and screw speed and moisture content (x3x4) were also observed.The drag flow developed by screw rotation inside extruder and the pressure developed due to constriction at the die influences the mass flow rate Ludewig, . Multiplp < .05) increased the MFR. Such behavior is expected as there is a proportional relationship between drag flow in extruder and the screw speed. For this reason, higher screw speeds means higher mass flow rate and there is greater ability for the material to move along the extruder barrel  increased the MFR. High moisture content aides in gelatinization of the dough and thus apparent viscosity decreases which has been observed in this study. This explains the increase in MFR with increase in moisture content of the dough. No significant effect (p>.05) of FDDG on the MFR was observed.The MFR varied between 1.67 and 2.28 kg/h. The effects of screw speed and moisture content on MFR are shown in Figure  Harper, . Increas3.3YSME) in terms of coded levels is shown in Table x1), temperature (x2), screw speed (x3), and moisture content (x4) had significant effects (p < .05) on SME. Temperature had a significant negative quadratic effect (p < .05) on SME. SME increased with increase in temperature, while excessive increase in temperature resulted in decrease in SME. The fitted quadratic model had a coefficient of determination (R2) of 0.89. The model (Table p < .05), whereas lack of fit was not significant (p > .05).A multiple linear regression equation of SME  in terms of coded variable is given in Table p < .05) by negative linear effects of FDDG (x1), temperature (x2), screw speed (x3), and moisture content (x4) suggesting that increase in the levels of these variables resulted in decrease in torque. Temperature also had significant negative quadratic effect (p < .05) on torque indicating excessive increase in temperature reduced the torque of the extruder. Interaction effects of FDDG and screw speed (x1x3) and temperature and moisture content (x2x4) were also observed. The responses were analyzed using ANOVA and the data are presented in Table R2 equal to 0.95 for the torque. The linear model was significant (p < .05), whereas lack of fit was not significant (p > .05) for torque.The multiple regression equation for torque (Torque increases with increase in FDDG level Figure a which a4The apparent viscosity, MFR, torque, and SME were shown to be significantly influenced by the extruder operating conditions. FDDG level and extrusion temperature significantly affected the apparent viscosity, torque and SME. Apparent viscosity increased with the increase in FDDG content. Higher feed moisture and higher extrusion temperature reduced the viscosity. With increase in the screw speed and feed moisture content the MFR also increased. Increasing the moisture of the blends and the extrusion temperature resulted in a decrease in torque. High SME was observed at high screw speed and low extrusion temperature.None declared.


An approximately 24-h biological timekeeping mechanism called the circadian clock is present in virtually all light-sensitive organisms from cyanobacteria to humans. The clock system regulates our sleep–wake cycle, feeding–fasting, hormonal secretion, body temperature, and many other physiological functions. Signals from the master circadian oscillator entrain peripheral clocks using a variety of neural and hormonal signals. Even centrally controlled internal temperature fluctuations can entrain the peripheral circadian clocks. But, unlike other chemical reactions, the output of the clock system remains nearly constant with fluctuations in ambient temperature, a phenomenon known as temperature compensation. In this brief review, we focus on recent advances in our understanding of the posttranslational modifications, especially a phosphoswitch mechanism controlling the stability of PER2 and its implications for the regulation of temperature compensation. This clock network entrains physiological processes including the sleep–wake cycle, liver metabolism, and body temperature  is the master oscillator. The SCN in the hypothalamus of brain is entrained by the light–dark cycle through the eye and neuronal retinal ganglion cells. Synchronized highly interconnected neurons in the SCN oscillate and transmit their rhythm to peripheral oscillators such as liver, lung, and kidney perature –3. At thperature –3.via retinal ganglion cells that communicate with the SCN via the retinal–hypothalamic tract. Homeothermic animals such as mammals maintain a nearly constant body temperature with a narrow range of fluctuations in most part of the body, whereas poikilotherms such as frogs have body temperature, which can vary in wide range  retained a 24-h rhythmicity in total darkness over a temperature range of 16–26°C  undergoes alternative splicing that is temperature sensitive. The resulting two isoforms have opposing effects on clock speed and was once proposed to underlie temperature compensation  is to underlie the short-period phenotype of the tau hamster  by CK1ε/δ  site. This explains how CK1εtau is a gain of function on phosphorylation at S478 and further supports the phosphoswitch between the two sites (the FASP and the β-TrCP site) , 3735, 3y CK1ε/δ , 37. They CK1ε/δ . Accordiy CK1ε/δ . FurtherCP site) .tau mutant hamsters have significantly impaired temperature compensation (LucPer2 mouse embryonic fibroblasts (MEFs). It was found that at higher temperatures, increased FASP site phosphorylation by the priming kinase leads to slow second-phase degradation and more accumulation of PER2, eventually lengthening and compensating period length. Similarly, LucPer2 MEFs at 30°C showed a marked decrease in second-phase degradation, whereas first-stage degradation remained intact. These findings underscore the importance of the relative rates of phosphorylation of the two phosphoswitch sites in temperature compensation  mechanism (There are a number of unresolved issues. The priming kinase has not been identified yet. It also remains unclear what happens to PER2 phosphorylation over the full 24-h day, in part because the methods to study this in mammalian systems are not suitably sensitive. This is relevant to another unsolved question: how PER2 is degraded in the third phase of three phase decay, when neither CK1 nor proteasome inhibitors impact PER2 loss? Moreover, further study is necessary to understand whether fluctuations in body temperature, which can entrain the clock, do so in part echanism , 45. FinDrosophila and Neurospora as well (It is remarkable that the complex yet robust phenomenon of temperature compensation is regulated by subtle differences in phosphorylation of the same protein at different sites. Notably, this finding is in general agreement with predictions of earlier mathematical models that suggested that opposing outputs with switch-like mechanisms might control temperature compensation , 17. In  as well –48.RN and DV were involved in conceptualizing, researching and writing this review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vinpocetine, a semi-synthetic derivative of vincamine, is a popular dietary supplement used for the treatment of several central nervous system related disorders. Despite its wide use, no pharmacokinetic drug interaction studies are reported in the literature. Due to increasing use of dietary supplements in combination with conventional drugs, the risk of adverse effects is on the rise. As a preliminary step to predict a possibility of drug interaction during concomitant use of vinpocetine and conventional drugs, this study was carried out to evaluate the effects of vinpocetine on three main regulators of pharmacokinetic drug interactions namely, cytochromes P450 (CYPs), P-glycoprotein (P-gp), and Pregnane X receptor (PXR).Inhibition of CYPs was evaluated by employing recombinant enzymes. The inhibition of P-gp was determined by calcein-AM uptake method in transfected and wild type MDCKII cells. Modulation of PXR activity was monitored through a reporter gene assay in HepG2 cells.50 8 μM) and a moderate inhibition of recombinant CYP3A4 and CYP2D6 (IC50 2.8 and 6.5 μM) with no activity towards CYP2C9, CYP2C19 and CYP1A2 enzymes. In HLM, competitive inhibition of CYP3A4 (IC50 54 and Ki 19 μM) and non-competitive inhibition of CYP2D6 (IC50 19 and Ki 26 μM) was observed. Activation of PXR was observed only at the highest tested concentration of vinpocetine (30 μM) while lower doses were ineffective.Vinpocetine showed a strong inhibition of P-gp (ECStrong inhibition of P-gp by vinpocetine is indicative of a possibility of drug interactions by altering the pharmacokinetics of drugs, which are the substrates of P-gp. However, the effects on CYPs and PXR indicate that vinpocetine may not affect CYP-mediated metabolism of drugs, as the inhibitory concentrations are much greater than the expected plasma concentrations in humans. Vinca minor (lesser periwinkle) plant. Chemically, it is known as ethyl apovincaminate , CYP3A4/7-benzyloxy-4-trifluoromethylcoumarin (BFC), CYP2D6/3-[2--ethyl]-7-methoxy-4-methylcoumarin (AMMC), CYP2C19/3-Cyano-7-Hydroxycoumarin (CHC), and CYP2C9/CEC high throughput inhibitor screening kits were from BD Gentest . Transwell® plates  were from Costar Corp. . Troleandomycin was from Santa Cruz Biotechnology, Inc. . Testosterone, 6β-hydroxytestosterone, vinpocetine, magnesium chloride, corticosterone and all other chemicals were purchased from Sigma-Aldrich . NADPH regeneration solution A and B were purchased from Corning Discovery Labware . HPLC grade acetonitrile, methanol and formic acid were from Fisher Scientific . The purity of the test compounds and positive controls used in the study was ≥95%. The final concentrations of DMSO and methanol did not exceed 0.1% and 0.5%, respectively, in all the experiments.Mixed gender pooled human liver microsomes (HLM) prepared from 25 individual donors  were obtained from Corning Life Sciences  and hMDR1-MDCK-II (transfected) cell lines were a gift from Dr. Gottesman . Dulbecco’s Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Hanks Balanced Salt Solution (HBSS), HEPES, Trypsin EDTA, Penicillin-streptomycin and Sodium Pyruvate were from GIBCO BRL . Fetal bovine serum (FBS) was from Hyclone Lab Inc. . CYP1A2/3-Cyano-7-Ethoxycoumarin  supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% 50 values (co-incubation assay) were obtained from concentration-response curves generated by plotting log concentration versus % control.The assay conditions for determining reversible and time dependent inhibition of CYPs were similar as reported earlier ,12. Test50 values (pre-incubation assay) were obtained as above. The IC50 shift fold was calculated as the ratio of IC50 (co-incubation)/IC50 (pre-incubation).For TDI activity of vinpocetine, the initial incubation mixture (180–190 µL), consisting of test sample or positive control, recombinant enzyme, control protein (0.05 mg of protein/mL), cofactor mix, G6PDH, and 50 mM potassium phosphate buffer (pH 7.4), was pre-incubated for 30 min followed by addition of respective fluorescent substrates (10–20 µL) and further incubation for 15 (CYP1A2), 30 (CYP3A4) or 45 (CYP2D6) min. The reaction was terminated and fluorescence was measured as described above. IC+, glucose-6-phosphate, and G6PDH) in 0.1M potassium phosphate buffer (pH 7.4). Vinpocetine or positive controls (100–0.05 µM) were added to the reaction mixture and pre-warmed for 10 min. The reaction was initiated by the addition of HLM (0.6 mg/mL protein). After further incubation of 30 min (for CYP3A4) or 20 min (for CYP2D6) at 37 °C, reaction was terminated by addition of ice-cold acetonitrile containing 5 μL corticosterone  or 5 μL niflumic acid . The mixture was centrifuged at 10,000 rpm for 5 min and the supernatant (200 μL) was transferred into vials for HPLC (CYP3A4) or UHPLC (CYP2D6) analysis. IC50 values were obtained from concentration-response curves generated by plotting log concentration versus % of control by using GraphPad Prism 6 .The reversible inhibition of CYP3A4 and CYP2D6 was determined in terms of the metabolic conversion of testosterone to 6β-hydroxytestosterone and dextromethorphan to dextrorphan, respectively, according to a procedure described previously . The inc−1vs. substrate−1). Apparent inhibition constant (Ki) was calculated from the x-intercept of the Dixon plot (slope of the primary plot vs. inhibitor concentration), which was fitted by liner regression using GraphPad Prism 6 .The type of inhibition (competitive or non-competitive) was predicted by Lineweaver-Burk Plot   and the 50 value, defined as the concentration that caused an increase of 50% in calcein-AM uptake, was obtained from dose curves generated by plotting % increase in calcein-AM uptake versus log concentration using GraphPad Prism.The assay was performed by following reported method . MDCK-IITestosterone and 6β-hydroxytestosterone were quantified according to the previously described method . The HPLm/z = 272.1998 [M + H]+, 258.1844 [M + H]+, and 283.0676 [M + H]+ for dextromethorphan, dextrorphan, and niflumic acid, respectively. The LOD and LOQ of dextromethorph and dextrorphan, were 10 ng/mL and 5 ng/mL, respectively.The quantification of dextromethorphan, dextrorphan and niflumic acid  was done by using Agilent LC-quadrupole time-of-flight (QToF) (model 6230)-mass spectrometry . The UHPLC system consisted of a binary pump, autosampler, degasser, and a column compartment. The mass detector was equipped with an electron spray ionization interface, which was connected to Agilent MassHunter Work Station (B.05.00). Dextromethorphan and dextrorphan were analyzed using an Agilent Zorbax SB-C8 RRHD column . A column temperature of 35 °C and sample temperature of 15 °C were maintained during analysis. The mobile phase consisted of water (A) and Acetonitrile (B), both containing 0.1% formic acid, which were applied in the following linear gradient elution: 0 min, 80%A: 20%B in next 10 min to 30%A: 70%B and in next 2 min to 100% B, at a flow rate of 0.2 mL/min. Separation was followed by a 2 min washing procedure with 100% B and re-equilibration period of 3.0 min. The total run time for analysis was 10 min. The injection volume was 2 µL. Compounds were confirmed in positive ion mode with p < 0.05 was considered to be statistically significant.The data were analyzed by one way ANOVA, followed by Dunnett’s multiple comparison tests using GraphPad Prism Version 6, . etc. [in vivo [in vitro studies to identify compounds that may have potential to cause drug interactions. These studies are primarily focused on evaluating the modulatory effects of an investigational compound/drug on CYPs, P-gp or PXR [Pharmacokinetic drug-drug interactions have led to withdrawal of prescription medications like astemizole, mibefradil, and terfenadine due to life threatening side effects. Follow-up studies have shown that these drug interactions were primarily due to inhibition of CYP enzymes. As a result, the plasma concentrations in humans reached toxic levels when taken in combination with drugs  that were potent inhibitors of CYP3A4 . St. Johetc. . Further[in vivo . Consequp or PXR .50 values of 2.80 ± 0.98 and 6.5 ± 1.1 µM, respectively  and quinidine (IC50 = 0.05 µM) , as shown in 50 shift fold ratios of the positive controls for TDI, troleandomycin (5.9) and paroxetine (5.4)  used in the assays. The catalytic site of CYP3A4 enzyme has been shown to possess two different binding sites and thereby structurally diverse compounds are known to bind to the enzyme [i value of 32 µM, as shown in i value of 26 µM (50 and Ki) observed in this study will be achieved in vivo to inhibit CYP3A4 and CYP2D6 and thus the possibility of CYP mediated drug interaction seems to be remote.As part of our ongoing studies on safety of herbal dietary supplements, this study was carried out to evaluate the effects of vinpoectine on CYPs, P-gp and PXR activity and thereby determine its pharmacokinetic drug interaction potential. Using recombinant CYPs and specific fluorescent substrates, reversible and TDI of CYPs by vinpocetine was evaluated. It showed reversible inhibition of both CYP3A4 and CYP2D6 enzymes with ICectively . No inhi0.05 µM) , suggestne (5.4)  were sime enzyme . Also, Be enzyme , which mof 26 µM . It shou50 value of 8.0 ± 0.45 µM, which was 2.7-fold lower than positive control verapamil (22.0 ± 0.6 µM), as shown in 50 values below 10 µM in Calcein-AM transport assay are further recommended for in vivo studies [The presence of P-gp on different tissues and its ability to efflux diverse range of substrates has made it the primary transporter to regulate the pharmacokinetic properties of xenobiotics . Calcein studies . While tin vivo.The nuclear hormone receptor PXR is a known transcriptional regulator of several CYP enzymes  and efflux transporters (P-gp) . Since ain vitro report studying the pharmacokinetic drug interaction potential of vinpocetine. CYP3A4 and CYP2D6 inhibitory activities by vinpocetine were moderate in recombinant enzymes; however, weak inhibition was seen with HLM. The P-gp inhibition was stronger than verapamil, implicating the possibility of potential drug interactions with other drugs that are substrates for P-gp. Further studies are needed to see if the bioavailability and distribution of P-gp substrates are altered in vivo in presence of vinpocetine. No significant activation of PXR was seen with vinpocetine.To our knowledge, this is the first preliminary


Single usage of BTP showed worst prediction (r2 = 0.67) within models with only one biomarker. Subgroup analyses  demonstrated a slight additional benefit of including BTP into the prediction model for diabetic, hypertensive and lean patients. Among BTP-containing GFR equations the Inker BTP-based equation showed superior performance. Especially the use of cystatin C renders the addition of BTP unnecessary.Despite intense research the optimal endogenous biomarker for glomerular filtration rate (GFR) estimation has not been identified yet. We analyzed if ß-trace protein (BTP) improved GFR estimation in elderly. 566 participants aged 70+ from the population-based Berlin Initiative Study were included in a cross-sectional validation study. BTP, standardized creatinine and cystatin C were measured in participants with iohexol clearance measurement as gold standard method for measured GFR (mGFR). In a double logarithmic linear model prediction of mGFR by BTP was assessed. Analyses with BTP only and combined with creatinine and cystatin C were performed. Additionally, performance of GFR estimating equations was compared to mGFR. We found that the combination of all three biomarkers showed the best prediction of mGFR (r It can either be estimated (eGFR) using mathematical equations based on endogenous biomarkers or it can be measured (mGFR) using invasive methods based on the injection of exogenous markers. At present, in clinical routine the majority of health care providers use serum creatinine levels to calculate eGFR. Recently, it has been shown that, especially in older adults and children, adding cystatin C as another renal biomarker to creatinine level for assessing GFR increases the accuracy of eGFR as compared to the use of single-biomarker eGFR4. A rising number of novel biomarkers have emerged as alternative to creatinine and cystatin C6, one of them ß-trace protein (BTP), a heterogeneous low molecular weight glycoprotein produced in the central nervous system7. Concentrations in serum however are found to be lower8. Since BTP’s discovery about 20 years ago research has focused on its possible ability to shed additional light on assessing kidney function beyond serum creatinine (Scr) and serum cystatin C (SCys)9. With regard to its renal metabolism, BTP has been shown to be freely filtered through the glomerular basement membrane with little if any tubular reabsorption or non-renal elimination7. Also, ß2-microglobulin (B2M), another low molecular weight protein, has been found to be highly correlated with measured GFR (mGFR) and like BTP, is less affected by age and sex11. Other research groups have investigated the approach of “biomarker-panels” using several, still unspecified, markers simultaneously, although these markers are not yet established for standard laboratory analysis12.Glomerular filtration rate (GFR) is considered the best indicator of kidney function14. In this respect, BTP has been shown to have potentially superior properties for assessing kidney function, especially in renal transplant recipients16. Whether BTP may increase the accuracy of GFR estimates when combined with existing markers applied to older adults is still unknown.For the estimation of GFR it is important to know whether the renal marker in use performs equally well in different patient populations, such as children, older adults, individuals with liver cirrhosis or muscle wasting, and transplanted patients. Factors that are known to influence a renal biomarker’s potential to correctly estimate GFR are called the non-GFR determinants . Although creatinine-based GFR equations give reliable estimates in adult patients with reduced kidney function, it is known that both, for children and for older adults, continuous change in muscle mass leads to unreliable Scr levels for GFR estimationIn the present study we hypothesized that in old age adding BTP to single or the combination of current biomarkers improved GFR estimation compared to using Scr or Scys alone. In a double logarithmic linear model prediction of mGFR by BTP only and combined with creatinine and cystatin C was assessed. Additionally, we evaluated current BTP-containing eGFR equations against measured GFR and in comparison to combined creatinine/cystatin C-based equations in a population of older adults.2). Median serum concentration of BTP in the total cohort was 0.68 mg/l with 0.73 mg/l in males and 0.62 mg/l in females. For creatinine it was 0.91 mg/dl with 1.02 in males and 0.79 in females and for cystatin C it was 1.05 mg/dl with 1.1 mg/dl in males and 0.99 mg/dl in females. Figure In the BIS iohexol population with measured BTP Table , mean ag2 = 0.632) within the models with only one biomarker. The age, gender, and BMI-adjusted combination of BTP, creatinine and cystatin C showed the best prediction of mGFR (r2 = 0.833), whereat the adjusted combination of creatinine and cystatin C provided only minimally diverging results (r2 = 0.828). Additionally, the combination of creatinine and BTP (r2 = 0.789) did not outperform the combination of creatinine and cystatin C.All filtration markers were negatively correlated with measured GFR (mGFR)  (range) of 86 (14–163) ml/min/1.73 m2 was found with the White(BTP/Crea) equation compared to less biased BTP-based eGFR results of 58 (15–105), 63 (13–112), and 64 (15–117) for the Inker(BTP), the Pöge(BTP/Crea) and the Pöge(BTP) equations, respectively. Figure 2 the Inker(BTP) equation showed the closest estimate with the smallest interquartile range. Both equations by Pöge slightly overestimated median mGFR. The estimate of the White(BTP/Crea) equation largely overestimated mGFR and showed the widest interquartile range. Figure Mean mGFR (range) was 60.4 16–117) ml/min/1.73 m6–117 ml/(BTP) equation and worst for the White(BTP/Crea) equation. When comparing the new BTP-based equations with the established creatinine/cystatin C-based CKD-EPI(Crea/CysC), BIS2 and FAS equations it was surprising that the Inker(BTP) equation showed a better performance of bias (−2 versus 7 ml/min/1.73 m2) and accuracy (P10 of 45% versus 40% and P30 of 91% versus 88%) than the CKD-EPI(Crea/CysC) equation. All in all the BIS2 showed the best performance, which is not surprising since it was developed from the BIS dataset, and the FAS was relatively equal to Inker(BTP) and superior to CKD-EPI(Crea/CysC). Both Pöge equations did not outperform the established creatinine/cystatin C-based equations.Table Figure Subgroup analysis in individuals with arterial hypertension, diabetes mellitus, low or high body mass index demonstrated a slight additional benefit of including BTP into the prediction model for patients with arterial hypertension and diabetes as well as lean individuals (Supplement Table Improving the accuracy of glomerular filtration rate (GFR) estimating equations has been a long-standing aim in order to optimize clinical decision making with regard to diagnosing chronic kidney disease (CKD), medication dose-adjustment, contrast media application, initiating renal replacement therapy, evaluation of potential organ donors as well as prognostication of CKD-associated CV risk - all scenarios very relevant in the elderly.17 most probably due to its independence of height, age, gender and muscle mass. While the appropriate renal biomarker in old age has yet to be established, this raised the question whether the addition of BTP could further optimize GFR assessment in elder patients to improve patient management in geriatric nephrology.In the present study we investigated ß-trace protein (BTP), a relatively novel filtration marker for estimating kidney function, for the first time exclusively in older adults. This was motivated by the fact that introduction of cystatin C into current GFR estimating equations a couple of years ago has been shown to increase accuracy and precision especially in older adultsOur analysis was performed in 566 individuals aged 70 years and above with iohexol plasma clearance measurements. We demonstrated for the first time that when BTP was included into measured GFR (mGFR) predicting regression models, neither the single biomarker with or without age and gender was superior to creatinine or cystatin C, nor did BTP in combination with creatinine and/or cystatin C increase the regression model’s fit significantly.(BTP)18 equation showed the best performance. No additional benefit was found when evaluating the performance of BTP-based GFR estimating equations18 in our population of older adults compared to BIS2 and FAS(Crea/Cys) equations4. Therefore, using the two “established” standardized biomarkers creatinine and cystatin C for estimating GFR in individuals above the age of 70 is still superior to the currently available BTP-based equations. When comparing the Inker(BTP) to the creatinine/cystatin C-based CKD-EPI(Crea/Cys)2 equation we found that the Inker(BTP) equation showed a substantially smaller bias and better precision. This was very surprising to us since in our mGFR predicting regression models we found that the combination of creatinine and cystatin C was clearly superior to BTP as a single biomarker in older adults. Therefore, we would have expected the established CKD-EPI(Crea/Cys) equation to outperform the Inker(BTP) equation. One possible explanation why this was not the case might be that the development population of both equations were not the same: amongst others, the Inker(BTP) study population included individuals with a higher mean age and reported a diabetes prevalence of 21%, both of which corresponds more closely to the characteristics of our BIS population compared to the CKD-EPI study population. Also, one prominent non-GFR determinant in old age certainly is inflammation by which BTP is known to be less influenced compared to cystatin C11. This could be another possible explanation why the Inker(BTP) equation performed better than the combined creatinine/cystatin C- based equation.When comparing all BTP-containing GFR equations to mGFR the Inker19, our data showed that mean BTP concentration increases by 14% between the age of 70 and the age of 90 years and above, leading to a higher prevalence of GFR <60 ml/min/1.73 m2 in the higher age strata. The same phenomenon has been shown for creatinine and cystatin C20, and has been interpreted as a sign of physiological kidney senesence21. Cystatin C is said to be more sensitive to earlier GFR decrease22 in contrast to creatinine, which detects changes in kidney function not before a decrease of at least 50%. Priem et al. as well as Filler et al. have found the same advantage for BTP with regard to the “creatinine-blind” range24; in our population of elderly we did not find such a correlation (data not shown).Interestingly, in contrast to former analyses that found no independent association between age and BTP26 since a certified assay standardization has not yet been successfully established27, a fact that may also contribute to the rather disappointing results.We further investigated BTP for assessing kidney function in hypertensive, diabetic, lean or obese elderly patients and found a slight additional benefit of including BTP into the prediction model for patients with arterial hypertension and diabetes as well as lean individuals compared to the combination of creatinine and cystatin C alone. All in all our data did not show that BTP, in addition to creatinine and cystatin C, contains significant additional information on kidney function enabling a better distinction between disease-related and age-related “physiological” change of kidney function. Besides, the accuracy of BTP measurement is lower compared to creatinine and cystatin C28. As a purely cross-sectional analysis our focus exclusively lay on GFR prediction.In 2016, Foster and colleagues have shown that BTP may contribute valuable risk prediction information beyond the current biomarkers creatinine and cystatin C18. Since in our population we did not measure B2M our observations are limited to BTP only or in combination with creatinine and/or cystatin C. Secondly, our study explicitly focused on older adults and BTP’s potential benefit in estimating GFR in this selected population. Even if we cannot simply expand our results to other populations they support former findings where BTP performed neither superior in children29 nor in adults31 when compared to creatinine and cystatin C. Also we cannot comment on the impact of race, since we investigated a purely Caucasian population. Thirdly, the BTP-based equation by Pöge16 and White15 were derived in and developed for adult kidney transplant recipients; these equations were expected to perform inferior in our elderly non-transplanted study population.Several limitations of our study deserve mentioning. First, BTP has been shown to perform superior in combination with beta-2-microglobulin (B2M)In summary, BTP alone or the addition of BTP does not outperform current biomarkers such as creatinine and cystatin C for GFR estimation in older adults. Especially the use of cystatin C renders the addition of BTP unnecessary. A slight improvement might be present in diabetic, hypertensive and lean individuals, whether this is clinically relevant remains to be proven. Further research is necessary to evaluate whether BTP is a useful renal marker in risk prediction for older adults and whether it outperforms creatinine and/or cystatin C in this respect.32. In the iohexol subpopulation of 570 out of 2,069 BIS baseline participants we performed iohexol plasma clearance for exact measurement of GFR. Out of these 570 participants we had 566 with measured serum ß-trace protein (BTP) as well as the two common renal biomarkers creatinine and cystatin C; in four patients BTP analysis was not performed due to lack of biobank material. The methodology used to measure GFR has been described elsewhere33. Briefly, an eight time point plasma iohexol measurement was performed over a five hour time period after i.v. injection. Iohexol was analyzed with high performance liquid chromatography (HPLC) method. Measured GFR (mGFR) was calculated with the area under the receiver-operating characteristic curve of plasma concentration over time. External quality control was provided by Equalis . Iohexol results were indexed for body surface area according to the DuBois and DuBois formula34. All procedures involving participants and data were in accordance with the revised Helsinki Declaration of 2000, concerning ethical principles for medical research involving human subjects. We confirm that the study protocols and patient information were approved by the local’s ethics committee of the Charité University.The study includes a subpopulation (n = 570) of the longitudinal population-based Berlin Initiative Study (BIS) cohort . Participants, all living in Berlin, were recruited through one of the largest German statutory health care insurance company (AOK Nordost – Die Gesundheitskasse). Details on inclusion and exclusion criteria as well as study design and goals can be found elsewhereBTP measurement was done from frozen samples (−80 °C) at Labor Limbach Heidelberg, Germany, using the particle enhanced nephelometric (PENIA) N Latex® assay on the BN™ II System  in 2015. The interassay coefficients of variation for serum BTP levels were 3.45% at concentrations of 1.76 mg/L.Cystatin C measurements were performed from frozen samples at Charité laboratory, Labor Berlin, Germany, using the particle-enhanced nephelometric assay on the BN ProSpect nephelometer  in 2011. The interassay coefficient of variation for serum cystatin C levels were 1.5%, 3.5% and 2.4% at mean concentrations of 0.8, 2.3, 7.4 mg/L, respectively. The manufacturer’s reference interval for healthy subjects is 0.59–1.05 mg/L, after standardization of cysC according to ERM – DA 471/IFCC for BN Systems. All reagents and control material were provided by Siemens Health Care Diagnostics.All creatinine samples were analyzed directly after the study visit at the Synlab MVZ Laboratory, Heidelberg, using the standardized isotope dilution mass spectrometry-traceable enzymatic method  on a Roche modular analyzer P-Module between 2010 and 2011. The interassay coefficient of variation for serum creatinine were 2.3% and 3.4% at mean concentrations of 0.99 mg/dL and 3.75 mg/dL, respectively.(BTP) equation18: eGFR = 55 × BTP0.695− × 0.998age Inker(BTP) equation16: eGFR = 47.17 × BTP−0.7933Pöge(BTP/Crea) equation16: eGFR = 974.31 × BTP0.2594− × creatinine0.6−Pöge(BTP/Crea) equation15: eGFR = 167.8 × BTP0.758− × creatinine−0.204 .WhiteThe performance of the four currently published GFR estimating equations  was calculated:3, (6) CKD-EPI(Crea/CysC)2 and (7) FAS(Crea/CysC)4 equations.For comparison purposes we also calculated eGFR values with the combined creatinine and cystatin C based (5) BIS22) to quantify to which extent the inclusion of BTP would improve the fit of the model of mGFR. To investigate whether interaction between endogenous biomarkers and BMI was significant we defined three BMI categories  and included them and their interaction terms with respective biomarkers into regression models adjusted for age and gender.Descriptive analysis includes means, SDs, ranges or medians for continuous variables; absolute frequencies and percentages for categorical variables. We assessed Pearson and Spearman correlation coefficients of the log of each marker to the log of mGFR. In double logarithmic linear models prediction of mGFR (reference standard) by BTP only and in combination with serum creatinine and/or cystatin C were performed . Subgroup analyses were performed in hypertensive (AHT), diabetic (DM), lean (BMI ≤ 23), and obese (BMI > 30) elderly individuals. We calculated the coefficient of determination  were calculated via bootstrap resampling.Additionally, confidence limits for RTo examine how well the four current BTP-based GFR estimating equations performed in our population of older adults we assessed bias as the mean and median difference between eGFR and mGFR, with positive values indicating an overestimation of mGFR. Precision was assessed as interquartile range for the difference and SD of the bias, and accuracy as the percentage of estimates within 10% and 30% of mGFR . Mc Nemar test were applied to compare P30 values between equations.35.Additionally, for comparison of estimated GFR versus measured GFR, we used Bland-Altman analysis including limits of agreement taking into account random measurement errorsStata Statistical Software: Release 14. College Station, TX: StataCorp LP).The analysis was done using STATA (StataCorp. 2015. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.We confirm that informed consent was obtained from all participants and/or their legal guardian/s.Supplementary Material


S-transferases (GSTs) play important roles in insecticide/drug resistance and stress response. Medically, GSTs of house dust mites  and German cockroach (Blattella germanica) are human allergens. In this study, classes, isoforms and B-cell and allergenic epitopes of GST of American cockroach, Periplaneta americana, the predominant species in the tropics and subtropics were investigated for the first time. Enzymatically active native and recombinant P. americana-GSTs bound to IgE in sera of all P. americana allergic patients that were tested. By gel-based proteomics and multiple sequence alignments, the native GST comprises three isoforms of delta and sigma classes. All isoforms interacted with serum IgE of the cockroach allergic subjects. Molecularly, the protein contains six B-cell epitopes; two epitopes located at β1-α1 and β4-α3 regions bound to patients’ serum IgE, indicating that they are allergenic. P. americana are ubiquitous and their GST can sensitize humans to allergic diseases; thus, the protein should be included in the allergen array for component resolved diagnosis (CRD) of allergic patients, either by skin prick test or specific IgE determination. The GST is suitable also as a target of environmental allergen detection and quantification for intervention of cockroach sensitization and allergic morbidity.Insect glutathione  Although worldwide prevalence of the CR allergy is relatively less than the house dust mite (HDM) allergy3 clinical manifestations caused by CR are usually more prolonged and severe and often require emergency-room visit, hospitalization and/or intensive care4. The CR allergens are prevailed in the environment especially in the infested areas6. In Thailand, the principal CR species causing human allergic sensitization and morbidity is Periplaneta americana (American CR)7. Currently, officially recognized P. americana allergens include Per a 1 (a protein that exists in multiple variants containing different numbers of repeated amino acid domains) which elicit 93% skin reactivity among CR allergic patients and bound to IgE in all patients’ sera10; Per a 2 or aspartic protease-like protein (42 kDa) bound to IgE in sera of 63% of allergic rhinitis or asthma and rhinitis patients11; Per a 3 (Cr-PI), an insect hemolymph or insect storage protein related to arylphorin which caused skin reaction in 93% of CR allergic patients12; Per a 6 or troponin C, a 17 kDa protein that bound to IgE in sera of 54% of atopic patients14; Per a 7 or tropomyosin (a 37 kDa protein with high homology to other invertebrate and vertebrate tropomyosins) which bound to IgE in sera of 41% of atopic and 57% of CR allergic patients17; Per a 9 or arginine kinase which is a 43 kDa pan-insect protein that reacted to IgE of all CR allergic patients tested18; native troponin T (47 kDa) bound to IgE in sera of 17% of P. amaricana allergic patients19; Per a 10 or serine protease, a 28 kDa protein that elicited skin reactivity in 82% of CR allergic patients20; and Per a 11  and Per a 12  derived from midgut of P. americana which have been found to react to sera of 83 and 63.8% of allergic patients in immunoblot analysis21.Cockroaches (CR) are ubiquitous. They are pestiferous source of human pathogens and allergensS-transferases (GSTs) is a large family of intracellular enzymes of aerobic prokaryotes and eukaryotes that the host organisms use for protection against oxidative stress, degenerative conditions, aging, and cancers and also detoxify endogenous and xenobiotic electrophiles such as drugs, herbicides, insecticides23. GSTs are classified according to their cellular localizations into three major families, i.e., cytosolic, mitochondrial/peroxisomal, and microsomal GSTs24. All insect GSTs are cytosolic and further subdivided into at least six classes including delta, epsilon, omega, sigma, theta, and zeta, based on phylogenetic analysis23. Delta and epsilon GSTs are arthropod specific25. Each GST subunit is approximately 21–28 kDa and may exist as either homo- or hetero-dimeric form26.Glutathione 27. It was reported that increased levels of GST activity contributed to increase detoxification capacity and resistance of the insects to several insecticides31. For medical aspect, GST of many organisms have been reported as human allergens including Dermatophagoides pteronyssinus (Der p 8)33, Bromia tropicalis (Blo t 8)34, Blattella germanica or German CR (Bla g 5)35, Alternaria alternata36, Ascaris lumbricoides and Ascaris suis (Asc s 1 and Asc s 3)37. The B. germanica GSTs, i.e., sigma BgGSTS1 and delta BgGSTD1 have been reported as potent human allergens38. Data on allergenicity and several other attributions of P. americana GSTs are lacking. Therefore in this study, P. americana GST classes, isoforms, allergenicity, and B cell epitopes were investigated.Most research on insect GSTs focused on their role in insecticide/drug resistanceP. americana extract and other allergens, but positivity to the P. americana extract was more pronounced. Serum specific IgE levels to American CR extract were measured by using ImmunoCAP . Serum samples of five subjects who were negative by SPT, IgE ImmunoCAP and IgE-binding ELISA to the P. americana and other extracts served as non-allergic  controls. A pool of sera of 10 P. americana allergic patients was prepared by mixing 0.5 ml of individual samples.This study was approved by Siriraj Ethical Committee (COA no. SI268/2008), Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok. All methods were performed in accordance with the relevant guidelines and regulations by International Ethical Guidelines for Health-related Research Involving Humans. Informed consent was obtained from each subject. Serum samples were isolated from clotted blood aliquots collected from 15 patients who visited Allergy Clinic, Department of Oto-Rhino-Laryngology, Siriraj Hospital, Bangkok. All patients were multiply positive by skin prick test (SPT) to crude P. americana were kept frozen at −80 °C until use. Frozen CR was ground to fine pieces in liquid nitrogen and total RNA was isolated from the powder (100 mg) by using TRIzol reagent . After checking RNA integrity by agarose gel electrophoresis, cDNA was synthesized from the RNA and used as a PCR template for amplification of full-length GST coding sequence (gst). The PCR primers were designed from GenBank database ; forward: 5′-CCG GAT ATC ATG ACC ATC GAC TTC TAC-3′; reverse: 5′-CGA AAG CTT TCA CTT CTT GGC GAG GTT-3′. The PCR reaction mixture was: 1 μl cDNA, 10 μM each of the forward and reverse primers, 2.5 μl of 10× buffer, 3 μl of 25 mM MgCl2, 2 μl of 2.5 mM dNTP, 0.2 μl of 5 units/μl DNA polymerase , and 40.3 μl ultrapure distilled water (UDW). The thermal cycles were: initial denaturation at 94 °C, 5 minutes; 30 cycles of 94 °C for 30 seconds, 50 °C for 30 seconds, and 72 °C for 40 seconds; and final extension at 72 °C for 7 minutes. The PCR product was verified by DNA sequencing before cloning into pKRX-T , subcloned into pET20b+ expression vector, and put into BL21 (DE3) E. coli. A transformed E. coli colony was grown in isopropyl β-D-1-thiogalactopyranoside (IPTG)  conditioned-Luria-Bertani (LB) broth . The recombinant protein was purified from the bacterial lysate by using HISTrap FF affinity chromatography  and verified by SDS-PAGE and Coomassie Brilliant Blue G-250 (CBB) staining and LC-MS/MS. Nucleotide and deduced amino acid sequences of the P. americana rGST was subjected to phylogenetic analysis together with GSTs of other closely related insects to determine percent identity and GST class.Adult cockroaches were caught from houses in Bangkok. They were identified entomologically and only the ® P sonicator, Germany) in ice-bath at 20 kHz, 2 minutes pulse-on, 3 minutes pulse-off for a total of 15 minutes and then centrifuged at 10,000 g at 4 °C for 15 minutes. The clear supernatant was collected, filtered through a sterile 0.45 µm filter, and protein content was quantified by Bradford’s method  using bovine serum albumin (BSA) standard curve for calibration. The preparation was loaded onto a GSTrap FF affinity column (GE) that had been equilibrated with the binding buffer. The column was washed thoroughly with the binding buffer to eliminate unbound materials. The column-bound protein was eluted in 1 ml-fractions with a total of 10 ml elution buffer  at a flow rate of 1–2 ml/min. Fractions containing the eluted protein were verified by SDS-PAGE and CBB staining, pooled, dialyzed against distilled water, and lyophilized. The protein was verified by LC-MS/MS.Five ml of binding buffer  were added to dissolve the CR powder (15 mg). The preparation was sonicated (Sartorius LABSONIC17. A 4% stacking and 12.5% separating polyacrylamide gels were used in the SDS-PAGE which was performed in a Mini-PROTEAN® 3 Cell (Bio-Rad). Separated proteins in the gels were either stained or electro-blotted onto a nitrocellulose membrane (NC) for WB. For WB, empty sites on the blotted NC were blocked with 3% BSA in PBS before placing the membrane in a solution of mouse anti-6× His tag . Anti-mouse immunoglobulin-alkaline phosphatase (AP) conjugate  and BCIP/NBT substrate  were used to reveal the 6× His-tagged-rGST band.SDS-PAGE and WB were performed as described previously39. The generated ion spectra of the peptides from tryptic-digested GSTs were interpreted by using the Turbo SEQUEST algorithm in the BioWorksTM 3.1SR1 software package (Thermo Fisher Scientific) and the nr.fasta database. The protein search parameter was performed as described previously39, which included mass tolerance of 1.25 amu, a fragment mass tolerance of ±0.4 amu, methionine (M) oxidation, and threonine (T) or serine (S) phosphorylation. The identified peptides were further evaluated using charge state versus cross-correlation numbers (Xcorr). The criteria for a positive identification of the peptides were Xcorr > 1.5 for singly charged ions, Xcor > 2.0 for doubly charged ions, and Xcorr > 2.5 for triply charged ions. A delta correlation (∆Cn) of >0.08 was used as a cut-off for peptide acceptance.Native and recombinant GSTs were verified by LC-MS/MS as described previouslyS-transferase assay kit  which the 1-chloro-2,4-dinitrobenzene (CDNB) was used as the enzymatic substrate. GST standard (0.25 mg/ml) was provided with the test kit. Active GST catalyzes the conjugation of L-glutathione to CDNB via a thiol group of the glutathione. The reaction product, GS-DNB conjugate, could be detected at absorbance 340 nm (A340nm). The amount of the product is directly proportional to the GST activity in the sample. In a reaction mixture, 2 µl (0.5 µg) of native, recombinant, and standard GST were mixed individually with 1 ml of substrate solution containing 980 µl Dulbecco’s PBS, 10 µl of 200 mM reduced L-glutathione, and 10 µl of 100 mM CDNB in a 1-ml cuvette. Absorbance 340 nm of the reaction mixture was determined by spectrometer against blank  using BioMateTM3 series spectrophotometer (Thermo Fisher Scientific). A kinetic program was set for every 30 seconds over a period of 5 minutes after a lag time of 1 minute. Specific catalytic activities of both nGST and rGST (µmol of GST/ml/minute) were calculated.Enzymatic activities of the nGST and the rGST were determined by using glutathione 17. The nGST and rGST  were added to separate wells (100 μl/well) of a microtiter plate  and kept at 37 °C until dried. All GST-coated wells were washed and blocked with 200 μl of a blocking solution (1% BSA in PBS) before adding with serial two-fold dilutions of individual sera and the plate was incubated for 3 hours. Wells added with only the serum diluent served as blank. All wells were washed and added with 100 μl of mouse anti-human IgE-biotin conjugate . Streptavidin-horseradish peroxidase (HRP) conjugate (Dako Cytomation) and ABTS substrate solution (KPL) were used for color development. Absorbance at 405 nm (OD405nm) of the content in each well was determined  against the blank. Cut-off OD405nm between positive and negative IgE-ELISA was arbitrarily set at ≥mean OD405nm of non-allergic sera + 2 standard deviations (SD).Allergenicities (IgE binding frequencies) of the nGST and the rGST were determined by IgE-ELISA. The assay was performed as described previously39. For the first dimensional electrophoresis, 7 cm-IPG strips and 0.5% pH 3–10 IPG buffer  were used. The electrophoresed-IPG strips were then subjected to 12% SDS-PAGE and proteins in the gel were stained by CBB. Gel pieces containing proteins of ~21 kDa were excised from the stained gel and subjected to in-gel tryptic digestion and LC-MS/MS, respectively. Protein orthologues were identified by comparing the peptide sequences of the P. americana-GST generated from the mass spectrometry with the Arthropoda/Insecta database sequences.Isoforms of nGST were determined by a gel-based proteomics. Purified nGST was subjected to 2DE as described previouslyThe nGST isoforms were checked for their reactivity to IgE in the pool of CR allergic patients’ sera by 2DE IgE-immunoblotting. The 2DE-separated nGST was electro-transblotted onto an NC, blocked with BSA, and the blot was allowed to react with the CR allergic patients’ serum pool. After keeping at 4 °C overnight, the NC was washed with TBS-T before placing in a solution of appropriately diluted mouse anti-human IgE-biotin conjugate (Southern Biotech) and kept at 25 °C on a rotating platform for 3 hours. Spots of the nGST isoforms bound by the specific serum IgE were revealed by using streptavidin-AP conjugate (Dako Cytomation) and BCIP/NBT substrate (KPL).P. americana-GST were predicted by using BepiPred 1.0 server. The amino acid sequence of P. americana-GST was submitted to the server. Three methods available at the server, i.e., BCPred40, AAP41, and FBCPred42 were used for the epitope prediction. Specific threshold and the epitope length were set at 85% and 20–25 residues, respectively. All predicted peptides obtained from individual methods were aligned with the P. americana-GST sequence to obtain consensus sequences (predicted linear B-cell epitopes). Peptides containing the potential linear B-cell epitopes of P. americana-GST were synthesized. Moreover, in the case that two of the predicted epitope sequences were close to each other (peptides 2 and 4 of this study), the overlapped peptide between the two sequences was also synthesized (peptide 3 of this study). Binding of the synthetic peptides to antibodies in a pool of P. americana sensitized human sera (for determining B-cell epitopes) and to IgE in individual allergic patients’ sera  were determined by dot-ELISAs.Linear B-cell epitopes of For detecting binding of the synthetic peptides to antibodies in the pool of the CR allergic patients’ sera, individual synthetic peptides (1 μg) were dotted onto one cm-NC squares. PBS was used as negative peptide control. The NC pieces were blocked with 1% BSA in PBS-T and then placed in the allergic patients’ serum pool. After keeping at 25 °C for 3 hours, the membranes were washed and allowed to react with goat-anti-human IgG-AP conjugate and BCIP/NBT substrate, respectively, with appropriate incubation and washing with PBS-T between the steps. The enzymatic reaction was stopped by rinsing the membranes with distilled water. Color appeared at the peptide-dotted spots indicated that the peptide contained B-cell epitope.For determining the IgE reactivity of the synthetic peptides, individual peptides were dotted separately onto NC squares (1 μg/dot) and let air-dried. They were blocked with 1% BSA in PBST and probed with individual serum samples (diluted 1:4 in PBST). Serum samples of normal subjects were included in the experiments. After incubating and washing, all NC squares were reacted sequentially with biotin-labeled-goat-anti-human IgE, streptavidin-AP conjugate, and BCIP/NBT substrate, respectively. The peptides that gave the typical colored spots indicated that they contained allergenic (IgE binding) epitopes.P. americana GST was submitted to I-TASSER server43. The templates used for GST 3D modeling (selected by the server) were PDB ID: 3WYW, 3VK9, 3AY8 and 4PNF. The allergenic peptides were mapped on the 3D modeled structure of the P. americana-GST.Deduced amino acid sequence of the cloned P. americana allergic patients and 5 normal subjects as well as clinical diagnosis and results of the skin prick tests and the specific IgE levels among the patients are shown in Table Dermographic data of the 15 P. americana-GST (648 bp) was cloned into pKRX-T cloning vector and subcloned into pET20b+ protein expression vector. Supplementary Fig. P. americana-GST of this study (accession number MG255130). Fig. P. americana-GST and GSTs of homologous and heterologous insects. Alignment  and percent identity of amino acid sequence of P. americana-rGST of this study (accession number MG255130) with GSTs of other insects are shown in Supplementary Fig. P. americana-rGST was purified from the gst-pET20b+-transformed E. coli , which the molecular mass was 24606 Da and the pI was 6.44. This protein has possibility of 14 phosphorylation sites and a possible O-glycosylation at residue 116. Peptides of spot no. 3 matched with sigma GST of Locusta migratoria (migratory locust) (accession number Gi 565341529) with molecular masses of 23459 Da and pI 6.19. This protein also has 14 possible phosphorylation sites; albeit most sites are different from the delta GST of spots 1, 2, and 4. Peptides of spot no. 5 matched with peptides of B. germanica sigma GST (accession number Gi 359326585) which has a molecular mass of 23377 Da and pI 6.84 with possible 16 phosporylation sites. Thus, the nGST of P. americana of this study has three isoforms which belong to delta (one isoform) and sigma classes (two isoforms). By the 2 DE-IgE immunoblotting, all three isoforms of the nGST bound to IgE in a serum pool of the P. americana allergic patients  located between β1 and α1 and peptide 3 (61GFCLWESRAILSYLADQYGK80) located between β4 and α3 of the P. americana-GST molecule  and tropics , especially in crowded and unhygienic urban cities where their infestation is enhanced44. Several P. americana-derived proteins have been recognized as important (major) allergens as they sensitized >50% of cockroach allergic subjects. Glutathione S-transferases (GSTs) are indispensable enzymes which insects use to protect themselves against oxidative damage and stress and insecticide toxicity30. Most interest on the insect GSTs has been focused on their role in insecticide and drug resistance while other attributions of this protein family seem to be neglected. In this study, three P. americana-GST isoforms belonging to delta and sigma classes were identified. The roles of the protein in causing human allergy, i.e., allergenicity (specific serum IgE-binding frequency) as well as B-cell epitopes and allergenic epitopes were investigated. To our knowledge, this is the first report on the allergenic attribution of the American cockroach, P. americana-GST. The protein that was cloned from the GenBank database  has been designated Per a 5 (Per a 5.0101) by the IUIS Allergen Nomenclature. The other isoforms reported in this study were based on the tryptic digested peptides generated from the 2DE protein spots that matched with the orthologous proteins of the database. Because the complete amino acid sequences of these isoforms were not available, they were not submitted to the IUIS Allergen Nomenclature.American cockroach, P. americana-GSTs were produced. On the equal weight of both proteins (0.5 μg), the native protein was about 12.5 times more active than the recombinant counterpart. The difference may be because the nGST contains several isoforms belonging to different GST classes whereas the recombinant one is produced from only one cDNA sequence. The multiple isoforms in the nGST might confer additive or synergistic enzymatic activity. Besides, the rGST produced from the transformed BL21 (DE3) E. coli contains additional 6× His tag (useful for subsequent protein detection and purification) but lacks putative post-translational modifications such as glycosylation and disulfide bridge formation which might impact on the protein folding compared to the native state and hence the less enzymatic activity. After subjecting the nGST and rGST to SDS-PAGE and protein staining, the molecular masses of the native and recombinant proteins were ∼23 and ∼25 kDa, respectively. The larger size of the latter should be from the plasmid franking regions and the 6× His tag. The rGST produced in this study (accession number MG255130) contained 216 amino acids which are similar in residue number to the cloned 24614 Da B. germanica GSTD145. The P. americana-rGST has 99.5 and 98.1% sequence identity to the previously reported P. americana-GSTs (UniProt ID G8XWU4 and Q1M0Y4) and 32.9–82.4% identity to the delta GSTs of other organisms and less so to the other GST classes. Thus, the recombinant P. americana-GST (accession number MG255130) should belong to the delta class.Enzymatically active native and recombinant P. americana GSTs reacted with IgE in sera of all cockroach allergic patients when tested by indirect ELISA, indicating that the GST is a novel and important (major) allergen of the P. americana. The ELISA using rGST as antigen gave higher background signal than when the nGST was used. This could be due to the contamination of residual proteins of the E. coli used as the rGST expression host in the rGST preparation which reacted to the anti-E. coli that exists naturally in the human sera.Both native and recombinant 47. Several methods have been used for gaining information on B-cell and allergenic epitopes of an allergen. These include the use of overlapping synthetic peptides or fragments of recombinant allergens48; mimotope mapping49; peptide microarray immunoassay51; X-ray crystallography and nuclear magnetic resonance techniques55, computerized prediction58 and specific monoclonal antibody binding and IgE competition assay60. In this study, a combination of in silico methods and synthetic peptide based-immunoassays was used for predicting and determining B-cell and allergenic epitopes of the P. americana-GST. For the in silico prediction of B cell epitopes, three different methods of the BepiPred 1.0 server, i.e., BCPred, AAP, and FBCPred were used. All methods gave a conformed prediction of a peptide located at α2-β3-β4; the AAP and PBCPred methods predicted another peptide at α3-α4; the BCPred and the FBCPred methods predicted another peptide at α5-α6; and only the AAP method predicted two more peptides at β1-α1 and α8-α9. The different results so-obtained suggest that several in silico methods should be used for increasing the possibility of finding the potential B-cell epitopes of a particular protein. From the in silico prediction, consensus peptides encompassed the potential B-cell epitopes were synthesized and used in the dot-ELISAs for detecting peptides bound by antibodies (B-cell epitopes) and IgE  in sera of P. americana sensitized subjects. By using the dot-ELISAs, all six synthetic peptides that contained predicted GST B-cell epitopes reacted with antibodies in a serum pool of P. americana exposed-subjects, implying that the six peptides were part or contained GST B-cell epitopes. Among them were two IgE-binding epitopes located at the β1-α1 and β4-α4 regions of the GST molecule.Data on the allergenic repertoire of a protein are useful for understanding the patients’ allergenic response, cross-reacting allergenic determinants among allergens, as well as for properly designing of an engineered therapeutic allergen vaccine/diagnostic material, particularly for personalized immunotherapy and component resolved diagnosis (CRD)P. americana-GST beyond the previously established physiologic roles of the protein in the host defense against toxic substances and stress conditions. Both native and recombinant GSTs of the P. americana were bound by IgE in sera of cockroach allergic subjects and thus the GST is a novel and major P. americana allergen. Because cockroaches are ubiquitous, attention should be paid on reducing the insect derived-allergenic proteins from human environment for intervention of allergic sensitization of the naives and clinical aggravation of the sensitized subjects.In summary, this study provides an insight into characteristics and medically important role of the Supplementary Figures


Drosophila melanogaster exposed to chlorpyrifos, as characterized by western blotting assays.Exposure to organophosphate compounds, such as chlorpyrifos, has been linked to disturbances on cell signaling pathways. Mitogen activated protein kinases (MAPK) are a family of protein kinases involved in a range of cellular processes, including stress response, apoptosis and survival. Therefore, changes in the activation state of these kinases may characterize key mechanisms of toxicity elicited by xenobiotics. Here we report data on the phosphorylation of p38MAPK and JNK, members of the MAPK family, in  Specifications TableValue of the data•The data describe changes in the phosphorylation of p38MAPK and JNK after exposure of Drosophila to organophosphate compound, chlorpyrifos, by means of western blotting techniques.•in vivo.The data can be used as a reference regarding the phosphorylation state of these protein kinases as a response to organophosphate compound poisoning in a complex biological system •Taking into consideration the involvement of mitogen activated protein kinases (MAPK) in a range of cellular processes, including stress response, apoptosis and survival 1p<0.05) in the phosphorylation of p38MAPK . Control flies received 1% sucrose only. It was observed a significant increase  in adult female flies (1.182 ppm), which was previously determined by our group Adult female flies (1–4 days) were left, overnight, in glass tubes containing filter paper soaked in 1% sucrose for acclimation. Subsequently, 30 flies per group were kept in glass tubes containing filter paper soaked with 250 µL of each treatment solution. The experimental groups were: Control (received 1% sucrose solution only) and Chlorpyrifos (CP) 0.75 ppm (diluted in 1% sucrose solution). The CP concentration used here was based on the LC