Although the detrimental influence of parenting stress on child problem behavior is well established, it remains unknown how these constructs affect each other over time. In accordance with a transactional model, this study investigates how the development of internalizing and externalizing problems is related to the development of parenting stress in children aged 4–9. Mothers of 1582 children participated in three one-year interval data waves. Internalizing and externalizing problems as well as parenting stress were assessed by maternal self-report. Interrelated development of parenting with internalizing and externalizing problems was examined using Latent Growth Modeling. Directionality of effects was further investigated by using cross-lagged models. Parenting stress and externalizing problems showed a decrease over time, whereas internalizing problems remained stable. Initial levels of parenting stress were related to initial levels of both internalizing and externalizing problems. Decreases in parenting stress were related to larger decreases in externalizing problems and to the (stable) course of internalizing problems. Some evidence for reciprocity was found such that externalizing problems were associated with parenting stress and vice versa over time, specifically for boys. Our findings support the transactional model in explaining psychopathology. Adolescent internalizing and externalizing problems that persist throughout adulthood are often rooted in childhood  parenting stress is related to each of these problem clusters. Fourth, we will test whether there are gender differences in any of these models. The following hypotheses are tested. First, we expect no decrease or increase in internalizing problems, as these problems remain relatively stable during childhood  in the first assessment. At the subsequent assessment 979 mothers participated (67 %), including 95 ‘new’ parents who did not participate in the baseline assessment for different reasons. In the third assessment wave, 819 (61 %) parents participated in the study, including 148 ‘new’ parents who did not participate in the baseline assessment. We used Mplus allowing us to estimate missing cases, leading to a final sample size of 1582. At baseline, mothers had a mean age of 36.61 (SD = 4.41), the majority was of Dutch origin (92.4 %) and were part of a two-parent household (89.1 %). Most mothers, 44.6 %, were highly educated with a college or university degree, 37.8 % finished vocational education, 13.7 % finished a low level of Dutch secondary school, and 4 % finished a different form of education. Attrition analyses showed that families who completed three waves (n = 817) did not differ from the dropouts (n = 522) in child age, gender, maternal educational level, family structure, internalizing, and externalizing problems, and parenting stress. Families that missed at least on of the three waves were more likely to be of non-Dutch origin .Mothers of children aged 4–7 from 29 primary schools throughout the Netherlands were recruited for the Dutch “Kind in Zicht” study, in which mothers of 1339 children participated , containing 2558 children in two kindergarten classes, Grade 1 and 2. Schools received €1000 for participation. Teachers handed out information and consent letters to parents. Passive consent of 2360 (92.3 %) parents was obtained. Only mothers were allowed to participate in the study in order to decrease gender bias in responding. In all waves, mothers completed questionnaires either digitally or by paper and pencil.www.sdqinfo.com. Higher scores indicate more problem behaviors. Cronbach’s alphas were .63, .67, .65 for emotional symptoms at T1, T2, and T3 and .48, .47, .55 for conduct problems at T1, T2, and T3. Alternative indicators of reliability based on Structural Equation Modeling – are known as Jöreskog rho or McDonalds OmegaH. Because this indicator is suggested to be more accurate when scale distributions are skewed, as is often the case in instruments measuring problem behavior, like the SDQ (Jöreskog h). Omega values were .79, .80, .81 at T1, T2, and T3 for the emotional symptoms scale, and .71, .75, .77 at T1, T2, and T3 for the conduct problems scale. Emotional symptoms and conduct problems were positively skewed and leptokurtic. In line with Tukey’s . The degree of mental health of the mothers during the past 4 weeks was measured at the first wave with a short version of the General Health Questionnaire . Subsequently, we tested whether the separate growth parameters of parenting stress and internalizing problems and externalizing problems, respectively, were related to each other by combining the univariate growth models . In this step, gender differences were again tested by multi-group modeling.Means, standard deviations and bivariate correlations of all study variables were calculated. Regarding the main analyses, first we investigated the growth of parenting stress, and internalizing and externalizing problems by employing univariate Latent Growth Curve modeling, leading to an intercept and slope for each of the three constructs (the growth parameters). Gender differences in these growth parameters were investigated by comparing a freely estimated model to a model wherein parameters were constrained to be equal for boys and girls multi-group modeling. If a significantly worse fit to the data was found for the constrained model, we employed a stepwise approach, such that each of the parameters were tested separately for gender differences by means of the χ2 difference test. All models were controlled for maternal mental health and age.To evaluate direction of effects of the associations of parenting stress with internalizing problems on the one hand, and externalizing problems on the other over time, we tested two cross-lagged path models, using Mplus version 5 (Muthén and Muthén χ2 (0) = 0.00), therefore we did not report fit indices for these models. Although this procedure is employed frequently, we acknowledge there is debate on the question whether saturated models should be interpreted. We follow Muthén’s recommendation that significance of pathways within the model can be interpreted without fit indices (see for a discussion: http://www.statmodel.com/discussion/messages/11/2127.html?1397836729).Since children from the same classes may share common behaviors , intraclass correlations (ICC) were calculated to determine the effects of class clustering. The ICC’s for internalizing problems were .019, .00, and .003 at T1, T2, and T3, respectively, .00, .047, and .016 at T1, T2, and T3 respectively, for externalizing problems, and .031, .056 and .017 at T1, T2, and T3 respectively for parenting stress. This indicates that only a small proportion of the variance could be explained by a clustering effect and therefore we decided to run the analyses without adjusting for clustering. Model fit was assessed with various fit indices, including robust chi-square with estimated degrees of freedom (df), comparative fit index  = 10.76, p = 0.013; CFI = 0.987; RMSEA = 0.041 (CI = 0.017–0.069); TLI = 0.957). No gender differences were found regarding initial level (intercept), change over time (slope), the variance around the initial level and change over time, or covariance among initial level and change over time, indicating that boys and girls do not differ in their development of internalizing problems (Δχ2(5) = 4.50, p > .90). Internalizing problems remain stable over time, which was indicated by a non-significant slope. Significant inter-individual differences in initial level, but not in change of these problems were found, suggesting that children differ in their level of internalizing problems at baseline. The initial level of, and change in internalizing problems were not related, suggesting that the level of internalizing problems is not associated with change in these problems.Growth parameter estimates for the basic growth curve models are reported in Table χ2(3) = 3.92, p = 0.27; CFI = 0.998; RMSEA = 0.014 (CI = 0.00–0.048); TLI = 0.994). Several gender differences were found regarding initial level and change in externalizing problems over time. Boys showed higher levels of externalizing problems at baseline than girls (Δχ2(1) = 5.43, p < .025), and girls showed a steeper decrease in externalizing problems than boys (Δχ2(1) = 8.09, p < .005). No gender differences were found regarding the variance around the initial level and change over time, or covariance among initial level and change over time (Δχ2(3) = 4.86, p > .90). Significant inter-individual differences in initial level, and in change of these problems were found, suggesting that children differ in both their level of externalizing problems at baseline and the decrease in these problems. The initial level of, and change in externalizing problems were negatively related, such that children with higher levels of externalizing problems at baseline showed smaller decreases of externalizing problems over time.Regarding externalizing problems, again an adequate fit to the data was found (χ2(3) = 18.68, p = 0.000; CFI = 0.978; RMSEA = 0.059 (CI = 0.035–0.086); TLI = 0.928). No gender differences were found regarding initial level, change over time, the variance around the initial level and change over time, or covariance among initial level and change over time (Δχ2(5) = 2.98, p > .90). Significant inter-individual differences in initial level, and in change of parenting stress were found, suggesting that mothers differ in both their level of parenting stress at baseline and the decrease in the parenting stress. The initial level of, and change in parenting stress were negatively related, such that mothers with higher levels of parenting stress at baseline showed smaller decreases of parenting stress over time.Regarding parenting stress, a satisfactory fit to the data was found (χ2(11) = 50.53, p = 0.000; CFI = 0.974; RMSEA = 0.049 (CI = 0.036–0.063); TLI = 0.933). No gender differences were found regarding interrelations between internalizing problems and parenting stress (Δχ2(3) = 1.54, p > .10). Higher levels of baseline levels of internalizing problems were associated with higher levels of parenting stress and the course (i.e. stability) of internalizing problems was related to stronger decreases in parenting stress. The level of internalizing problems was not related to the decrease of parenting stress. Also, the level of parenting stress was not related to the course (i.e. stability) of internalizing problems.Correlations between growth parameter estimates of parenting stress and internalizing problems, and externalizing problems respectively are reported in Table χ2(11) = 35.34, p = 0.000; CFI = 0.984; RMSEA = 0.038 (CI = 0.025–0.053); TLI = 0.961). Higher baseline levels of externalizing problems were associated with higher baseline levels of parenting stress and decreases in externalizing problems were related to larger decreases in parenting stress. The level of externalizing problems was not related to the decrease of parenting stress; also, the level of parenting stress was not related to the decrease of externalizing problems. No gender differences were found regarding interrelations between externalizing problems and parenting stress (Δχ2(3) = 6.210, p > .10).Fit statistics for the model investigating the interrelation of parenting stress and externalizing problems were satisfactory , as did parenting stress . Internalizing problems and parenting stress were positively concurrently related at each time point, indicating that more internalizing problems are related to more parenting stress. Cross-lagged paths from internalizing problems to parenting stress were all non-significant, indicating that there were no child effects of internalizing problems on parenting stress. Parenting stress at T1 was related to internalizing problems at T2, and a statistical trend was found for the cross-lagged path of parenting stress at T1 to internalizing problems at T3, such that more parenting stress was related to more subsequent internalizing problems. Parenting stress at T2 was not related to internalizing problems at T3. No gender differences were found regarding cross-lagged paths (Δχ2(6) = 4.735, p > .10).The model in which the direction of effects of parenting stress on internalizing problems and vice versa was tested was saturated. Internalizing problems showed moderate to strong stability throughout childhood . Externalizing problems and parenting stress were positively concurrently related at each time point, indicating that more externalizing problems are related to more parenting stress. The cross-lagged paths differed across gender (Δχ2(6) = 12.865, p < .05). For boys, significant child-on-parent effects were found. Externalizing problems at T1 were related to parenting stress at T2 and externalizing problems at T2 were related to parenting stress at T3, such that more externalizing problems were related to more subsequent parenting stress. Also, parent-on-child effects were detected, such that parenting stress at T1 was related to externalizing problems at T2, and parenting stress at T2 was related to externalizing problems at T3. Thus, more parenting stress was related to more subsequent externalizing problems. For girls, externalizing problems at T1 were related to parenting stress at T3, such that more externalizing problems were related to more subsequent parenting stress. This relation was not found at the other time points. Parenting stress at T1 was related to externalizing problems at T2, such that more parenting stress was related to more subsequent externalizing problems. Again, this relation was not found at other time points.The model in which the interrelations between parenting stress and externalizing problems were tested was also saturated. Externalizing problems showed moderate stability throughout childhood (The present study examined the developmental patterns of parenting stress, internalizing and externalizing problems and the relations between parenting stress and internalizing and externalizing problems over time in children aged 4–9 from a large community sample. Additionally, we examined how parenting stress was related to each of these problem clusters. These associations were compared between boys and girls and we controlled for maternal mental health.It is well known that externalizing problems tend to decrease during childhood (Maughan et al. Regarding developmental course, children varied in their decrease rate regarding externalizing, but not regarding internalizing problems. This is interesting, as distinct trajectories in the development of internalizing problems have been reported previously (Sterba et al. Only one study investigated the developmental course of parenting stress (Williford et al. The current study expanded on Williford et al. , by inveAs for directionality, consistent patterns in parenting stress and externalizing problems were found for boys above and beyond stability of these problems and while taking into account concurrent relations. For girls, the pattern was less consistent but still in accordance with a bidirectional model, as parenting stress at baseline impacted externalizing problems 1 year later, and externalizing problems at baseline impacted parenting stress 2 years later. These results fit well with the theoretical notion that both parents and children affect each other’s development, as described in the bidirectional model Sameroff . HoweverFirst, given the consistent empirical finding that informants tend to disagree regarding problem behavior and parenting (De Los Reyes and Kazdin This study employed a developmental perspective to investigate the interrelatedness between parenting stress and internalizing and externalizing problems. Given the limited studies investigating these interrelations dynamically, the current study is an important contribution to the extant literature. We showed that parenting stress is not a static construct and decreases during early childhood, similarly for boys and girls. In addition, we showed that internalizing problems remain stable in early childhood, and replicated findings regarding decreasing externalizing problems. Finally, parenting stress and internalizing and externalizing co-evolve, thereby mutually influencing each other. Regarding internalizing problems, however, child effects were not found. This finding calls for the need for future studies to further disentangle the interrelatedness of parenting and internalizing problems Figs. , 2 and 3


When membrane and membraneless EBCs are compared, MPD of the membraneless EBC that is run at the similar operating condition to EBC including membrane is speculated as about 134 μW · cm−2.Mediatorless and membraneless enzymatic biofuel cells (EBCs) employing new catalytic structure are fabricated. Regarding anodic catalyst, structure consisting of glucose oxidase (GOx), poly(ethylenimine) (PEI) and carbon nanotube (CNT) is considered, while three cathodic catalysts consist of glutaraldehyde (GA), laccase (Lac), PEI and CNT that are stacked together in different ways. Catalytic activities of the catalysts for glucose oxidation and oxygen reduction reactions (GOR and ORR) are evaluated. As a result, it is confirmed that the catalysts work well for promotion of GOR and ORR. In EBC tests, performances of EBCs including 150 μm-thick membrane are measured as references, while those of membraneless EBCs are measured depending on parameters like glucose flow rate, glucose concentration, distance between two electrodes and electrolyte pH. With the measurements, how the parameters affect EBC performance and their optimal conditions are determined. Based on that, best maximum power density (MPD) of membraneless EBC is 102 ± 5.1 μW · cm Enzymatic Biofuel cells (EBC) are device that converts chemical energy into electrical energy using (i) enzyme as biocatalysts and (ii) glucose/alcohol as biofuels since it was reported in 196411345678Regarding the enzyme immobilization strategy, although physical adsorptions like physical entrapment and chemical bonding like covalent coupling and enzyme cross-linking were attempted, using Layer-by-Layer (LbL) has been recently emerged. According to the LbL structure, (i) multiple repetitive layers are stack together by electrostatic interaction between oppositely charged species and (ii) the stack layers are formed on the nano-sized supporters like carbon nanotube (CNT)21011122 to H2O without production of intermediate H2O2 can be considered the meritAs the possible enzyme catalysts, glucose oxidase (GOx) catalyzing glucose oxidation and laccase (Lac) catalyzing oxygen reduction have been considered14To fabricate the viable enzyme-included LbL catalysts, using proper conducting polymer (CP) and/or cross-linker can be affordable option. In this study, poly(ethylenimine) (PEI), glutaraldehyde (GA) and CNT were used as the CP1719Regarding cost and size of EBC system, optimizing role of membrane included in the EBC may be critical. For doing that, we evaluate two things; first, inspecting optimal membrane thickness and second, removing membrane although the latter is likely to be more effective because the removal of membrane can reduce the size and cost of EBC in a more straightforward way. The membrane usually plays two different roles as separator (i) to alleviate occurrence of mixed potential and (ii) to increase ohmic resistance so that it should be designed (i) what is appropriate membrane thickness if the membrane is utilized and (ii) how to modulate associated parameters if membrane is not used.2/CNT] biocatalyst that is an enzyme structure based on our previous research for glucose oxidation reaction (GOR) and (ii) cathode consisting of three different biocatalysts including GA, Lac, PEI and CNT for oxygen reduction reaction (ORR)In this study, we fabricate EBC system using (i) anode consisting of GA/[[GOx/PEI]2/CNT] used as a catalyst for anode and (ii) Lac-based three different catalysts  used as catalysts for cathode. Regarding the activity of anodic catalyst, GOR occurring at flavin adenine dinucleotide (FAD) that was deep inside GOx molecules was investigated by CV measurements in N2-state and air-state (with O2)  GA/[[GOx/PEI]with O2) . For the2-state. It is due to occurrences of reduction reaction of O2  and redox reaction of FAD (GOx (FAD) + 2H+ + 2e− ↔ GOx (FADH2), existence of redox reaction peaks appeared at −0.469 V vs. Ag/AgCl)2 decreased with decrease in cathodic current. In this case, O2 was consumed for redox reaction of FAD (GOx (FADH2) + O2 → GOx (FAD) + H2O2), not for reduction reaction of O2, producing cathodic current. Due to the change in role of O2, CV curve downshifted before supply of glucose was upshifted with increase in glucose concentration2224There are two observations about the CV measurements. First, according to the 2-state (without O2) was also measured . In the N2-state, redox reaction peaks of cupric ions within Lac molecules of the Lac-based three different catalysts were observed at 0.272, 0.274, and 0.282 V vs. Ag/AgCl. The small reaction peaks were included in inset of the 2 was supplied, current densities of the three catalysts were down-shifted from 0.1 V vs. Ag/AgCl, while redox peak of cupric ions within Lac molecules was shifted to positive direction and appeared around 0.6 V vs. Ag/AgCl. These observations indicate that ORR of the catalysts takes place appropriatelyIn contrast, regarding the activity of cathodic catalyst, ORR occurring at cupric ions within Lac molecules of the three different Lac-based catalysts was also measured. For comparison, the reaction occurring at Nss) and apparent Michaelis-Menten constants (Kms) were measured using Laviron’s formula and Michaelis-Menten plot, respectively 2227s of GA/[[GOx/PEI]2/CNT] catalyst was 12.7 s−1, while that of GA/[Lac/PEI/Lac/CNT] catalyst was 8.6 s−1, (Kss of Lac/CNT and Lac/PEI/Lac/CNT were 6.5 and 8.4 s−1).To investigate and compare reaction rate of the catalysts, their electron transfer rate constants  . There are two noticeable things for 22s of the catalysts was higher than that of other similar catalysts, meaning that the catalysts played their role well and second, we swept the entire potential ranges using very slow potential scan rate (5 mV·s−1)22Even in comparison of K2/CNT] and the three different Lac-based structures were measured. Also, to inspect effects of enzyme catalysts on EBC performance, those of EBCs adopting catalysts fabricated without enzyme molecules  were measured (−1 (anode) and 100 cc · min−1 O2 gas was provided (cathode). There were two things to mention about the results. First, regarding EBC performance, maximum power density (MPD) of EBC adopting GA/[[GOx/PEI]2/CNT] and GA/[Lac/PEI/Lac/CNT] catalysts was highest . It indicates that GA plays a critical role in immobilizing more Lac molecules and activating ORR, followed by improving EBC performance. Second, based on −2 respectively), it was proved that both enzymes (GOx and Lac molecules) were well acted as catalysts for EBC.To evaluate performances of EBCs including membrane between anode and cathode electrodes, polarization curves of the EBCs adopting GA/[[GOx/PEI]measured . For thects) of the EBCs used in 2 gas. According to the Nyquist plots, when enzyme catalysts were included, Rct was highest when GA/[Lac/PEI/Lac/CNT] was employed in EBC, while the value was lowest when Lac/CNT was employed . On the other hand, when enzyme catalysts were not included, Rct of the EBC was very low .To further investigate effect of enzyme catalysts on charge transfer of EBC, charge transfer resistances  because the Lac molecule is assemble of proteins that have non-conductive propertyct trend of ss) of EBCs are not affected by catalyst types .These results make sense. As reported previously, R2/CNT] catalyst for anode) and 2/CNT] catalyst was measured, while cupric ion redox reaction peaks of Lac-based catalysts were measured for the It is also important to investigate whether our enzyme catalysts can work well for a long time. To evaluate the long term stability of enzyme catalysts, catalytic activities of the enzyme catalysts were measured every week for four weeks and the results are represented in 2/CNT] catalyst was maintained to 87% of initial value, while those of Lac-based catalysts were maintained to 89, 75 and 64% of initial value. It can be explained that (i) long term stabilities of all the catalysts are better than that of other similar catalysts, implying that EBCs adopting these catalysts may also keep their stabilities22According to the Figs S4a,b, the peak current density of GA/[[GOx/PEI]The results mentioned above discernibly mean that enzyme structures suggested for both anode and cathode electrodes play their role as catalyst. It is now time to determine how performances of membraneless EBCs adopting the enzyme catalysts are affected by four main parameters like glucose flow rate, glucose concentration, interval between two electrodes and electrolyte pH. For doing that, polarization curves of the corresponding EBCs were mainly measured.2 fuels are properly acted for GOR and ORR, inducing superior OCP and MPD. However, as glucose flow rate increases, the fuels become easy to access to the opposite electrode, lowering ORR potential and producing self-discharge current by occurrence of mixed potential. Such decreased ORR potential induces decrease in OCP, followed by decrease in MPD of membraneless EBC. More specifically, when glucose flow rate was 0.5 cc · min−1, MPD of EBC was 34 ± 2.6 μW · cm−2, while the MPD was plummeted to 10 ± 0.9 μW · cm−2 with the glucose flow rate of 90 cc · min−1. This result was well matched with one reported by Jayashree et al., who studied effect of fuel flowrate on performance of microfluidic membraneless fuel cellet al., who investigated effect of fuel flowrate on performance of proton exchange membrane fuel cell (PEMFC) previously reported and their result was also compatible with oursRegarding the glucose flow rate effect, according to polarization curves , as gluc−1. Their polarization curves are presented in As another parameter affecting performance of membraneless EBC, glucose concentration was regarded and its effect on membraneless EBC performance was evaluated. For the EBC tests, glucose concentrations of 10, 25, 40, 100 and 200 mM (pH 7.4) were provided with glucose flow rate of 0.5 cc · minet al., who investigated effect of ethanol concentration on performance of direct ethanol fuel cell (DEFC)2 concentration .In detailed evaluations, it is clear that under different glucose concentrations, performances of EBCs measured by polarization curves mainly depend on the difference in current density region occurred by electrolyte resistance and mass transfer with little change in OCP of the EBCs . According to the Effect of distance between anode and cathode on performances of membraneless EBCs was also critical and related polarization curves were measured . For the−2). The linear relationship among ohmic resistance, distance between two electrodes and MPD of the EBC is explained that the ohmic resistance loss is probably one of dominant factors to determine MPD of membraneless EBC because MPDs of membraneless EBCs follow up trend of ohmic resistance loss in all three different distances of 1, 2 and 3 mm.Second, as a distance between two electrodes was reduced, MPDs of the EBCs increased in a linear progression  using different MEA thickness, as distance between electrodes in DMFC decreased (decrease the membrane thickness), its performance increased due to reduction of ohmic resistance between electrodesSuch results are reasonable and compatible with previously reported ones−1 and glucose concentration of 40 mM. Their polarization curves are represented in −2). Generally, GOx  was mostly activated near to pH 7~8, while activity of Lac  was best near to pH 3~5. Thus, it means that the electrolyte pH effect is mostly dominated by catalytic activity of Lac molecules, confirming that performance of this membraneless EBC depends on cathodic reaction.Effect of pH of electrolyte (glucose solution) on performance of membraneless EBCs was also investigated because the pH of glucose solution filling empty acrylic separator placed between two electrodes affected the EBC performance. For the EBC tests, glucose solutions showing five different pHs like 2, 3, 5, 7.4 and 9 were adopted with distance of 1 mm, glucose flow rates of 0.5 cc · min−) controlled by electrolyte pH. When the pH is higher than 5, OH− ions also increase with activated reduction reaction. Such increased OH− ions then make activity of Lac molecules weak because the OH− ions are stuck to the active sites of Lac molecules, preventing O2 molecules from reacting at the active siteset al. and Tan et al.40This pH dependence on performance of membraneless EBC may be ascribed to change of hydroxide anion (OH−2) is also pretty excellent value compared to that of other membraneless EBCs. For instance, MPD of membraneless EBC consisting of GOx/PPy/CNT (anode) and Lac/PPy/CNT (cathode) was 30 μW · cm−2 −2 −2 −2 It is noticeable that the best MPD of the EBC (102 ± 5.1 μW · cm−2. Although the MPD is still less than that of EBC including membrane (171 ± 5.5 μW · cm−2), it is obviously viable result.As we already explained, EBCs including membrane and membraneless EBCs showed their own performances. However, since there is a difference in the distance between two electrodes , it is not possible to compare performances of the two EBCs in the status quo. To compare them in a proper manner, we estimated the regression curve between MPDs of the membraneless EBCs gained under the condition of 0.04 M glucose solution (pH 3) and distance between two electrodes . Based o−2). Although further efforts are required to improve the stability, it is reasonable to say that our membraneless EBC system is relatively stable.Stability of membraneless EBC was also measured for four weeks by tracking variance in its MPD. For the tests, optimal conditions of the aforementioned parameters were used and three samples were experimented for determining MPD. According to the MPD results represented in −2), meaning that (i) GA promoted immobilization of more Lac molecules, followed by activation of the ORR and (ii) cathodic reaction (ORR) was a rate determining step to affect performance of EBC.In this study, performances of mediatorless/membraneless EBCs adopting enzyme catalysts and catalytic activities of the enzyme catalysts were investigated. When the catalytic activities of catalysts for GOR and ORR were evaluated, they showed reasonable reaction trend with excellent activity values. In EBC tests, performances of EBCs adopting Nafion 117 membrane were evaluated. According to the EBC tests, performance of EBC adopting GA/[Lac/PEI/Lac/CNT] as cathodic catalyst was best , 40 mM (glucose concentration), 1 mm  and pH 3 were determined.When performances of membraneless EBCs were measured, four different parameters like glucose flow rate, glucose concentration, interval between two electrodes and electrolyte pH were considered. In the polarization curve measurements, how the parameters affected the performances of EBCs was clarified and the best condition of the parameters was obtained. As a result of that, MPD of membraneless EBC was reached to 102 ± 5.1 μW · cm−2 was calculated by the extrapolation equation. Although this value is still lower than that of EBC including membrane, it is clearly promising result.To compare performances of EBCs including membrane and membraneless EBC in equivalent state, MPD of the membraneless EBC was expressed as distance between electrodes. With that, a linear progression of MPD = −0.037 mm + 0.14 was obtained and the MPD value of 134 μW · cm−1 solid), Laccase , glutaraldehyde (GA) solution, and polyethylenimine  were purchased from Sigma Aldrich . Sodium-acetate buffer pH 5 was made from mixture between sodium acetate and acetic acid and that was purchased from Sigma Aldrich .Multiwall carbon nanotubes (MWCNT) (its purity is higher than 90%) were obtained from NanoLab . Glucose oxidase  were mixed. The mixture was further dissolved into 0.4 mg·mL−1 Lac solution under 0.1 M sodium-acetate buffer (pH 5). The mixture was then incubated for 2.5 h and centrifuged at 13,000 for 10 min. Lac/PEI/Lac/CNT catalyst was prepared by additional process of the Lac/CNT catalyst. The suspended Lac/CNT catalyst was mixed with 2.5 mg·mL−1 PEI solution (in 0.5 M NaCl (pH 5)). Supernatant of the mixture was then removed and washed with DI water to remove excess PEI. 0.4 mg·mL−1 Lac solution (in 0.1 M sodium-acetate buffer pH 5) was added to the mixture and the final mixture underwent the same incubation process to Lac/CNT catalyst. GA/[Lac/PEI/Lac/CNT] catalyst was prepared by additional process of the Lac/PEI/Lac/CNT catalyst. The suspended Lac/PEI/Lac/CNT catalyst was mixed with 0.5 w/v % GA solution. The mixture was then centrifuged at 13,000 rpm for 10 min. The excess GA was removed by using DI water 2 times to avoid further cross-linking process. All the catalysts were dipped in sodium-acetate buffer (pH 5) at 4 °C when not in use.Three different types of catalysts including Lac molecules were prepared for cathode. The catalysts are denoted as Lac/CNT, Lac/PEI/Lac/CNT, and GA/[Lac/PEI/Lac/CNT], respectively. Same to anodic catalyst, by carrying out preliminary optimization process, it was confirmed that 0.4 mg·mL2O and then, 10 μm of catalytic ink was dropped on the GCE. The catalytic ink-loaded working electrode was dried for 45 min. After drying, 5 wt % Nafion solution was coated on the working electrode to complete configuration of working electrode. As for electrolyte, 0.01 M PBS (pH 7.4) was considered.A computer connected potentiostat  was used for electrochemical measurements. For the half-cell tests like CV, Pt wire and Ag/AgCl (soaked in 3.0 M KCl) were served as counter and reference electrodes respectively, while catalysts loaded on glass carbon electrodes (GCE) were acted as working electrode. For loading of catalysts, catalytic powder was mixed with 1 mL of Hs/Rct), new EBC kit was designed with active surface area of 1.0 cm2. When membrane was included, the kit was consisted of catholyte reservoir, cathode electrode, membrane, anode electrode and anolyte reservoir. On the other hand, when used as membraneless EBC kit, membrane was removed and instead, acrylic separator was inserted. As the membrane, Nafion 117 (150 μm thick) was chosen.For measuring polarization curve and membrane/charge transfer resistances  and placed in cathode part of the EBC kit. After the cathode was ready, it was stuck to the membrane, while GA/[[GOx/PEI]s/Rct) in the corresponding EBC single cells, its Nyquist plot was measured. Nyquist plot produced is available for anode and cathode, indicating that semicircle of the Nyquist plot reflects Rct of the full cell, while intercept of x axis stands for Rs.To measure polarization curves from the EBC single cell, Bio-Logic  potentiostat was used. It was also connected with a frequency response analyzer (FRA). By coupling FRA with the potentiostat, the power output was determined. The EIS measurements were performed at the open cell potential condition of the EBC. The impedance spectra were decided in the frequency range between 2 MHz and 10 Hz with 10 steps per decade and modulating potential was fixed as 10 mV. To attain the membrane/charge transfer resistances .


The Psychological Record, 64(3), 433–440. doi:10.1007/s40732-014-0052-9, (2014) demonstrated the novel finding that the magnitude effect for medical outcomes does not reverse across delay and probability discounting as it does for monetary outcomes. We suggest that a possible reason for the lack of a reverse magnitude effect in nonmonetary outcomes is incomparable divisibility of discounted alternatives.Research by The Psychological Record, 64(3), 433–440. doi:10.1007/s40732-014-0052-9, (2014) was marginally not significant, although it was directionally consistent with their prediction of steeper discounting of small medical outcomes  both in time and probability discounting. Our manipulation by substituting a divisible outcome  for an indivisible one (brain cancer) did not, however, bring expected results. We discuss the explanations and possible implications of introduced division for divisible and nondivisible medical outcomes.To test whether the lack of a reverse magnitude effect in probability discounting of medical outcomes is due to incomparable divisibility of treatment effects, 4 studies were conducted. In the replication study, the effect observed by  Choices in which at least one payment is delayed are referred to as intertemporal choices. In such situations, the decision maker chooses between a smaller earlier or larger later payment. Choices in which at least one payment is only probable are referred to as choices under risk. In such situations, the decision maker considers the choice of certain payment or payment with a given probability  compared to a situation where one winner takes everything (a nondivisible result). Similarly, having one ski probably has as much utility as having no ski at all, since, when compared to having two skis, having one ski is almost useless, and its utility is close to zero. This can be understood in terms of a dramatically different curvature in the utility function for different goods. Having half of an initial monetary value gives you more or less half of the initial utility (depending on your money-utility function), while there are some goods where utility is either all or nothing. A similar type of reasoning is probably also applicable in many domains not involving money or goods .Weatherly and Terrell  used acnH1. In the case of a nongradable disease (brain cancer), the magnitude effect for medical outcomes does not reverse between delay and probability discounting as it does for monetary outcomes.Thus, we suggest that the lack of divisibility of treatment effects in the case of brain cancer resulted in Weatherly and Terrell’s failure to obtain the reverse magnitude effect for nonmonetary outcomes. We hypothesized that replacement of brain cancer with a disease whose treatment effects are divisible  would restore the reverse magnitude effect for nonmonetary outcomes . We therefore aimed to replicate Weatherly and Terrell’s  finding H2: In the case of a gradable disease , the magnitude effect for medical outcomes reverses between delay and probability discounting, as it does for monetary outcomes.We also aimed to demonstrate that the effect disappears in the case of a gradable disease  and hypothesized that:The study protocol was approved by the Institutional Review Board at Kozminski University. To test H1 and H2, four studies were conducted . All participants received a payment of $0.40 USD.All four studies were conducted in parallel using the Amazon Mechanical Turk (MTurk) and the LimeSurvey platform. Only people with at least a 95 % prior approval rate were invited to participate. Shortly after giving their informed consent, participants were randomly distributed to one of the four studies mentioned in Table Study 1 was an exact replication of the experiments done by Weatherly and Terrell . In StudSuppose you were suffering from acne . Your physician informs you that you will need to wait X (time) before obtaining a treatment that is 100 % effective. However, you could immediately begin a treatment that has a lesser chance of success. What minimum percent chance of success would you be willing to accept for the different treatment in order to choose it rather than waiting X (time)?When medical treatments were discounted participants read the following instructions:If you won $100  and were not going to get the money for X (time), what is the smallest amount of money you would accept today rather than having to wait X (time)?When monetary outcomes were discounted, participants read the same in both studies:Similarly to the original study, the question was repeated 5 times in succession to gather answers for 5 delay periods , for each outcome size , for each domain . Thus, in total 20 questions were asked at this stage (5 × 2 × 2). The order of particular delay periods was randomized within each of four blocks ; also, the blocks themselves were presented in random order. Participants were able to move to another block only after answering all five questions from a previous block; however, at any stage participants were allowed to use a “back” button to inspect and correct previous answers. We used a slider-type scale, with end points labeled 0 and 100 % for the medical conditions and 0 to $100 / $100,000 for the monetary conditions; 2 % increments  were used to gather answers instead of the radio button cafeteria-type scale used in the original study. In an online approach, including 50 radio buttons within one column would require scrolling to access higher values (the possibility of anchoring therefore existing), while breaking things into two columns could have resulted in anchoring answers with respect to the top of each column.Study 3 was an exact replication of the experiments performed by Weatherly and Terrell . In StudYou are a finalist in a national sweepstake. You have a X% chance of winning $100 . However, if your number is not called, you do not receive anything. The organization running the sweepstake is willing to guarantee to pay you a certain amount of money if you would agree to remove your name from the sweepstake. What is the smallest amount of money you would be willing to accept rather than having a X% chance of winning $100 ?When monetary outcomes were discounted, participants read the following instructions:Suppose you were suffering from acne (brain cancer). Your physician informs you that there are two treatment options. The first one completely cures the disease, but it only works for X% of the patients who choose it. The second treatment is guaranteed to work, but it only partially treats the disease. You can only afford to choose one treatment. What is the minimum percentage of success that the second treatment would need to guarantee for you to choose it over the first treatment?When medical treatments were discounted, participants read the same in both studies:Again, we used a slider-type scale, with endings labeled 0 and 100 % for the medical conditions and 0 to $100 / $100,000 for the monetary conditions; 2 % increments were used . Similar to the original study, each question was repeated 5 times in a row to gather answers for five probability levels – 1 %, 10 %, 50 %, 90 %, and 99 % – all presented in random order, for each outcome size , for each domain . Thus, again, in total 20 questions were asked at this stage (5 × 2 × 2). The order of particular probability levels was randomized within each of the four blocks , and the blocks themselves were also presented in random order. Again, participants were able to move to the next block only after answering all five questions from a previous block; however, at any stage participants were allowed to use a “back” button to inspect and correct previous answers.Exclusionary criteria were identical to those used by Weatherly and Terrell . We iden2 – a1)[(b1 + b2)/2], where standardized a1 and a2 are consecutive delays in months (or probabilities) while b1 and b2 represent consecutive subjective values of delayed (or uncertain) payoffs. This method allows calculation of the speed of a value’s decrease as the delay (or uncertainty) increases. The lower the AUC, the greater the diminution from the starting value because of delaying (or making more uncertain) the starting value.Data for participants whose choices met the criterion for systematic discounting were analyzed by calculating the area under the curve  reported in Table 2Figure F = 31.11; p = .001; η2 = .431. However, the difference in discounting strength between the smaller and larger magnitude medical outcomes (acne vs. brain cancer) was marginally nonsignificant, F = 2.94; p = .094; η2 = .067. In Study 2 we found a higher discount rate for smaller monetary outcomes than larger outcomes, F = 24.09; p = .001; η2 = .359, as well as for the smaller as compared to the larger magnitude medical condition, F = 7.41; p = .009; η2 = .147.In all groups, the larger outcome was discounted more steeply than the smaller outcome. In Study 1, a repeated measures ANOVA on rates of discounting identified a statistically significant difference in discounting strength between the smaller and larger monetary amounts, F = 24.53; p = .001; η2 = .374, and in Study 2: $100,000 vs. body paralysis, F = 5.84; p = .020; η2 = .120. This suggests that large medical conditions are discounted more strongly than large monetary outcomes. However, this was not the case for small outcomes: In both Study 1, F = 3.50; p = .068; η2 = .079, and Study 2, F = .24; p = .627; η2 = .006, differences in discounting strength for small magnitude medical outcomes (acne) and small monetary outcomes ($100) were nonsignificant (p > .05).Moreover, the results showed statistically significance differences between the large monetary and large magnitude medical outcomes in Study 1: $100,000 vs. brain cancer, The original sample of participants consisted of 143 people enrolled from Amazon’s MTurk worker pool. After employing the exclusionary criteria, the final sample across both studies consisted of 97 participants (see Table The exclusion rates for the probability discounting studies Studies 3 and 4) reported in Table  and 4 re3Figure F = 8.09; p = .006; η2 = .142. Although from Figure F = 3.38; p = .072; η2 = .064. In Study 4, participants more strongly discounted larger monetary outcomes when compared to smaller monetary outcomes, F = 19.65; p = .001; η2 = .299. For medical outcomes , however, the difference in discounting strength was not significant, F = .04; p = .847; η2 = .001.In Study 3, participants more strongly discounted larger monetary outcomes when compared to smaller monetary outcomes, F1, 9 = 8.09;F = 33.95; p = .001; η2 = .409, and Study 4 , F = 39.98; p = .001; η2 = .465. Finally, significant differences were also found between small magnitude medical treatment outcomes and small monetary outcomes in Study 3 ($100 vs. acne), F = 12.548; p = .001; η2 = .204, and Study 4 ($100 vs. acne), F = 34.113; p = .001; η2 = .426. These results all suggest that monetary outcomes are discounted more strongly than medical outcomes when comparing small–small and large–large magnitude outcomes.Results also showed statistically significant differences between large magnitude monetary and large magnitude medical treatment outcomes in both Study 3 , After the 20 discounting-related questions, participants were presented with manipulation check questions involving the rating of worry  for items mentioning both small intensity illness  and large intensity illness . Thus, participants had to rate 3 medical conditions × 2 intensity states, making a total of six questions presented in random order to participants. We assumed that the reduction of worry (between high and low intensity) should be higher for a divisible illness as compared to an indivisible one. The results confirmed our expectations . Thus, we conclude that in general receiving treatment that partially treats brain cancer results in less happiness than receiving treatment that partially treats (by the same proportion) body paralysis or acne.Indeed, participants rated high-intensity cancer higher on the worry scale than low-intensity cancer ; they also did the same for body paralysis (by 16.36 points) and for acne . However, the decrease in worry between the high and low conditions was the highest for the easily graduated condition of acne, smaller for body paralysis, and lowest for brain cancer  = 3.95, p = 0.050, η2 = 0.046”, had a very small effect size associated with it and that p was close to  a nonsignificant value. We replicated Weatherly and Terrell’s study and demonstrated that, for money discounting, small amounts are discounted more steeply than larger amounts, but that a similar phenomenon does not occur for medical outcomes when large and small are defined as cancer and acne, respectively, F = 2.94; p = .094; η2 = .067. However, when using divisible medical outcomes , so that the situation vis-a-vis divisibility was comparable to that for money, we found an effect similar to that obtained for monetary outcomes, with a higher discount rate for smaller than for larger medical outcomes, F = 7.41; p = .009; η2 = .147. This confirmed our expectation that divisible medical outcomes would behave in the same way as divisible monetary outcomes.Weatherly and Terrell  argued tF = 4.94, p = 0.028, η2 = .037,” again indicated a very small observed effect, and the effect was not observed at all in our replication study, F = 3.38; p = .072; η2 = .064. After substituting nondivisible cancer with divisible paralysis, the effect was negligible, F = .04; p = .847; η2 = .001, with large medical outcomes discounted at the same rate as small medical outcomes. Hence, the manipulation introduced did not alter probability discounting in the manner expected.For probability discounting, Weatherly and Terrell observed differences in the discounting of monetary and medical outcomes. Large monetary values were discounted more steeply than small values, but small medical outcomes were discounted more steeply than large outcomes. However, the effect size associated with the repeated measures ANOVA for medical outcomes  on p. 438 of Weatherly and Terrell’s paper, “The manipulation-check part of the study showed that the (dis)utility drop associated with declining illness levels (from high to low intensity) is smaller for some severe illnesses (such as cancer) than for other severe illnesses , with the former being less divisible than the latter. Therefore, people would probably behave differently when (as in Studies 3 and 4) choosing between two treatments, one of which completely cures the disease but works for only a certain percentage of the patients who choose it, and one of which is guaranteed to work but only partially treats the disease (as expressed by a certain percentage). Having a small brain tumor is almost equally as bad as having a large one: a person can die from either. Therefore, partial tumor reduction would probably be avoided in favor of even a small chance of full recovery, and a disproportionately greater percentage chance of success would be demanded in the case of the latter treatment to compensate for even a small chance of full recovery offered by the former treatment. Ultimately, in this situation, a patient should be less impulsive compared to a patient suffering from a divisible illness such as paralysis. Thus, although Weatherly and Terrell’s  testing We believe that a particularly complicated answering paradigm could at least partially explain such results. Respondents usually have trouble when scaling their attitudes on ratio scales: when asked open ended questions such as “What is the smallest amount of money (minimum percentage of success) you would be willing to accept,” they could treat the whole situation as a bargaining process and make high declarations because more money (or a greater chance of success) is always better than less. In consequence, scale sensitivity deteriorates significantly. Thus, in further replication studies other choice based methods should be used, such as (1) adjusting procedures . Because of the age difference, our participants may have been more sensitive to medical outcomes (the relevance of this topic to participants would be likely to increase with age) or generally more patient. Also, it has been suggested that in general AMT workers are more rational than samples drawn from other populations , our sample consisted of a (more diverse) group of AMT volunteers. Moreover, our sample was older and better balanced in terms of gender (In the interests of fully replicating Weatherly and Terrell’s  study we


AL) amyloidosis is a life‐threatening protein deposition disorder; however, effective therapy can dramatically improve the prognosis of AL patients. Therefore, accurate diagnosis of the underlying hematologic disease is important. Multi‐parameter flow cytometry (MFC) is a reliable method to analyze lymphatic neoplasias and to detect even a small lymphatic clone. We analyzed the presence of clonal plasma cell (PC) and B cells in the bone marrow of 63 patients with newly diagnosed AL amyloidosis by MFC. We compared the results with the levels of monoclonal protein, the histopathology and cytogenetic results. As reference of light chain restriction, we used the immunohistochemical results of κ or λ positive amyloid deposits in various tissues. MFC identified underlying clonal lymphatic cells in all but two patients . Sixty‐one patients harbored malignant PCs, whereas B‐cell lymphomas were identified in two patients. Furthermore, MFC indicated at least one putative immunotherapeutical target  on malignant PCs in all but one patient. These results demonstrate that MFC is a reliable tool for an accurate diagnosis of the underlying hematologic disease and the detection of potential immunotherapeutical targets in patients with AL amyloidosis.Systemic amyloid light chain ( Given the different underlying diseases and therapeutic options in systemic AL amyloidosis, the identification of only the paraprotein in the serum and urine is insufficient. The amyloidogenic clone must also be detected and characterized for optimal therapeutic strategy Multicolor flow cytometry (MFC) is a widely available diagnostic technique that allows the differentiation of PC and B‐cell disorders while simultaneously detecting a broad array of surface and intracellular antigens at a high level of sensitivity. Compared with histopathological evaluation, BM aspirates are easier to obtain than biopsies, and MFC analysis is significantly faster than immunohistochemistry (IHC) techniques.+ BM PCs. This study aimed to demonstrate the utility and sensitivity of MFC in the identification and characterization of underlying clonal cells in AL amyloidosis and, in particular, to evaluate the accuracy of this method in the detection of light chain restriction on a retrospective cohort of well characterized patients with AL amyloidosis.We retrospectively analyzed 63 consecutive patients with AL amyloidosis who were referred to our Amyloidosis Center from March 2014 to May 2015 and were thoroughly assessed using IHC for the amyloid deposits in organs, serum as well as urine immunofixation (IF), serum FLC test, BM cytology, BM MFC, BM IHC, and interphase fluorescence in situ hybridization (iFISH) on sorted CD138We retrospectively analyzed 63 patients who were newly diagnosed with systemic AL amyloidosis from May 2014 to May 2015 at our Amyloidosis Center at the University Hospital in Heidelberg, Germany. BM smears for cytomorphological quantification of PCs, IHC of BM biopsies and serum and urine IF measurements were performed as a part of the clinical routine. All patients gave written informed consent for the study. Approval was obtained by the Ethics Committee of the University Heidelberg.λ‐light chain, κ‐light chain, fibrinogen, Aβ amyloid  λ‐light chain , and κ‐light chain (AK3) The diagnosis of AL amyloidosis was confirmed histologically in every patient. Amyloid was detected by Congo red‐staining viewed under polarized light showing green birefringence and Congo red fluorescence. Immunohistochemical classification of amyloid was carried out as described elsewhere with commercially available monoclonal antibodies directed against AA amyloid and polyclonal antibodies directed against amyloid P‐component, μL aliquots. To detect malignant B cells, immunophenotyping was conducted with antibodies against k light chain , λ light chain , CD19 , and CD45 . For PC immunophenotyping, an eight‐parameter flow cytometry analysis was performed using cytoplasmic anti‐κ, cytoplasmic anti‐λ, CD19, CD20, CD22, CD27, CD30, CD38, CD45, CD52, CD56, CD81, and SLAMF7 antibodies . Before measurement, the cells were washed twice and resuspended in 500 μL of phosphate‐buffered saline . The measurement was performed on a FACSCanto™ II cell analyzer . The compensation matrix was calculated using BD CompBead particles , and the compensation setup tool in BD FACSDiva™ software was used. Data were analyzed using the Infinicyt™ software .For immunophenotyping, fresh BM aspirates were obtained as a part of the clinical routine. The BM aspirates were separated into 100 Aberrant B cells were identified based on the expression of CD19 and intracellular light chain restriction. PCs were identified based on the coexpression of CD38 and CD138 antigens. Malignant and normal PCs were differentiated based on intracellular light chain restriction and an analysis of aberrant CD45, CD19, and CD56 expression. When an intracellular light chain restriction was not evident, a back‐gating strategy of CD45− and CD19‐negative as well as CD56‐positive PCs was performed. Overall, positivity was defined when the antigen was expressed on ≥20% of analyzed PCs.κ light chain, 5.7–26.3 mg/L for λ light chain, and 0.3–1.6 for κ/λ ratio The concentrations of serum FLCs were measured by a latex‐enhanced immunoassay  on a Behring 2 nephelometer as described elsewhere + BM PCs were purified by auto‐magnetic‐activated cell sorting with anti‐CD138 immunobeads. For iFISH analyses, a panel of two‐color probe sets for the detection of numerical changes for the chromosome loci 1q21/13q14, 5p15/5q35, 8p21/19q13, 9q34/15q22, and 11q22.3/17p13, the IgH‐translocations t, t, t as well as an IgH‐breakapart probe were used. After hybridization according to the manufacturer's instructions , a minimum of 100 interphase nuclei per probe was evaluated using an automated iFISH spot counting system . Hybridization efficiency was validated on interphase nuclei obtained from BM of a healthy donor and the thresholds for gains, deletions, and translocations were set at 10%. Hyperdiploidy was defined according to the criteria described by Wuilleme et al., which require trisomies of at least two of the three chromosomes 5, 9, and 15 Molecular cytogenetic testing was performed as described previously t‐test and the chi‐squared test. A P‐value ≤0.05 was considered statistically significant.The results are presented as median, maxima, and minima. Data were compared using student's We included 63 patients  with newly diagnosed systemic AL amyloidosis. The median age was 62 (range 40–77) years. The demographic and disease characteristics are summarized in Table n = 63). We identified an aberrant PC population in 61 and a light chain‐restricted B‐cell population in two of the 63 patients.To detect the amyloidogenic clone, we performed an MFC analysis of the BM aspirates of all patients . In these cases, serum and urine IF identified a consistent light chain restriction in 78% (40/51) and 86% (44/51) of samples, respectively. The serum FLC test was positive in all κ  and all but four λ AL  amyloidosis cases. MFC detected the amyloidogenic clone in all κ  and in 95% (38/40) of patients with λ AL amyloidosis  of the examined cases. Figure To evaluate the accuracy of MFC in detecting PC light chain restriction, we compared the MFC results with serum and urine IF, the serum FLC, and the IHC of BM biopsies Table . We refen = 9, Table In a second step, we focused on AL amyloidosis cases in which the amyloid type was not analyzed or identified by IHC . Translocation t was the most frequent aberration and was found in 38 patients (63%). Deletion, del13q14 was present in 16 cases (27%). Multiple myeloma (MM) high‐risk aberrations were found in five patients (8%). Although translocation t and del17p13 were identified in two cases, respectively, t was found in only one case. Hyperdiploidy defined according to the criteria described by Wuilleme et al. was found in nine AL amyloidosis patients (15%). Other aberrations were present in 32 cases (53%). Overall, typical chromosomal aberrations of PC dyscrasias were found in 95% of AL amyloidosis patients (57/60) with a light chain‐restricted BM PC population identified by MFC as the amyloidogenic clone : CD20 positivity was observed in 54% (20/37) of t positive and only in 5% (1/20) of t negative cases.To characterize PC targets for potential treatment options in systemic AL amyloidosis with an aberrant PC population as the amyloidogenic clone, we evaluated the expression levels of SLAMF7, CD52, CD30, CD22, and CD20 on BM PCs by MFC in an increased number of patients  AL amyloidosis patients. This result is in agreement with that of Paiva et al., who demonstrated the utility of MFC in AL amyloidosis patients with a malignant PC clone and identified aberrant PCs in 97% of patients with AL amyloidosis We used in this series also iFISH results as a marker for clonality. We could identify a typical PC disorder aberration in 95% of the patients, which confirmed our previous reports Furthermore, we demonstrate that MFC provides useful information to specify immune‐chemotherapy in systemic AL amyloidosis. SLAMF7, CD20, and CD52 were expressed in 98%, 38%, and 25% of AL amyloidosis cases with light chain‐restricted PCs as the amyloidogenic clone, respectively. The high percentage of CD20‐positive cases is consistent with the immunophenotyping analysis performed by Deshmukh et al., who noted that CD20 was expressed in approximately 40% of the analyzed AL amyloidosis patients κ/λ based on serum IF and positive FLC test in these patients and confirms the finding of an amyloidogenic B‐cell clone by MFC. Based on this finding, the two patients received lymphoma‐tailored therapy instead of a PC disorder‐adapted therapy.In addition, it was possible to discriminate between malignant PC and B cells, so we identified two AL amyloidosis patients with a clonal B‐cell population in our study. This low frequency is consistent with the observation that AL amyloidosis rarely occurs (2–4%) in the setting of a NHL Overall, we demonstrate the great practicability and applicability of BM MFC in patients with AL amyloidosis as a part of clinical routine to detect light chain‐restricted lymphatic clones. We also demonstrate that the MFC analysis of BM aspirates is a very sensitive method to distinguish B‐cell lymphomas from PC clones, making a decisive contribution to diagnosis. Regarding recent results about the necessity of minimal residual disease analysis in MM, MFC‐based analysis might also open the door in AL amyloidosis for detection of small malignant clones. In the long run, achieving a complete remission in AL amyloidosis to prevent the further production of amyloid might be even more important than in MM. In addition, MFC could detect a potential target for immunotherapeutic approaches on the surface of the malignant PC  in approximately all patients.Katharina Lisenko: no conflict of interest. Stefan Schönland: received financial support for participation of congresses and research projects by Janssen and honoraria for talks by Janssen and Celgene. Anna Jauch: no conflict of interest. Mindaugas Andrulis: no conflict of interest. Christoph Röcken: no conflict of interest. Anthony D. Ho: Research funding from, and Membership on Advisory Board of Genzyme/Sanofi‐Aventis. Hartmut Goldschmidt: Research Support (Institution): Celgene, Janssen, Chugai, Novartis, BMS, Millennium; Advisory Boards (Institution): Celgene, Janssen, Novartis, Onyx, Amgen, Takeda, BMS; Honoraria: Celgene, Janssen, Novartis, Chugai, Onyx, Millennium. Ute Hegenbart: received financial support for participation of congresses by Janssen and honoraria for talks by Janssen and Celgene. Michael Hundemer: received financial support for participation of congresses by Baxalta, Celgene, and Amgen, financial support for research projects by Celgene, Chugai and Genzyme and honoraria for talks by Celgene.


Cortisol is a hormone involved in many physiological functions including fetal maturation and epigenetic programming during pregnancy. This study aimed to use hair cortisol as a biomarker of chronic inhaled corticosteroid (ICS) exposure and assess the potential effects of asthma on the hypothalamic-pituitary-adrenal (HPA) axis in pregnant women. We hypothesized that pregnant women with asthma treated with ICS would exhibit lower hair cortisol concentrations, indicative of adrenal suppression, compared to women with asthma not using ICS and women who do not have asthma.n = 56) and without (n = 31) ICS treatment, and pregnant women without asthma (n = 31). Hair samples were segmented based on the growth rate of 1 cm/month and analyzed by enzyme immunoassay to provide cortisol concentrations corresponding to preconception, trimesters 1–3, and postpartum. Hair cortisol concentrations were compared within and among the groups using non-parametric statistical tests.We performed an observational retrospective cohort study. Hair samples were analyzed from pregnant women with asthma, with (P = 0.03 for Controls vs. ICS Treated and Controls vs. No ICS). ICS Treated women taking more than five doses per week had hair cortisol concentrations 47 % lower in third trimester than Controls. Linear regression of the third trimester hair cortisol results identified asthma as a significant factor when comparing consistent ICS use or asthma as the predictor  = 9.7, P = 0.005, R2adj = 0.257).Hair cortisol concentrations increased across trimesters for all three groups, but this increase was dampened in women with asthma  contains supplementary material, which is available to authorized users. Cortisol has many important functions in the body including metabolism, regulation of blood pressure, and roles in the inflammatory and stress responses. When required, the hypothalamus and pituitary gland are activated to release a series of hormones that signal the adrenal cortex to release cortisol. Asthma is a common chronic inflammatory disease for which the recommended therapy for long-term control is inhaled corticosteroids (ICS) . ICS binr = 0.33, p = 0.04) and multiple saliva samples collected over 7 days  [Classically, the matrices used to measure a patient’s cortisol level are blood, saliva, or urine. However, these matrices do not account for the diurnal nature of cortisol secretion and only reflect the point in time when sampling is conducted . More re = 0.03) . Since h = 0.03) , p. 2), Hair cortisol has previously been used to assess the effects of various medical conditions on the hypothalamic-pituitary-adrenal (HPA) axis, including psychological and physical stressors. It is already known that cortisol concentrations increase in relation to a stressful event, and these changes were reflected in the hair of children fearful of beginning school and individuals who recently experienced a traumatic event –9. SimilIn 2001 in the United States, the prevalence of asthma in pregnancy was 8 % . Only onWe performed a retrospective observational cohort study. Three groups of pregnant women were recruited from June 2012 to December 2014: women with ICS-treated asthma (ICS Treated), and two comparison groups consisting of women without asthma (Controls) and a disease-matched group of women with asthma not treated with ICS (No ICS). The sample size was not formally determined due to the unavailability of data on hair cortisol concentrations in pregnant women with and without asthma when the protocol was created. Participants were recruited in person in obstetric clinics at the British Columbia Women’s Hospital and Health Centre in Vancouver, British Columbia, and St. Michael’s Hospital in Toronto, Ontario. Additionally, women were recruited through the Hospital for Sick Children’s Motherisk Program teratology information service via telephone and mail. All pregnant women were eligible provided they could read and understand English, did not use any corticosteroid products on their scalp, or have any known medical conditions characterized by high cortisol levels such as Cushing’s syndrome. Women were recruited at any time during pregnancy up to 6 months postpartum. Research ethics board approval was obtained from each institution and informed written consent was obtained from all participants.Relevant clinical information was obtained through medical record review and/or patient interview and included asthma and ICS treatment history and concomitant medications. Factors reported to affect hair cortisol levels, such as frequency of hair washing, days since last washing and chemical treatment (color or relaxer), were also collected . Additio2 at 37 °C, reconstituted with 125 μL of phosphate buffered saline, and vortexed for one minute. Initially, samples were reconstituted with 250 μL, but this resulted in some samples having results below the quantitation limit (0.33 pmol/mL or 0.12 ng/mL).A lock of hair approximately 3 mm thick  was cut from the vertex posterior region of the head as close to the scalp as possible. Based on an average growth rate of 1 cm/mo, each hair sample was further cut into segments 2–3.6 cm in length (the majority were 3 cm long) to correspond to preconception (PC), trimesters 1–3 , and immediate postpartum (PP) time points as available depending on the hair length and collection date  [The samples were analyzed using the Salimetrics High Sensitivity Salivary Cortisol Enzyme Immunoassay (EIA) kit  following the manufacturer’s instructions. All of the % cross-reactivity reported by the manufacturer is less than 0.6 % for other steroid hormones, such as prednisolone, prednisone, or cortisone, except for dexamethasone, which was 19.2 %. Additionally, the cross-reactivity was determined for each of the ICS available in Canada using an 8000 ng/mL solution run six times on two different EIA plates. The results were not detectable for fluticasone propionate, budesonide and ciclesonide, 0.01 % for beclomethasone dipropionate, and 0.03 % for mometasone furoate. Hair cortisol concentrations measured using this kit have been correlated to two different liquid chromatography-mass spectrometry methods with a Spearman rho of ≥ 0.92 , respectively, were reanalyzed or reprocessed based on sample availability. Cortisol concentrations are reported as a ratio to the hair sample weight (pmol/g).post hoc analyses were performed using the unpaired t-test with Welch’s correction, and Mann–Whitney U test with Bonferroni correction for multiple comparisons to calculate the adjusted p-values (Padj), as needed.Statistical analysis was performed using Graphpad Prism software, version 5.0c  and SPSS, version 22.0 . Comparisons between the groups for hair cortisol concentrations, demographic information, and clinical information were performed using one-way analysis of variance with Tukey’s multiple comparison test, the Kruskal-Wallis test with Dunn’s multiple comparison test, and Chi-square test or Freeman-Halton extension of the Fisher’s exact probability test as appropriate for normally or not normally distributed, and categorical data. Further post hoc analysis for the Friedman test was performed using the Wilcoxon matched-pairs signed rank test. A natural log transformation was required for the hair cortisol data prior to performing the Wilcoxon tests to best satisfy statistical assumptions. The Holm-Bonferroni correction was applied to p-values to correct for multiple comparisons. Additionally, the linear regression for median hair cortisol concentrations from PC to T3 for each group were compared using analysis of covariance with the Bonferroni correction for multiple comparisons to calculate the adjusted p-values. Univariate and multiple linear regressions were performed with the variables “consistent ICS use” (defined as use of ICS for ≥ 5 doses per week), “intranasal corticosteroid use” (yes/no), and “asthma” (yes/no) for T3 to determine if there was any influence of these variables on hair cortisol concentrations in that trimester.The Friedman test was used to compare hair cortisol concentrations for the five time points within each group. Because most women did not have results for all five time points, post hoc when the segment lengths were finalized and some deviated from 3 cm.Spearman correlations were calculated for the hair cortisol results with previously published confounders, including the age of hair sample, body mass index (BMI) (pre-pregnancy and at time of hair collection), number of hair washes per week, days since last washing, and PSS score. Because hair chemical treatment is a binary outcome, a point biserial correlation was performed using natural log transformed hair cortisol concentrations with concentrations ≥ 276 pmol/g removed to best satisfy the assumption of normally distributed data, which was unsuccessful for the PC concentrations in the treated asthma group. Correlations with the hair segment length were added p-values were calculated and considered significant if ≤ 0.05.In all cases, two-tailed i.e., if the hair segment was less than 2 cm) or >6 months postpartum. Some of the first hair samples analyzed had cortisol concentrations below the quantitation limit (0.33 pmol/mL) but could not be repeated due to insufficient quantity of the original hair sample; therefore, these results were recalculated using the quantitation limit. This recalculation predominantly affected the No ICS group (n = 5 patients/12 segments) compared to the Controls (n = 3 patients/3 segments) or ICS Treated (n = 2 patients/3 segments).Hair samples were analyzed for 118 pregnant women, consisting of 31 Controls, 31 No ICS, and 56 ICS Treated. Fourteen additional hair samples from the ICS Treated group could not be analyzed due to insufficient quantity, inaccurate segmentation, ICS being used to treat a condition other than asthma, or hair being collected too early in T1  = 9.6, P = 0.028, n = 4) and increased from 7.9 pmol/g  in PC to 21.1 pmol/g  in T3. The ICS Treated group also showed overall significant changes in cortisol (χ2(4) = 21.4, P < 0.001, n = 7) as well as an increase between PC  and T3 . Only the No ICS group did not have a significant overall change in cortisol across all time points (χ2(4) = 2.1, P = 0.768, n = 3) but did show a similar trend of increasing cortisol from PC  to T3 , although the increase from PC to T3 was also not significant (χ2(4) = 2.0, P = 0.583, n = 8). Generally, post hoc analyses revealed significant differences over the course of pregnancy between hair cortisol concentrations during PC and T1 compared to T2 and onward for the Controls and ICS Treated  = 14.8, P = 0.005)  = 22.6, Padj = 0.026) and No ICS  = 20.2, Padj = 0.033), whereas there was no significant difference between the No ICS and ICS Treated  = 0.1, Padj = 2.29). When the subgroup of women with samples with concentrations ≥ 276 pmol/g were excluded as potential confounders, the comparison between the Controls and No ICS was no longer significant  = 13.5, Padj = 0.064)  Fig. . Compari64) Fig. .Fig. 2Coχ2(2) = 5.1, P = 0.078)  = 2.189, P = 0.036). Furthermore, when women who reported ICS use less than five doses per week and concentrations ≥ 276 pmol/g were excluded as potential confounders, the T3 median hair cortisol concentration for the ICS Treated group was significantly lower (47 %) than the Controls   = 9.7, P = 0.005, R2adj = 0.257). When the samples with concentrations ≥ 276 pmol/g were included (with ICS use still restricted to five or more doses per week), the median hair cortisol concentrations at T3 for ICS Treated group was still 48 % lower than the Controls, but the difference was no longer significant   Fig.  and perf29) Fig. . Univari86) Fig. .Fig. 3BaThis study is the first to investigate the use of hair cortisol as a biomarker to assess asthma and the potential effects of ICS on the HPA axis in pregnant women. The 2- to 3-fold increase in serum or salivary cortisol over the course of normal pregnancy compared to a non-pregnant state is well-documented –22. Our Our results are in contrast to findings reported previously that did not find a significant difference in serum cortisol concentrations among pregnant women with asthma, with and without ICS treatment, and pregnant women without asthma . LimitatBased on previous reports of the association between decreased cortisol production and ICS therapy , 12, it P = 0.029). However, this significance did not remain when the regression model was adjusted for all other significant variables considered by the researchers . Nevertheless, our research and that of Braig et al. supports the premise that hair cortisol is a useful tool for future research to ascertain whether there is an association between decreased maternal cortisol and pregnancy outcomes. We were unable to find any further research reporting on pregnancy outcomes for women with lower-than-normal cortisol production suggesting that the condition has possibly gone undetected until now, thus indicating an advantage of hair cortisol as a biomarker of adrenal function during pregnancy.The prevalence of adrenal insufficiency or suppression in pregnancy is currently not well documented. This is in part due to the dynamic and significant increases of cortisol during pregnancy potentially masking any deficit , and a lin utero were more likely to experience ‘endocrine, metabolic disorders’ (hazard ratio (HR) = 1.84, CI95% 1.13–2.99) and digestive system diseases  [adj = 1.43, CI95% 1.34–1.52), nervous system , and digestive system  [The surges in cortisol from fetal and maternal sources during pregnancy both likely contribute to fetal maturation. Multiple mechanisms, such as the release of placental corticotropin releasing hormone and adrenocorticotropic hormone, result in increased maternal cortisol as pregnancy progresses, and the surge in cortisol in late pregnancy is involved in epigenetic processes that program fetal cardiovascular, neurologic, endocrine, and metabolic systems , 31. If 18–2.02) . Seconda04–1.32) , all of 04–1.32) , 30, 31.Limitations of our study include its relatively small sample size and possible discrepancies in how the hair was collected . Given the observed expected change in hair cortisol concentrations over the course of pregnancy, any inaccuracy due to improperly collected samples is not obvious, nor expected to be significant. Additionally, all medication use was self-reported, either to the study personnel or a healthcare provider. Generally, women tend to reduce their ICS use during pregnancy , 39, andOur findings suggest that hair cortisol may be a useful biomarker of HPA axis function during pregnancy and sensitive enough to detect the effects of asthma, both with and without ICS treatment, on systemic cortisol levels. Using hair cortisol analysis, we are the first to show that pregnant women with asthma are potentially unable to mount the expected cortisol response seen in later pregnancy regardless of ICS use. Perinatal outcomes that are known to be associated with maternal asthma may thus be a result of decreased maternal cortisol that adversely impacts fetal maturation and epigenetic programming. Building upon our current work, future research on the effects of maternal cortisol levels on pregnancy outcomes could benefit from using hair cortisol analysis as an assessment tool.n, sample size; Padj, adjusted p-value; PC, preconception; PP, postpartum; PSS, Perceived Stress Scale; SD, standard deviation; T1, first trimester; T2, second trimester; T3, third trimesterBMI, body mass index; cm/mo = centimetres per month; EIA, enzyme immunoassay; HPA, hypothalamic-pituitary-adrenal axis; HR, hazard ratio; ICS, inhaled corticosteroids; IQR, interquartile range;


Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species.Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is  Escherichia coli and Salmonella34The earliest microbiologists were astonished when they observed bacterial motility under their rudimentary microscopes, as this trait undoubtedly indicated to them that such tiny entities are living beingsEnsifer meliloti, Rhizobium lupini, and R. leguminosarum, possess peritrichous flagella that rotate in the CW direction only. In these bacteria, runs are produced when flagella rotate co-ordinately in a bundle, while a tumble occurs when individual flagella stop or slow down, causing bundles to fall apart78Caulobacter crescentus possesses a monotrichous polar or subpolar flagellum, and its switch from the CW to CCW rotational sense cause reversals of swimming with steep changes in direction caused by Brownian effects11Recent investigations have described alternatives to the RT modelBradyrhizobium diazoefficiens USDA 110, a soil α-proteobacterium that fixes atmospheric N2 in symbiosis with soybean, possesses two different flagellar systems. These systems are described as subpolar flagella based on the Salmonella modelC. crescentus, Rhodopseudomonas palustris and Rhodospirillum rubrum, while the secondary system is related to flagella of E. meliloti, Mesorhizobium loti and R. etliVibrio spp. the most studied. However, these γ-proteobacteria are taxonomically distant to B. diazoefficiens, and their flagellar systems seem unrelated15B. diazoefficiens may produce both flagellar systems simultaneously in liquid medium131718Shewanella putrefaciens being the only other known example with this capabilityB. diazoefficiens mutants lacking either the primary or secondary flagellar filaments did not allow for the assignment of clearly distinct functions to each one in swimming, swarming or competition for soybean nodulation18B. diazoefficiens here, we intended to assess their contributions to swimming performance in liquid and semisolid media, attachment to glass, and chemotaxis.Studies of B. diazoefficiens flagellar systems is shown in The cellular disposition of Bradyrhizobium species, as well as from R. palustris, C. crescentus, E. meliloti, Sinorhizobium fredii, Agrobacterium radiobacter, Mesorhizobium sp. and M. loti.  and the other lacking the subpolar flagellar filament (ΔfliC), were tracked at 30 frames s−1 in videos of 5–6 s of duration. Because bacterial cells could be tracked only while remaining in the focal plane, the duration of individual trajectories varied within videos. The mean durations  for each strain were 3.44 ± 0.17 s (n = 121) for the wild-type, 3.22 ± 0.20 s (n = 110) for ΔlafA and 4.26 ± 0.09 s (n = 115) for ΔfliC. Hence, the total time recorded was 6.93 min for the wild-type, 5.90 min for ΔlafA and 8.16 min for ΔfliC.A total of 346 swimming cells of fliC (24−1), starting again in the forward direction, which provoked a “flick” at low speed (15.5–18.7 μm·s−1), and finally resuming swimming at normal speed with RR/RT changes of direction. The average run speeds  in smooth trajectories were 28.8 ± 1.3 (n = 48) for the wild-type, 26.9 ± 2.0 (n = 50) for ΔlafA, and 17.2 ± 0.8 (n = 28) for ΔfliC.The trajectory shapes that were obtained were complex, all of which are shown together in fliC . In somefliC. In addition, the frequency histogram showed that the frequencies of curved and irregular trajectories were similar between the wild-type and ΔlafA, but there were clear biases toward circular trajectories in the wild-type and toward linear trajectories in ΔlafA. We sorted irregular trajectories according to their predominant changes in direction in RR, RRF and RT in the wild-type and ΔlafA and tight RT in ΔfliC, which we further divided into rough  based on known quantitative descriptors of path shape. The curvature of smooth trajectories may be estimated by their radius of curvature or may be reflected in the net-to-gross displacement ratio (NGDR)x-y coordinate space to the gross distance that is actually travelled between these same points, according to the expression:To find an objective and quantitative classification of trajectories, we built a x and y are the coordinates of the position, i represents the i-th position, 0 represents the initial position, and n is the number of successive positions that were considered. Therefore, persistently circular trajectories tend to present NGDR values between start and end points near 0, while linear trajectories have NGDR values near 1. With this criterion, in NGDR were <0.25, linear when their NGDR were >0.75, and curved when their NGDR were in between those values. The NGDR has the advantage over the radius of curvature that it can be used with any trajectory shape, curved or not. In addition to circular trajectories, those with significant changes in direction (low persistence) may also give NGDR values near 0 between start and end points. The persistency of trajectories may be evaluated through the rate of change of NGDR, which is low in smooth trajectories. In contrast, in trajectories with low persistence, sudden changes in direction are reflected in high absolute values of the slope of NGDR against the time of successive positions (NGDR (s):where ositions . Hence, registered in a trajectory is a good indicator of its persistence.αi) between frames:All trajectories, either smooth or irregular, showed changes in direction frame by frame. Most of these changes in direction were due to translational and rotational diffusion, with average angle of 18 ± 3°, but some of them were due to active switching of bacterial motion and had higher angles. Therefore, we calculated the cosine of the angle (α>). Next, we looked for significant changes in direction using the relative change in direction index (RCDI) that was provided by the softwareRCDI values higher than 1,000 corresponded to x-y positions in which cos αi was lower than <cos α> minus two standard deviations, suggesting that peaks of RCDI higher than 1,000 would identify significant changes in directionc  as follows:With all these descriptors, we defined the trajectory index  TRAIN values distinguished irregular trajectories with an RRF mode of change in direction  from smooth circular  and linear , without differences among the wild-type and ΔlafA. The swimming of ΔfliC was different, with two main trajectory shapes: rough , and diffusive  −see also TRAIN values, we considered that the frequency distribution of TRAIN values is a more objective estimate of the trends of trajectory shapes in each strain than the classification that is made by eye, or assisted with NGDR  in ΔlafA and towards the lower (highly irregular trajectories) in ΔfliC. Furthermore, wild-type trajectories were more like each other than the ΔlafA or ΔfliC mutants, as indicated by their coefficients of variation .The frequency histograms of TRAIN values and trajectory durations , which lead us to question its influence on the distributions of TRAIN values in the frequency histograms. In particular, linear trajectories in ΔlafA seemed shorter than the others  as described in the Methods section. These numbers were 47.4 ± 12.1 for the wild-type, 144.2 ± 52.9 for ΔlafA, 11.5 ± 5.6 for ΔfliC, and 12.6 ± 9.1 for ΔfliC/ΔlafA. Thus, lateral flagella were not required, but seemed to interfere with the subpolar flagellum for attachment to glass.The observation that the wild-type swam mainly in circles, while in ΔPreviously, the chemotaxis of the bradyrhizobial strains USDA 110, 10 K and 643 b was studied with a wide variety of attractant substances (chemoattractants), which presumably are nutrients and signals present in the soybean root exudates28lafA behaved similarly to the wild-type, while ΔfliC did not respond to any of the four chemoattractants that were assayed (R) values of ΔfliC cannot be attributed solely to its slower swimming speed. Together, these results indicate that the subpolar flagellum is enough for a chemotactic response in this assay.In agreement with data in USDA 110  assayed . BecauselafA in soft agar (0.3% w/v) with mannitol as the sole carbon source was slower than the wild-type and similar to ΔfliC1718lafA but not that of ΔfliC, while at 80 mPa s, ΔlafA was completely halted, similarly to the ΔfliC/ΔlafA non-motile mutant  may be a mechanical signal for lateral flagella induction, which seems relevant in the soil.It is interesting that LafA was induced in soft agar (0.3% w/v) without the addition of a viscous agent, by difference to Vibrio spp. and Aeromonas spp. lateral flagella are used for swarming on surfaces1516B. diazoefficiens swarming is not as evident and the requirement of these flagella for swarming is not so specificB. diazoefficiens soil populations or to the special properties of soil inner surfaces is a matter of future research.The different roles of lateral flagella in adhesion and swimming with respect to model systems might be related with the different activities of lateral flagella in swarming. While in Bradyrhizobium bacteria can swim freely inside macropores, either near the channel surfaces or in the bulk liquid  is a derivative from LP 3004 in which 97.8% of the coding sequences of lafA12 (bases 7561160–7563387) were replaced with a streptomycin-spectinomycin-resistance cassette. LP 5843 (ΔfliC) is a derivative from LP 3004 in which 75.6% of the fliC1234-coding sequences (bases 6410728–6418519) were replaced by a kanamycin-resistance cassette. LP 6543 (ΔfliC1234/ΔlafA12) is a non-motile derivative from LP 6865 with the same mutation of LP 5843. B. japonicum USDA 6T, B. elkanii USDA 76T, B. liaoningense 2281T and B. yuanmingense CCBAU 10071T were kindly provided by Dr. Esperanza Martínez-Romero, UNAM, Mexico. Unless specified, strains were grown in liquid AG mediumhttp://img.jgi.doe.gov or http://www.ncbi.nlm.nih.gov. These sequences were concatenated by hand, and multiple sequence alignments were performed with Clustal WSequences of FlhA, FlhB, FliF, FliG and FliP from each organism were recovered from 500) of 1.0 and diluted in Götz minimal medium without a carbon source−1 in videos of 5–6 s of duration with a phase contrast Axioscope 2 Carl Zeiss light microscope  that was connected to a Sony Exwave HD video camera . Computer-assisted motion analysis of the digitized images was performed with CellTrak . From these videos, we determined the coordinate position of each cell in the x-y space.Bacteria were harvested at an optical density at 500 nm . Bacteria were collected from liquid cultures when their OD (500 nm) reached 1.0. To collect bacteria from soft agar plates, 8-day old 10–110 bacterial colonies  were transferred to one 50-ml centrifuge tube, and one volume of sterile distilled water was added. Afterwards, the tubes were kept for 15 min on ice, vortexed for 5 min, and centrifuged at 10,000 × g for 30 min at 4 °C. The supernatants, or the liquid cultures mentioned above, constituted the starting materials for flagellin enrichment. These fractions were incubated with 1.3% w/v polyethylene glycol 6000 and 166 mM NaCl for 2 h at 4 °C. This suspension was centrifuged at 11,000 × g for 40 min at 4 °C, and the pellet was resuspended in Laemmli bufferBacteria were obtained either from liquid cultures or from beneath the surface of swimming agar (0.3%) plates. The observations were carried out at the Microscopy Service, Faculty of Veterinary Sciences, UNLP as describedTRAIN values within samples, TRAIN was expressed as average ± SD. All other values, when presented to appreciate the differences between experimental means, are presented as mean ± confidence interval. Confidence intervals were obtained by multiplying the standard mean errors by the Student’s-t with α = 0.05. Factorial analysis was carried out with sets of 20 trajectories of each strain chosen at random and checked for normality with the Anderson-Darling test with α = 0.05. Sets that passed the Anderson-Darling test were analyzed in a 2 × 2 factorial design for the effects of subpolar flagella, lateral flagella and their interaction on the TRAIN values. Because the non-motile double mutant ΔfliC/ΔlafA was immobile under the microscope and therefore could not be tracked by the software, we assigned it TRAIN = 0 with one degree of freedom. We believe that this assumption is validated by the average TRAIN = 0.05 of ΔfliC diffusive trajectories, because average TRAIN of ΔfliC/ΔlafA should be necessarily lower.To appreciate the extent of variation of How to cite this article: Quelas, J. I. et al. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems. Sci. Rep. 6, 23841; doi: 10.1038/srep23841 (2016).