Chronic kidney disease (CKD) is a global epidemic, but efforts to reduce its burden have been less effective than for other non-communicable diseases. Between 1990 and 2017, the global age-standardized mortality rate fell by 41.3% for chronic obstructive pulmonary disease, 30.4% for cardiovascular disease, 14.9% for cancer, but by only 2.8% for CKD [post hoc analysis of a previously reported randomized, double-blind, placebo-controlled study that examined the systemic cardiovascular and renal effects of a selective endothelin A (ETA) receptor antagonist [Circulating cardiac troponin is a strong independent predictor of cardiovascular morbidity and mortality in individuals with and without CVD . We recetagonist . Endothetagonist  as well tagonist .A receptor antagonist sitaxentan, nifedipine once daily or a matched placebo for 6 weeks in addition to their usual medications. There was a minimum 2-week washout period between phases. All subjects completed all three phases of the study. Patient diagnoses were immunoglobulin-A (IgA) nephropathy (n = 14), focal and segmental glomerulosclerosis (n = 6), membranous nephropathy (n = 3), hypertensive nephrosclerosis (n = 2), reflux nephropathy, microhematuria of presumed glomerular origin (n = 1 for both) and one subject with an unknown cause for their CKD. The mean age was 48 ± 12 years, mean body mass index was 29.3 ± 4.6 kg/m2 and 23 participants were male. The mean estimated glomerular filtration rate was 54 ± 26 mL/min/1.73 m2 and mean proteinuria was 2.0 ± 1.7 g/day. Eighteen (67%) participants were on statins and 24 (89%) were on either an angiotensin converting enzyme inhibitor or an angiotensin receptor blocker.In a randomized, double-blind, three-way crossover study, 27 participants with optimally managed and stable non-diabetic proteinuric CKD received the selective ETHigh-sensitivity cardiac troponin I concentrations  were measured in stored plasma at baseline and at 3 and 6 weeks during each phase  <10% at 4.7 ng/L) . Low-denA antagonism reduced troponin concentrations by 1.6 ± 0.5 ng/L (27 ± 19%)  (P < 0.001 versus both) [At baseline, the median troponin concentration was 2.3 ng/L (interquartile range 1.6–4.4), the mean systolic BP was 125 ± 12 mmHg and the LDL cholesterol concentration was 103 ± 32 mg/dL. There were no differences in any of these parameters at the start of each phase . Whereas placebo and nifedipine did not affect cardiac troponin, selective ETA antagonism  study demonstrated the efficacy of atrasentan, an ETgression . Interes ongoing  and shouIn this proof-of-concept study, improvements in these risk factors were achieved despite high baseline use of statins and renin–angiotensin system blockers. Further studies are required to determine whether prolonged reductions in BP can alter left ventricular mass and reduce cardiac troponin concentrations and to establish the role of serial troponin testing in monitoring the response to novel and established cardiovascular therapies in those with and without CKD.This work was supported by the British Heart Foundation .A.A.S.V. has received honoraria from Abbott Diagnostics. D.J.W. has acted as a consultant for Idorsia. N.L.M. has acted as a consultant for Abbott Diagnostics, Roche and Singulex. N.D. has acted as a consultant for Retrophin. All other authors have no financial disclosures. The sponsors or funders had no role in the design and conduct of the study; in the collection, analysis and interpretation of the data; or in the preparation, review or approval of the manuscript. The results presented in this article have not been published previously in whole or part.


Kawasaki disease (KD) is a self-limited vasculitis with unknown etiologies, and coronary artery lesions (CALs) are the most common and serious complications. Retinol-binding protein 4 (RBP4) has been confirmed effects on vasodilation, platelet activation inhibition, and cardiovascular diseases by researches. Therefore, this study was aimed at investigating the relationship between RBP4 and inflammation as well as thrombogenesis in children with KD.  79 subjects were from 62 children with KD and 17 healthy controls (HCs). The KD group was divided into KD with CALs (KD-CALs) and KD without CALs (KD-NCALs), and the serum RBP4 levels were measured by enzyme-linked immunosorbent assay (ELISA). p < 0.05). RBP4, hemoglobin (Hb), and mean platelet volume (MPV) levels were higher, while platelet counts (Plt) and thrombin time (TT) levels were lower in the KD-NCALs group than in the KD-CALs group (p < 0.05). RBP4 had positive correlation with time point of intravenous immunoglobulin (IVIG), Hb, and percentage of leukomonocytes (L%) and negative correlation with the percentage of neutrophils (N%), MPV, C-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), prothrombin time (PT), fibrinogen (Fbg), and D-dimer (DD) in the KD group; RBP4 had positive correlation with the time point of IVIG and L% and negative correlation with N%, MPV, and NLR in the KD-NCALs group; and RBP4 had positive correlation with Hb and L% and negative correlation with N%, CRP, NLR, and PT in the KD-CALs group (p < 0.05). Multiple linear regression analysis confirmed that Hb and CRP in the KD group, MPV and N% in the KD-NCALs group, and PT and CRP in the KD-CALs group were independent predictors of RBP4 (p < 0.05).  Compared with the HC group, serum RBP4 levels in the KD group were significantly decreased ( Lower RBP4 was observed in the KD group than in the HC group, and RBP4 had associations with markers of inflammation and thrombogenesis in children with KD. Coronary artery lesions (CALs), thrombogenesis , 2, and Ls in KD –6.Retinol-binding protein 4 (RBP4), a newly discovered adipokine, is the main transport protein of retinol (vitamin A)  and is m62 KD patients as the KD group, 42 males and 20 females, average age 2.51 ± 0.99 years old, and 17 healthy children as the healthy control group (HC), 10 males and 7 females, average age 2.69 ± 0.70 years old, were enrolled in a consecutive manner from the Children's Hospital of Chongqing Medical University between November 2018 and August 2019. The present study was a cross-sectional design study. All of the KD patients were diagnosed in strict accordance with the criteria proposed by the Japanese Circulation Society Joint Working Group [Z score and CALs evaluation standard is as follows [Z score less than 2.5; small coronary artery aneurysm (CAA) or coronary artery dilation, Z score between 2.5 and 5; large CAA, Z score between 5 and 10; and giant CAA, Z score equal to or greater than 10. CALs included CAA or coronary artery dilation, large CAA, and giant CAA. According to inner diameter of coronary by echocardiography and the calculation standard of the Z score, the 62 KD patients were divided into two groups: KD without CALs  and KD with CALs . Blood samples were collected in tubes containing liquid EDTA for complete blood count (CBC), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) determination, in a tube with a clot activator (and gel separator) for procalcitonin (Pct), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase-MB (CK-MB) determination, and in a tube with sodium citrate for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fbg), thrombin time (TT), and D-dimer (DD) determination (coagulation test). White blood cell counts (WBC), platelet counts (Plt), hemoglobin (Hb), percentage of neutrophils (N%), percentage of leukomonocytes (L%), mean platelet volume (MPV), and platelet distribution width (PDW) were assessed by a Sysmex XE-2100 hematology analyzer ; CRP was assessed by a gold standard digital quantitative analyzer ; ESR was assessed by a VISION automatic dynamic erythrocyte sedimentation rate analyzer ; Pct was assessed by a Roche Cobas 8000 automatic electrochemiluminescence immunoassay analyzer ; AST, ALT, and CK-MB were assessed by a Vitros-350 automatic dry biochemical analyzer ; and PT, APTT, Fbg, TT, and DD were assessed by a Sysmex CS 5100 automatic blood coagulation analyzer . Blood samples were drawn before administration of IVIG and anticoagulants.Professional pediatricians collected baseline information including age, gender, growth and development history, medical history, and family history and performed comprehensive physical examinations for all subjects. Body weight and height were measured according to a uniform standard in all subjects. Echocardiography was performed by one experienced ultrasound expert on the KD patients within 2 weeks of the onset and before administration of intravenous immunoglobulin (IVIG) and anticoagulants. The echocardiographic diagnosis based on the calculation standard of  follows , 22: no Blood samples were collected from KD patients (before IVIG) and HC patients and then centrifuged at 3000 rpm/min for 10 minutes to separate the serum immediately. Serum samples were stored at -80°C. The concentration of serum RBP4 was detected by a sandwich enzyme-linked immunosorbent assay (ELISA) kit .Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were calculated as neutrophil and platelet counts divided by lymphocyte counts, respectively. Body mass index (BMI) was calculated by weight in kilograms dividing height in meters squared.Informed consent was obtained from the legal guardian of each patient. This research was approved by the ethics committee of the Children's Hospital of Chongqing Medical University. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee.T-test and applicable χ2 test were used to compare the baseline data. Mann-Whitney U test was used for statistical analysis of continuous data without normal distribution, and an independent sample T-test was used for statistical analysis of continuous data with normal distribution. Spearman's rho test was used to determine the correlations with the data of nonnormal distribution. Pearson's correlation test was used for normal distribution data. CRP and NLR were logarithmically transformed into normal distribution for Pearson's correlation analysis and multiple linear regression analysis. For multiple linear regression analysis, variables entered into the stepwise forward model were normally distributed, showed significant parametric correlations with RBP4, and showed no multicollinearity. RBP4 was a dependent variable of correlation analysis and multiple linear regression analysis. p < 0.05 was considered statistically significant.SPSS version 19  was used for analysis of the clinical data and laboratory variables. Normality of data was checked using the Shapiro-Wilk test. The measurement data conformed to a normal distribution and was expressed as means with standard deviations. The measurement data was consistent with the skewness distribution and described by the median (interquartile range). Independent sample χ2 = 0.472, p = 0.492) and age between the two groups (p > 0.05).There were no significant differences in the sex ratio (p < 0.05)  .p < 0.05). Moreover, the levels of Plt and TT were significantly lower in the KD-NCALs group than in the KD-CALs group (p < 0.05). However, there were no significant differences in age, gender, the time point of IVIG, WBC, N%, L%, PDW, CRP, ESR, Pct, AST, ALT, NLR, PLR, CK-MB, PT, APTT, Fbg, DD, and BMI between the two groups (p > 0.05)  .p > 0.05); positive correlation with the time point of IVIG, Hb, and L% (p < 0.05); and negative correlation with N%, MPV, CRP, ALT, NLR, PT, Fbg, and DD (p < 0.05) in KD patients ; and no significant correlation with WBC, Plt, Hb, PDW, CRP, ESR, Pct, AST, ALT, CK-MB, PLR, PT, APTT, Fbg, TT, DD, and BMI (p > 0.05). Serum RBP4 showed no significant correlation with the time point of IVIG, WBC, Plt, MPV, PDW, ESR, Pct, AST, CK-MB, PLR, APTT, Fbg, TT, DD, and BMI in the KD-CALs group (p > 0.05), positive correlation with Hb and L%, and negative correlation with N%, CRP, ALT, NLR, and PT in the KD-CALs group (p < 0.05)  .p < 0.001) and CRP  in the KD group; using the time point of IVIG, N%, L%, MPV, and NLR as independent variables, the stepwise multiple linear regression analysis indicated that MPV  and N%  in the KD-NCALs group; and using Hb, N%, L%, CRP, NLR, and PT as independent variables, the stepwise multiple linear regression analysis indicated that PT  and CRP  in the KD-CALs group were independent predictors of RBP4  . MoreoveCRP, an inflammatory biomarker, has an association with KD and CALs , and ourEndothelial cell inflammation and injury as well as a high level of platelet in KD patients result in hypercoagulability and thrombosis in the acute phase and are prone to myocardium infarction , 35. In Some limitations of this study warrant discussions. Due to the limited number of cases, the sample size was small, and expanding the observation of the sample size could help us to confirm the causal relationship between RBP4 and coronary artery lesions in KD patients. Second, the study that was cross-sectional could limit us to explore the association between RBP4 and coronary artery lesions in KD patients. Finally, the measurement of RBP4 levels was done by ELISA rather than by quantitative western blotting, which are standardized to the full-length RBP4 protein. However, the results of this study to some extent supported the hypothesis that RBP4 may have associations with KD and CALs.In summary, the levels of serum RBP4 were significantly lower in children with KD than in healthy controls, and the serum RBP4 levels were even lower in KD patients with CALs than in KD patients without CALs in our cross-sectional study. Meanwhile, RBP4 levels were negatively correlated with inflammatory indicators and coagulation indexes and positively correlated with Hb. Moreover, Hb and CRP in KD patients, MPV and N% in KD patients without CALs, and PT and CRP in KD patients with CALs were independent predictors of RBP4. Based on these findings, we can conclude that there may be a potential link between RBP4 and the development of CALs in KD, but the mechanism needs further study.


Negotiations and conflicts do not evolve smoothly but are discontinuous involving transitions, break-, and turning points that change the flow of the negotiation. Given that these departures may be decisive in determining whether the involved parties come to a successful conclusion, several scholars have pointed out the importance of investigating whether impasse and settlement dyads exhibit different turning point profiles. To address this question, we extended Druckman’s  turning point model by integrating interlocking action–reaction sequences that initiate and (dis)confirm the departure from zero-sum bargaining. Furthermore, we consider social signals as previously not addressed class of events triggering the turning point. We propose and show that social signals act as precipitants to substantive change at the offer level and that how negotiators enact the action–reaction sequences discriminates between successful and unsuccessful dyads. Negotiations are an inevitable and ubiquitous part of organizational life which take place not only during business deals between organizations but also when coordinating activities within the organization to meet organizational goals  conflicts or, when focusing on deal-making negotiations, in conjunction with (episodic) phase models  as well as social (expressed emotions) behavior. Using negotiation process analyses , represents the precipitant in which negotiator B sends a social signal to negotiator A by reacting with an emotional change. The second interact (TPt−1 to TPt) constitutes the actual turning point in which negotiator A reacts to the social signal by proposing a more favorable offer compared to the previous offer by negotiator B. In the final interact (TPt to TPt+1) negotiator B concludes the turning point by (dis)confirming the change introduced by A. According to our model, the (dis)confirmation occurs on two distinct levels. First, via social signals indicating (dis)approval of the progression on an emotional level and second by (mis)matching the turning point offer on a substantive level.We consider three interacts that introduce and conclude the turning point see Fig. . The firTurning points typically occur after a period of no progress, leaving negotiators prone to facing impasses, or after a period of crisis Druckman . This giExtending prior literature, we consider emotions as social signals that act as precipitants for substantive change (the departure). Accordingly, a social change at the emotional level is the precursor of substantive change at the offer level. Emotions are acute and intense psychophysiological changes in response to a significant event in a person’s environment motional responses in a bargaining situation are hardly irrational disturbances”  and by B receiving the offer (Other Utility). The joint utility of an offer proposed by party A to party B is given by These measures are commonly used in negotiation research to describe the quality of exchanged offers and outcomes in negotiation research . Negotiator A sends the message “Your prior proposal is not what we are looking for and we need to work on an agreement.” (TPt−2) to which negotiator B (TPt−1) reacts with one of the following two alternatives: (1) “Your current proposal is unacceptable and as we are a respected company providing high quality products we expect a better offer.” (2) “Your current proposal is unacceptable but I’m confident that if we work together on the problem we will be able to reach an agreement that is beneficial for both of us.”. Although not expressed directly, both messages (1) and (2) convey negative goal-oriented emotions  directed towards negotiator A’s offer (“the problem”). They serve as feedback mechanism for counterproductive behavior and signal that behavioral change at the substantive level is required .To generate the distance matrix serving as input for MDS we employed the subjective clustering method . To assure that the findings are grounded in theory, the interpretation has been performed in light of existing emotional models  occurred we made the respective adjustments (Greenhaus–Geisser) to take into account inflated F-statistics  × 2 (interact) ANOVAs, with repeated measures on the latter factor. A significant main effect for the interacts (within-subjects) indicates a transformational sequence, that is, the negotiators reacted to the counterpart with a significant change in behavior. When symmetry violations (Mauchly’s cs Bergh . We furtt−1 to TPt) indeed constitutes an interact with a significant departure from zero-sum bargaining and that before the turning point (TPt−2 to TPt−1) no such behavior can be observed.Before testing the proposed hypotheses, we conducted preliminary analyses testing that the turning point (TPt−2 to TPt−1), the 2 × 2 ANOVA revealed a significant main effect for the interacts with regard to joint utility, F = 57.78, p < 0.001, ηp2 = 0.54, Po = 0.96, contract imbalance, F = 55.56, p < 0.001, ηp2 = 0.53 Po = 0.95, and other utility, F = 68.97, p < 0.001, ηp2 = 0.58, Po = 0.98. Furthermore, the results also show significant group effects (successful vs. unsuccessful dyads) for joint utility, F = 16.09, p < 0.001, ηp2 = 0.39 Po = 0.78, contract imbalance, F = 13.45, p < 0.001, ηp2 = 0.35 Po = 0.67, and other utility, F = 15.77, p < 0.001, ηp2 = 0.39 Po = 0.76, as well as significant interaction effects for joint utility, F = 24.53, p < 0.001, ηp2 = 0.49 Po = 0.93, contract imbalance, F = 26.90, p < 0.001, ηp2 = 0.52, Po = 0.95, and other utility, F = 30.66, p < 0.001, ηp2 = 0.55, Po = 0.97. For own utility we found neither a significant interact, F = 0.02, p = 0.887, ηp2 = 0.00, Po = 0.05, nor a main effect for group, F = 1.06, p = 0.355, ηp2 = 0.04, Po = 0.06, and an interaction effect F = 0.95, p = 0.392, ηp2 = 0.04, Po = 0.06. Post-hoc paired t tests revealed that successful dyads are characterized by reciprocal sequences  and unsuccessful dyads exhibit negative transformational sequences for other utility , a slight increase of contract imbalance approaching significance  and reciprocal sequences for joint utility  and own utility . Overall, the results show that the action–reaction sequence prior to the turning point indicates no attempt for inducing a positive redirection of the negotiation with impasse dyads even showing a tendency for increasing zero-sum bargaining (see first interact in Table Prior to the turning point (TPt−1 to TPt), however, both successful and unsuccessful dyads exhibit a positive transformational sequence for joint utility  and contract imbalance  as well as a significant increase of other utility  and a significant decrease of own utility . Thus, we find a clear departure from zero-sum bargaining at the turning point after an action–reaction sequence of economically unfavorable offers. These results are confirmed by the 2 × 2 ANOVA that revealed significant interacts for joint utility, F = 72.91, p < 0.001, ηp2 = 0.57, Po = 0.97, contract imbalance, F = 173.63, p < 0.001, ηp2 = 0.76, Po = 1.0, own utility, F = 15.13, p < 0.001, ηp2 = 0.21, Po = 0.35, and other utility, F = 141.51, p < 0.001, ηp2 = 0.72, Po = 1.0. Except for own utility, however, the ANOVA also reveals significant main effects for group  and significant interaction effects . Thus, while both successful and unsuccessful dyads move towards value creation, successful dyads make a more favorable departure from zero-sum bargaining  = 3.48, p = 0.068, ηp2 = 0.06, Po = 0.07, and the post-hoc paired t tests show that both successful  and unsuccessful  dyads show a significant decline of goal-oriented emotions. The results resemble the pattern proposed in Fig. F = 0.69, p > 0.506, ηp2 = 0.02, Po = 0.05, however, a significant main effect for group, F = 5.11, p < 0.001, ηp2 = 0.16, Po = 0.19. While both impasse and settlement dyads decrease goal-oriented emotions, the former exhibit a more negative climate at the turning point . The ANOVA reveals an interact approaching significance, a. The ANF = 0.43, p = 0.515, ηp2 = 0.01, Po = 0.05, and submission-dominance F = 0.04, p = 0.834, Po = 0.05. Contrary to our expectations, successful dyads do not engage in a transformational sequence, however, they still maintain the level of other-oriented emotions via reciprocal sequences . Thus, no negative emotional signals on the relational level are conveyed. Impasse dyads, on the other hand, not only combine the negative transformational sequence of goal-oriented emotions with a matching sequence of solidarity-conflict  but also with a negative transformational sequence of submission-dominance . Thus, by reacting with a decline in other-oriented emotions they pair the emotional signal addressing goal achievement with a negative emotional signal addressing the status of the relationship (H1c). The ANOVA also revealed significant group effects for other-oriented emotions . Similar to goal-oriented emotions, impasse dyads exhibit a more negative emotional climate at the turning point—also with regard to other-oriented emotions . The ANOVA revealed no significant interact for solidarity-conflict b. The ANIn order to corroborate our results, we also performed regression analyses assessing the effect of the decline in goal-oriented emotions on contract imbalance and joint utility at the turning point and calculated the conditional effects of the likelihood goal-oriented emotions at the turning point have on reaching an agreement contingent on the level of other oriented-emotions.t−2 to TPt−1) and the utility variables (TPt−1 to TPt). The difference of the emotion variables reflects the change of emotional tone prior to the turning point with a higher positive value indicating a stronger decline in goal- and other-oriented emotions. According to our theorizing, the decline in goal-oriented emotions should be associated with an increase in joint utility  and a decrease in contract imbalance . Results of the regression analysis show that the decline of goal-oriented emotions is significantly associated with the decrease in contract imbalance  and, with a p-value of p = 0.055, the increase in joint utility (β = 16.987) at the turning point, also when controlling for the joint utility  and the contract imbalance  of the offers exchanged prior to the departure. Thus, the results indicate that decreasing goal-oriented emotions before the turning point is associated with mutually coming closer as expressed by the contract imbalance. The other-oriented emotions are significantly associated neither with the increase in joint utility  nor the decrease in contract imbalance  at the turning point. Note that other-oriented emotions, having the relationship as primary referent, are not expected to affect the substantive value of offers. However, they should be related to whether negotiators reach an agreement or not, compensating on a relational level for the potential detrimental effects of decreasing goal-oriented emotions.For the regression analyses, we calculated the difference of the emotion variables  and a lower contract imbalance β = 3.638, p = 0.010) and dominance-submission In order to corroborate the compensating effect of other-oriented emotions, we calculated the conditional effects of the likelihood that goal-oriented emotions at the turning point have on reaching an agreement contingent on other-oriented emotions. Results of the logistic regression show that a higher joint utility  (dis)confirm the departure and ultimately direct the negotiation towards or away from reaching a mutually satisfactory agreement (the consequence). As shown in Fig. Following Druckman’s  model, wF = 16.51, p < 0.001, ηp2 = 0.22, Po = 0.38, contract imbalance, F = 11.86, p < 0.001, ηp2 = 0.17, Po = 0.24, and other utility, F = 17.57, p < 0.001, ηp2 = 0.24, Po = 0.41. Furthermore, the results show significant group effects (successful vs. unsuccessful dyads) for joint utility, F = 17.26, p < 0.001, ηp2 = 0.38, Po = 0.79, contract imbalance, F = 29.99, p < 0.001, ηp2 = 0.51, Po = 0.96, and other utility, F = 29.86, p < 0.001, ηp2 = 0.51, Po = 0.96, as well as significant interaction effects for joint utility, F = 8.79, p < 0.001, ηp2 = 0.24, Po = 0.41, contract imbalance, F = 4.81, p = 0.012, ηp2 = 0.14, Po = 0.18, and other utility, F = 7.65, p < 0.001, ηp2 = 0.21, Po = 0.34. For own utility no significant interact, F = 0.03, p = 0.854, ηp2 = 0.00, Po = 0.05, group effect, F = 2.20, p = 0.120, ηp2 = 0.07, Po = 0.07, and interaction effect, F = 1.26, p = 0.291, ηp2 = 0.04, Po = 0.06, was found. Overall, the ANOVA shows that impasse and settlement dyads exhibit different interaction patterns to conclude the turning point. The paired t tests further support these results and provide a more detailed picture. As predicted (H2a), in successful negotiations the turning point is concluded by reciprocal sequences of joint utility  and contract imbalance  as well as own utility  and other utility   , inspecting the individual utilities tells a different story. Whereas successful dyads conclude the turning point with matching the individual utilities, impasse dyads counter the turning point offer by increasing the own utility  and decreasing the other utility . By employing negative transformational sequences and countering with zero-sum bargaining, they disrupt the redirection towards creating value introduced at the turning point and disconfirm the change.Conversely, impasse dyads fail to reinforce the positive departure as predicted (H2c) and conclude the turning point by increasing the contract imbalance with a negative transformational sequences  = 4.17, p = 0.046, ηp2 = 0.07, Po = 0.08, and submission-dominance, F = 5.55, p < 0.022, ηp2 = 0.09, Po = 0.1, as well as significant group effects for pleasure–displeasure, F = 21.82, p < 0.001, ηp2 = 0.43, Po = 0.89, and submission-dominance, F = 6.65, p = 0.003, ηp2 = 0.19, Po = 0.28. The interaction effects are not significant for pleasure–displeasure, F = 1.92, p = 0.156, ηp2 = 0.06, Po = 0.07, and approach significance for submission-dominance, F = 2.81, p < 0.068, ηp2 = 0.09, Po = 0.1. For solidarity-conflict no significant interact, F = 0.13, p = 0.718, ηp2 = 0.00, Po = 0.05, group effect, F = 2.03, p = 0.141, ηp2 = 0.07, Po = 0.08, and interaction effect, F = 0.62, p = 0.541, ηp2 = 0.02, Po = 0.05, was found. Paired t tests reveal that, consistent with our hypothesis (H2d), unsuccessful dyads match the emotional climate that precipitated the turning point with reciprocal sequences  rather than introducing emotional change with positive transformational sequences. Contrary to our prediction (H2b), we also find reciprocal rather than positive transformational sequences for successful dyads . However, successful dyads exhibit a more positive emotional climate during and after the turning point as evident by the significant group effects. They did not exhibit the decline in other-oriented emotions as observable in impasse dyads and additionally, when considering one more interact, an upwards trend of goal-oriented emotions is clearly observable .The emotional dimensions show a similar picture (see third interact in Table Overall, the results support the proposed interaction dynamics see Fig. . WhereasTurning points may be decisive in determining whether the parties involved in a dispute reach a mutually satisfactory solution or end up in an impasse Donohue . The aimPrevious research on turning points has shown that negotiators can initiate departures potentially directing the negotiation towards positive grounds by taking procedural and substantive actions Druckman . Our finHowever, in order to introduce the required change without endangering the outcome of the negotiation, it seems crucial that negotiators compensate the decline in goal-oriented emotions on a relational level rather than pairing the negative transformational sequence of goal-oriented emotions with a negative transformational sequence of other-oriented emotions. Prior research has shown that expressing negative emotions in negotiations may have serious detrimental effects (e.g., Friedman et al. Following Druckman’s  model, tIn settlement dyads, negotiators match their counterpart’s turning point offer in terms of contract imbalance, joint utility, and own- and other-utility. By employing reciprocal sequences, the negotiators reinforce the departure, signal shared understanding, and confirm the newly established pattern (Brett et al. Conversely, negotiators that failed to reach an agreement counter the turning point offer by increasing their own utility and decreasing the other’s utility, resulting in a rise of the contract imbalance. Thus, rather than reinforcing the newly established mutually beneficial pattern, the negative transformational sequences disrupt the departure and disconfirm the change introduced at the turning point (Brett et al. Overall, the following key findings and advice for negotiators emanate from the systematic comparison of interaction steps initiating and concluding the turning point between settlement and impasse dyads. Although negative emotions are often considered detrimental in negotiations, expressing negative goal-oriented emotions may aid in initiating the departure as they clearly signal dissatisfaction with the current progress and that change is required. However, it appears to be crucial to pair the negative transformational sequence of goal-oriented emotions precipitating the departure with positive other-oriented emotions in order to signal affiliative intent and not disrupt the relationship between the negotiators. Once a negotiator has introduced the departure from zero-sum bargaining towards creating value and equal offers, it is key that the counterpart confirms this newly established pattern by reciprocating with an equally favorable offer for both parties. Not responding in kind to the turning point offer via a reciprocal sequence may jeopardize a successful, mutually satisfactory conclusion of the negotiation.In line with related studies and theorizing (Brett et al. Furthermore, prior research has shown that the epistemic motivation and information processing needs (Van Kleef et al. A crucial question emerging from the present results is why some negotiators chose to embrace the departure and responded in kind via a reciprocal sequence while other negotiators failed to seize this opportunity for creating value and direct the negotiation towards positive grounds. A preliminary answer to this question is provided by Griessmair and Druckman . They shSimilarly, settlement dyads may have established a more cooperative-integrative climate prior to the turning point. Therefore, the turning point offer fell on fertile ground and was more likely to be reciprocated by the counterpart (Griessmair and Druckman Finally, following the tradition of negotiation process analyses (e.g., Brett et al.


Efforts directed at mitigating neurological disability in preterm infants with intraventricular hemorrhage (IVH) and post hemorrhagic hydrocephalus (PHH) are limited by a dearth of quantifiable metrics capable of predicting long-term outcome. The objective of this study was to examine the relationships between candidate cerebrospinal fluid (CSF) biomarkers of PHH and neurodevelopmental outcomes in infants undergoing neurosurgical treatment for PHH.Preterm infants with PHH were enrolled across the Hydrocephalus Clinical Research Network. CSF samples were collected at the time of temporizing neurosurgical procedure (n = 98). Amyloid precursor protein (APP), L1CAM, NCAM-1, and total protein (TP) were compared in PHH versus control CSF. Fifty-four of these PHH subjects underwent Bayley Scales of Infant Development-III (Bayley-III) testing at 15–30 months corrected age. Controlling for false discovery rate (FDR) and adjusting for post-menstrual age (PMA) and IVH grade, Pearson’s partial correlation coefficients were used to examine relationships between CSF proteins and Bayley-III composite cognitive, language, and motor scores.CSF APP, L1CAM, NCAM-1, and TP were elevated in PHH over control at temporizing surgery. CSF NCAM-1 was associated with Bayley-III motor score , with modest relationships noted with cognition  and language  scores. No relationships were observed between CSF APP, L1CAM, or TP and Bayley-III scores. FOHR at the time of temporization did not correlate with Bayley-III scores, though trends were observed with Bayley-III motor (p = 0.0647 and R = -0.2912) and cognitive scores (p = 0.0506 and R = -0.2966).CSF NCAM-1 was associated with neurodevelopment in this multi-institutional PHH cohort. This is the first report relating a specific CSF protein, NCAM-1, to neurodevelopment in PHH. Future work will further investigate a possible role for NCAM-1 as a biomarker of PHH-associated neurological disability. Post-hemorrhagic hydrocephalus (PHH) of prematurity is the most common cause of pediatric hydrocephalus in North America , 2 and rNeurosurgical management strategies for PHH in preterm infants may be broadly considered as temporizing procedures  and permanent procedures . There is wide variability in clinical pediatric neurosurgical practice in the timing and use of these procedures, with few Level I studies –17 and mCerebrospinal fluid biomarkers have been shown to accurately reflect real-time pathophysiology in a number of neurological diseases –25, inclie prior to PHH treatment, and neurodevelopmental outcome in PHH. To this end, CSF samples and clinical data were collected from consenting patients at participating Hydrocephalus Clinical Research Network (HCRN) sites, and CSF APP, NCAM-1, and L1CAM levels were related to neurodevelopmental outcomes measured using the Bayley Scales of Infant Development-III (Bayley-III) obtained in these same subjects at 15–30 months corrected age.The objective of the current study was to investigate the associations between the levels of CSF APP, NCAM-1, and L1CAM at the time of temporizing neurosurgical procedure, inclusion criteria: preterm neonates ≤ 34 weeks estimated post-menstrual age (PMA) with birth weight < 1500 grams, Grade III or IV intraventricular hemorrhage (IVH), frontal occipital horn ratio (FOHR) ≥0.50, and >72 hour life expectancy, admitted to an HCRN Clinical Center prior to surgical intervention for PHH; exclusion criteria: primary caregiver refusing participation. Written consent was obtained from the parents or guardians of the children who served as subjects of the study. This study ran in parallel with (though lagged by 10 months) the HCRN study Shunting Outcomes in Post-Hemorrhagic Hydrocephalus  and thus followed standardized SOPHH parameters for PHH diagnosis and treatment, as previously described  at 40.97 ± 5.13 weeks PMA with FOHR = 0.66 ± 0.09. Surgical outcomes for those with temporizing procedures (conversion to shunt or ETV ± CPC) and those with permanent CSF diversion procedures  are the subject of a separate, forthcoming study.CSF levels of APP, L1CAM, NCAM-1, and TP were measured in 16 controls and in 98 PHH subjects at the time of temporizing neurosurgical procedure. Each of the 3 specific CSF proteins and CSF TP were elevated at the time of the temporizing procedure . When coNeurodevelopment was assessed using Bayley-III at 15–30 months corrected age in 54 subjects who had CSF collected at their temporizing neurosurgical procedures  for PHH treatment across the HCRN Tables . Of noteRelationships between CSF APP, L1CAM, NCAM-1, and TP and Bayley-III composite scores are detailed in In recognition of substantial neurobehavioral impairments in infants with PHH, there is increased focus on efforts directed at enhancing neurodevelopmental outcomes. One major factor impeding advances in clinical care and clinical research in PHH is a dearth of tools capable of providing real-time input for the clinical management of PHH, from establishing the diagnosis of PHH to the timing of intervention, monitoring of therapeutic efficacy, and evaluating the impact of PHH treatment on long-term neurodevelopmental outcome. Such biomarkers of PHH would also be of tremendous value in stratifying patients and estimating long-term outcomes in clinical trials designed to establish evidence-based management approaches for the care of infants with PHH. The current study aimed to address this critical void by examining candidate CSF biomarkers of neurodevelopment in PHH. Notably, CSF NCAM-1 demonstrated a highly significant association with Bayley-III motor score as well as more modest relationships with Bayley-III cognition and language. This is the first report of a CSF protein acquired at the time of intervention demonstrating an association with long-term neurodevelopment in PHH. No significant relationships were observed between the other CSF proteins studied and Bayley-III scores.APP, L1CAM, and NCAM-1 were selected as candidate CSF biomarkers of PHH based on a body of work drawing on findings from a seminal proteomic analysis of PHH CSF . Among pCerebrospinal fluid APP, L1CAM, and NCAM-1 have been shown to correlate with ventricular size in individual human infants with PHH . MeasuriThe Bayley-III remains the gold standard for evaluating neurodevelopment in early childhood. The Bayley-III provides summary scores for cognition, expressive language, receptive language, fine motor skills, and gross motor skills. While the Bayley-III is a widely accepted tool for infants and young children, there are limitations in its application and interpretation, as several reports have suggested that the Bayley-III may underestimate developmental delay  and its In the current study, and similar to other recent reports in infant hydrocephalus, we observed low performance on the Bayley-III in our participants  3, 7, 1, 49–52. Relatedly, neurodevelopmental outcomes in PHH, whether measured using Bayley-III, as in this study, or other tools administered at later ages, would be expected to be affected by myriad factors, not the least of which are those related to the IVH itself  , 53–63 aSimilar to previous studies , 27, 63,Our group and others , 9, 64 hThe CSF samples included in this study were acquired prospectively from 7 different HCRN sites across North America and aggregated at the HCRN PHH CSF Repository at Washington University. The multi-institutional nature of this repository is both a significant strength and a limitation. Despite standard operating procedures, there was likely inherent variability in the handling, processing, storage, and shipping of CSF samples across sites. This variability enhances the generalizability of our results but also attenuates our ability to detect and quantify statistical relationships. To address the differences in access to research facilities across HCRN sites, our protocol specified for freezing CSF samples immediately after acquisition. While our quality control analysis identified no differences in the concentrations of NCAM-1, APP, L1CAM, or TP between these HCRN samples and samples centrifuged immediatedly after acqusition (see Methods above), it remains possible that cellular lysate or debris could have impacted our CSF protein analyses. Comparison of lumbar and ventricular CSF also creates a potential confound owing to possible rostro-caudal CSF protein gradients. We have addressed this thoroughly in prior reports , 34, oneS1 File(PDF)Click here for additional data file.


Urban ecosystems are increasingly viewed as an important component within strategies for wildlife conservation but are shaped as much by natural systems as they are by social and political processes. At the garden scale, attitudes and preferences govern design and maintenance choices including the decision to encourage or discourage specific faunal presence. At the global scale, charismatic taxa that are well-liked attract more conservation funding and volunteer stewardship. Amphibians are a class of animals that are both loved and loathed making them a suitable subject for comparing and unpacking the drivers of preference and attitudes towards animals. We conducted a mixed methods survey of 192 participants in three adjacent neighbourhoods in Cape Town, South Africa. The survey included both quantitative and qualitative questions which were analysed thematically and used to explain the quantitative results. The results revealed that attitudes formed during childhood tended to be retained into adulthood, were shaped by cultural norms, childhood experiences and the attitudes of primary care-givers. The findings are significant for environmental education programmes aimed at building connectedness to nature and biophilic values. With more than half the world’s human population urbanized , urban eAmphibians have ecological importance in many ecosystems around the world. They are an essential link in the natural food web and are important bio-indicators in determining wetland and river health whilst regulating invertebrate populations . They arCities are arguably the most altered sites of change. Urban environments are prone to urban warming, and local climate changes within cities have occurred at faster rates than surrounding areas  yet it wIn many cities around the world, retention ponds, attenuation ponds and rain-gardens, developed as components of stormwater systems, have been colonized by amphibians as breeding habitat –21. StudAmphibians are both loved and loathed, making them a suitable class for unpacking human attitudes . All oveet al. noted, “That the average amphibian receives 75% less funding than the average listed mammal, bird or reptile, and 90% less funding than the average listed fish reflecting the less-popular status of amphibians in general”  as a child I went to the garden, playing with water and a lot of tiny frogs popped out and I was so amazed and held them on my hands.” Respondent #93 said “looking for frogs on the sides of mountain pools (often around Disa uniflora) after a long hot walk on the mountain. If you could stay in the cold brown water long enough, we used to see how close we could swim to them before they jumped into the pool.”The thematic analysis of the narrative of a memory from childhood shows clear distinctions between those that find frogs ‘gross’ or ‘scary’ and those that find them ‘likeable’ or ‘OK’. Those that have no feelings did not reveal any clear consistency in themes, but 61% (n = 11) of them had no recollection to share. Catching tadpoles (n = 19) featured frequently as a theme amongst those that had an affinity for frogs. The second theme was childhood discovery (n = 14) recounting playing with and discovering frogs, seeing them or hearing them, often characterised by a sense of wonder. Respondent #129, who liked frogs said, ““My dad calling us all into the garden at night to show us a leopard toad by torchlight. It happened fairly often! And then I did not see one for years until about 12 years ago in our [place name] garden …a long space in between!”. Some (n = 5) reported trying to keep them as pets, and some reported playing with them more destructively, or using them to play practical jokes on their friends (n = 7) “I once found a frog and put it in my sister's room and she freaked out”, others remember listening to them during the rain or at night (n = 4), and lastly, there were those who witnessed the killing of frogs with some distress, implying that they were already familiar with them, were unafraid and held some empathy (n = 5). These themes and accounts had in common direct interaction, fond recollection and that the adults either facilitated, or allowed engagement with minimal interference or warning.Parental biophilia also featured among this group (n = 5), in which a primary care-giver would tell the child not to harm the animal or would be involved in facilitating the interaction, either by instruction or taking them frogging. Respondent #4 said “Where I come from, some people say frogs are sent by witchcraft, especially if it is not raining or it is unseasonably dry”. Additionally, those that had been chased with frogs or startled also featured (n = 6). Respondent #83 who thought frogs were ‘scary’ said "Someone put it on me and I ran away and that’s when I knew I was scared”.On the other hand, those who reported fear of frogs tended to hold beliefs about the ability of amphibians to harm them. Two main themes emerge. Firstly that they were told by an adult or parent, that touching them (or even looking at them in one case) can result in severe rashes or infections (n = 7) and secondly that frogs are associated with witchcraft (n = 6), Respondent #188 said “I remember living on an old farm in [place name] and one very rainy, stormy night we woke up to hundreds of frogs popping up from under the floorboards and trying to put on a pair of my mom’s high heels to avoid them jumping on my feet”.Respondents across the like-dislike spectrum described frogs coming into the house, out of the ground, or out of the drains in large numbers. One respondent who liked frogs said, Hyperolius horstockii was much more popular than other species and many people who were generally afraid of frogs said they thought it was ‘likeable’ and were more likely to leave it alone if they found it in their garden. This finding is consistent with other studies which found that tree frogs–visually similar to the reed frog presented here—were more popular than reptile species indicating its potential as a flagship species for conservation [This study examined the preferences of a Cape Town community towards amphibians and explored attitudes using a composite approach drawing from several sources. De Groot’s preference ladder was used as a theoretical framework for exploring preferences. The findings of this study were consistent with those of de Groot’s in that general preferences at the broad conceptual level can be different to the specific level . This stervation . Knight ervation . The aruervation . As the ervation ,45. Thoservation ,46. The ervation ,43 howevervation ,43.Connectedness To Nature (CTN) [Impacting the general preference level is more complex given the multiple social influences and individual life-experiences that shape human preferences towards nature. De Groot’s research closely associated a general preference for nature with a biophilic self-identity . Biophilre (CTN) . Althougre (CTN)  and thuslived experiences; encounters and conversations with passionate, caring or dedicated role models; cultural background; and prior knowledge” [Positive conservation efforts within the urban context would require a shift towards a culture of pro-environmental behaviour. A predictive relationship has been demonstrated between biospheric values and pro-environmental behaviour . Biospheowledge” . This stThis research has highlighted that the attitude of the carer, or adult facilitating these activities, has a prominent role to play in this trajectory. Individuals that were actively discouraged from playing with, observing or going near to amphibians in early childhood, retained their fear into adulthood, while those who were encouraged or facilitated by their parents showed affinity. This suggests that there are elements to human attitudes which are normative, learned and intergenerational.The role of a parent was often mentioned in the narrative results as someone who passed on an attitude of affiliation for nature, a superstitious outlook or a set of warnings. Klassen’s summary of connectedness to nature theory gives credence to encounters with passionate role models including friends, family, teachers, community members, social movement leaders and writers . Role moPhrynomantis bifasciatus) is common in the north-eastern parts of South Africa and the areas where Xhosa and Zulu are widely spoken. It secretes an irritating toxin which can result in rashes or vomiting if handled extensively by sensitive individuals [Cultural background includes cultural beliefs, values, attitudes and opinions of family and community members . It is rividuals . The preRecruiting community leaders or celebrities to champion pro-environmental behaviour can assist in fostering positive norms within a given society . EnvironThe knowledge results within this study showed a correlation between accurate knowledge and liking frogs. The group that disliked frogs had a smaller mean score for knowledge and beliefs. It is not clear if lack of accurate knowledge was driven by disliking frogs or if disliking frogs meant that individuals were disinterested in accurate knowledge. Many previous studies have determined that direct experiences of animals, pets, and nature increases the retention of knowledge about these creatures and improves attitudes . Those wWe did not seek to measure the impacts of educational strategies but rather to determine what factors were associated with a general attitude of liking frogs. We confirmed that there is a relationship between knowledge and liking frogs, which is echoed in studies which determined that attitudes are seated in what people believe (cognitive knowledge) and how they feel about the environment (affective knowledge) . We obseThis study used a traditionally unpopular group of animals to explore why people like or dislike amphibians and consequently what might motivate them to amphibian stewardship behaviours. It found that individual charismatic species can be championed amongst groups regardless of affinity towards the class of animals. However, positive general attitudes are shaped by a combination of complex social forces, most notably, cultural norms, and regular positive experiences of the species.S1 Fig(XLSX)Click here for additional data file.S2 Fig(SAV)Click here for additional data file.


Evaluating the molecular characteristics of brain metastases is limited by difficult access and by the blood–brain barrier, which prevents circulating tumor DNA (ctDNA) from entering the blood. In this study, we aimed to compare the sequencing results from cerebrospinal fluid (CSF) ctDNA versus plasma ctDNA, plasma circulating tumor cells (CTCs), and brain tissue specimens from patients with brain metastasis from non‐small cell lung cancer (NSCLC).This was a prospective study of 21 consecutive patients with NSCLC and brain metastasis diagnosed between April 2018 and January 2019. Samples of CSF and peripheral blood were obtained from all 21 patients. Brain tissues were obtained from five patients after surgical resection. Next‐generation sequencing was performed using the Ion system. Single nucleotide variants (SNVs) and small insertions or deletions (indels) were searched.EGFR) mutations in CSF ctDNA was 57.1% (12/21) whereas this rate was only 23.8% (5/21) in peripheral blood ctDNA and in CTCs. EGFR mutations were found in the CSF of 9 of 11 (81.8%) patients with leptomeningeal metastases, as compared with three of 10 (30%) patients with brain parenchymal metastases. Mutations were also detected in KIT, PIK3CA, TP53, SMAD4, ATM, SMARCB1, PTEN, FLT3, GNAS, STK11, MET, CTNNB1, APC, FBXW7, ERBB4, and KDR . The status of EGFR and TP53 mutations was consistent between CSF ctDNA and brain lesion tissue in all five patients.Mutations were detected in the CSF ctDNA of 20 (95.2%) patients. The detection rate of epidermal growth factor receptor (Sequencing of CSF ctDNA revealed specific mutation patterns in driver genes among patients with NSCLC and brain metastasis.In some small‐sample studies, the importance of cerebrospinal fluid in guiding the treatment of cancerous brain lesions has been verified in that it may reflect genomic mutations of brain tumors relatively accurately.Cerebrospinal fluid is a new form of liquid biopsy that can be helpful in improving the management of patients with brain metastasis from non‐small cell lung cancer by detecting genetic abnormalities specific to brain metastases. Brain metastasis is a common neurological complication of systemic cancer in which metastases from the primary extracranial tumor invade the brain, usually indicating end‐stage disease and short life expectancy.Examining the molecular characteristics of brain metastases may assist in guiding therapy as the tumor clone that metastasizes to the brain may be different from the primary tumor.et al.A study by Pan The importance of CSF in guiding the treatment of cancerous brain lesions has been verified in some small‐sample studies showing that it could relatively accurately represent the genomic mutation of brain tumors.This was a prospective study of 21 patients with NSCLC and brain metastasis diagnosed between April 2018 and January 2019. The study was approved by the Ethics Committee of Tianjin Huanhu Hospital. All patients provided their written informed consent prior to any study procedure.The inclusion criteria were: (i) diagnosis of NSCLC confirmed by histopathological examination; (ii) first‐ever diagnosis of brain metastasis by MRI and ThinPrep cytologic test; and (iii) no prior treatment for brain metastasis.Samples of CSF and peripheral blood were obtained from all 21 patients. Brain tissues were obtained from five patients after surgical resection. CSF was obtained by lumbar puncture before brain surgery. Blood (10 mL) was collected from an antecubital vein into EDTA tubes. CSF and blood samples were preserved with 10 mL of a patented cell preservation solution (patent #CN201710442744.2) for transport and to ensure high‐quality samples for sequencing. The LiquidBiopsy system focuses on increased capture and molecular analysis of CTCs, including cells undergoing epithelial–mesenchymal transition. CTCs in CSF and blood samples were isolated using this system.Total DNA was extracted from plasma and CSF using the QiAamp Circumstance Nucleic Acid kit , according to the manufacturer's instructions. Total DNA was extracted from brain lesion specimens using a DNA/RNA Isolation kit (#R6731‐00).The reference library for all three types of samples was constructed using the Ion AmpliSeq Library Kit 2.0 and Ion AmpliSeq Cancer HotSpot Panel v2  and the Ion Library TaqMan Quantitation kit , according to the manufacturer's instructions. PCR amplification was performed using either a Veriti Dx 96‐well Thermal Cycle . Samples were prepared using various kits of the Ion system, according to the manufacturer's instructions .Data were analyzed using Ion Proton software . Alignment of NGS reads was performed using TMAP 5.4 software (The ProtPlot Web site). Two types of mutation were screened forusing Torrent VariantCaller 5.4 software: single nucleotide variants (SNVs) and smallinsertions or deletions (indels). Variant annotation and filtering were performed usinghttps://www.oncomine.org/resource/login.html), COSMIC (https://cancer.sanger.ac.uk/cosmic), My Cancer Genome (https://www.mycancergenome.org/), and dbSNP (https://www.ncbi.nlm.nih.gov/snp) databases.Ion Reporter 5.2 software, based on the reference genome version GRCh37(hg19), and Oncomine  had positive tumors. A total of 14 (66.7%) patients had previously received treatment with tyrosine kinase inhibitors (TKIs).Among the 21 patients with NSCLC, 10 (47.6%) were male and 11 (52.4%) were female. Mean age was 59.7 ± 9.9 years. A total of 10 (47.6%) patients had leptomeningeal metastases (LM), 11 (52.4%) had brain parenchymal metastases (BPM), 5 (23.8%) patients had EGFR mutations in CSF ctDNA was 57.1% (12/21) whereas it was only 23.8% (5/21) in peripheral blood ctDNA and in CTCs  patients. Mutations were also detected in KIT, PIK3CA, TP53, SMAD4, ATM, SMARCB1, PTEN, FLT3, GNAS, STK11, MET, CTNNB1, APC, FBXW7, ERBB4, and KDR .Mutations were detected in the CSF ctDNA of 20 patients (95.2%), in the blood ctDNA of 14 patients (66.7%), and in the circulating tumor cells (CTCs) of eight patients (39.0%). The detection rate of EGFR mutations were found in the CSF of nine of 11 (81.8%) patients with LM, compared with three of 10 (30%) patients with BPM.Mutations were detected in the CSF of all 11 patients with LM (100%), as compared with nine of 10 (90%) patients with BPM. EGFR and TP53 mutation statuses were consistent in all five patients. There were two patients with lesions and CSF ctDNA PIK3CA mutation, but a PIK3CA mutation was detected in the CSF ctDNA of one additional patient. One patient had lesions and CSF ctDNA STK11 mutation, but a STK11 mutation was detected in the CSF ctDNA of an additional patient. Otherwise, CSF ctDNA mutations were detected in a number of genes, and none were found in brain lesion tissues , and the detection rate of EGFR‐driven genes, the results from brain lesion tissues were consistent with those from CSF, as supported by Li et al.EGFR mutations in CSF might also reveal uncommon EGFR mutations that could be targeted with specific treatments.EGFR, mutations in KIT, PIK3CA, TP53, SMAD4, ATM, SMARCB1, PTEN, FLT3, GNAS, STK11, MET, CTNNB1, APC, FBXW7, ERBB4, KDR, and other genes were also frequently detected in CSF and brain tissue. Alteration of the mTOR signaling pathway may be one mechanism in brain metastasis from lung cancer.MET among the primary tumor, peripheral lesions, and brain lesions might guide the treatment as some MET mutations lead to resistance to gefitinib but may still respond to crizotinib. The impact of the response in leptomeningeal metastases on NSCLC prognosis outweighs the impact of the responses in peripheral lesions.EGFR driver mutation is found, irrespective of the location.In terms of It is difficult, often impossible, to obtain brain tissue samples from patients with brain metastasis via nonsurgical methods. Therefore, the brain tissues in this study were all from patients with brain parenchymal metastasis and who underwent surgery. From the standpoint of physiology, when metastasis erodes the leptomeninges, it is easy to create direct contact with the CSF in the ventricle, releasing more ctDNA and even tumor cells. Therefore, it is considered that CSF has importance for guiding the diagnosis and treatment of leptomeningeal metastasis. Indeed, in the present study, the rate of mutation detection was higher in patients with leptomeningeal metastases than in those with brain parenchymal metastases.The sequencing results of brain tissue were inconsistent with the results of CSF in patients of brain parenchyma metastasis. The reason could be that noise might be present from germline mutations in normal tissue. In addition, for patients with brain parenchymal metastasis, a clinically important tumor mutation in CSF might be associated with high tumor activity, which may warrant close follow‐up. Importantly, the use of a cell preservation solution is crucial to ensure high‐quality liquid biopsy samples for sequencing, as the number of tumor cells and amount of ctDNA are very small and even a small degree of degradation could compromise the results.This study has several limitations. The number of patients was small and only basic descriptive statistics were used. In addition, the impact of each mutation or their combination on the progression of disease and prognosis were not investigated. Brain lesion tissues were not available for all patients and there was no follow‐up to assess the prognosis. The present results should be considered preliminary pending more in‐depth mechanistic studies.In conclusion, the present study findings suggest that sequencing of CSF ctDNA may reveal specific mutation patterns in driver genes in brain metastases among patients with NSCLC and brain metastasis. CSF is a new form of liquid biopsy that could help in improving the management of patients with brain metastases.All authors declare that they have no conflict of interest.


SLC20A2, PDGFRB, PDGFB and XPR1. A variety of progressive neurological and psychiatric symptoms have been described, including cognitive impairment, movement disorders, bipolar disorder, chronic headaches and migraine, and epilepsy. Here we describe a family with a novel SLC20A2 mutation mainly presenting with neurological symptoms including cortical myoclonus and epilepsy. While epilepsy, although rare, has been reported in patients with IBGC associated with SLC20A2 mutations, cortical myoclonus seems to be a new manifestation.Idiopathic basal ganglia calcification (IBGC) or primary familial brain calcification is a rare genetic condition characterized by an autosomal dominant inheritance pattern and the presence of bilateral calcifications in the basal ganglia, thalami, cerebellum and cerebral subcortical white matter. The syndrome is genetically and phenotypically heterogeneous. Causal mutations have been identified in four genes:  Idiopathic basal ganglia calcification (IBGC) or primary familial brain calcification (PFBC) is a rare autosomal dominant genetic condition characterized by the hallmark of bilateral calcifications in the basal ganglia, thalami, cerebellum and subcortical white matter. The clinical presentation can be widely heterogeneous even among members of the same family. While the imaging phenotype shows 100% penetrance , the clinical phenotype only shows 61% penetrance , 19, 24.SLC20A2, PDGFRB, PDGFB and XPR1 , suggesting that the sodium-dependent phosphate transporter-2 (PiT2) is essential for [Pi] export from the CSF, and thus regulation of CSF [Pi]  .SLC20A2 are associated with psychiatric conditions (56.9%) including bipolar disorder, mood disturbances and psychosis  suggesting that PiT2 is essential to export Pi from the CSF and thus to regulate the [Pi] in cerebrospinal fluid (CSF) [The mutation found in our proband affects rebellum . In the id (CSF) . Althougid (CSF) . Moreoveid (CSF) , 27.Future studies will help explain why calcification is limited to specific parts of the brain, and the functional impact of the disordered phosphate homeostasis in the brain”.SLC20A2 mutation and a possibly associated new clinical presentation including cortical myoclonus. The variety of neurological symptoms and the overlapping psychiatric manifestation can prove challenging for diagnoses and management. The potential co-occurrence of different paroxysmal events, including epilepsy, dystonia, tremor and cortical myoclonus requires meticulous neurophysiology assessment to elucidate the origin of the events and to offer the best management.In conclusion, we report one individual with


Apparently, non-genomic factors played a dominant role in the inter-individual variation of the CYP3A probe drug midazolam. A small intra-individual pharmacokinetic variation after repeated administration of midazolam was rated earlier as indication of high heritability of CYP3A activity, but according to present data that could also largely be due to constant environmental factors and/or heritability of liver blood flow. The higher heritabilities of 4ß-OH-cholesterol and of 1-OH-midazolam may deserve further research on the underlying factors beyond CYP3A genes.Human CYP3A enzymes (including CYP3A4 and CYP4A5) metabolize about 40% of all drugs and numerous other environmental and endogenous substances. CYP3A activity is highly variable within and between humans. As a consequence, therapy with standard doses often results in too low or too high blood and tissue concentrations resulting in therapeutic failure or dose-related adverse reactions. It is an unanswered question how much of the big interindividual variation in CYP3A activity is caused by genetic or by environmental factors. This question can be answered by the twin study approach. Using midazolam as CYP3A probe drug, we studied 43 monozygotic and 14 dizygotic twins and measured midazolam and its metabolite 1-OH-midazolam. In addition, endogenous biomarkers of CYP3A activity, 4ß-OH-cholesterol and 6ß-OH-cortisol, were analyzed. Additive genetic effects accounted for only 15% of the variation in midazolam AUC, whereas 48% was attributed to common environmental factors. In contrast, 73, 56, and 31% of 1-OH-midazolam, 4ß-OH-cholesterol and 6ß-OH-cortisol variation was due to genetic effects. There was a low phenotypic correlation between the four CYP3A biomarkers. Only between midazolam and its 1-OH-metabolite, and between midazolam and 6ß-OH-cortisol we found significant bivariate genetic correlations. Midazolam AUC differed depending on the Clinical Trial Registration:ClinicalTrials.gov: NCT01845194 and EUDRA-CT: 2008-006223-31. The cytochrome P450 (CYP) enzymes 3A4 and 3A5 play a key role in the biotransformation of about 40% of currently used drugs . CYP3A eExpression and activity of human CYP3A enzymes in the gut epithelia and liver cells is highly variable both inter- as well as intra-individually . CYP3A ain vivo probe drugs are the most important tools for the study of individual CYP3A activity. Numerous CYP3A probe drugs have been applied. Currently, midazolam is the most widely used  may be due to additive genetic factors . HoweverThus, at present, it is not clear how much of the inter-individual variation in CYP3A4 activity is in fact genetically determined. Twin studies performed in monozygotic (MZ) and dizygotic (DZ) twins allow to distinguish between additive (A) and dominant (D) genetic factors on the one hand, and constant (C) and unique environmental (E) factors on the other hand. We used this approach with midazolam as CYP3A4 probe drug and endogenous biomarkers of CYP3A activity. To allow comparison with the repeated drug administration approach , each CYClinicalTrials.gov (NCT01845194) and in the European clinical trials database (EUDRA-CT number: 2008-006223-31).The study was a repeated dose pharmacokinetic/pharmaco- genetic study on monozygotic (MZ) and dizygotic (DZ) twin pairs. The ethics committee of the University of Göttingen and the German Federal Drug Administration (BfArM) approved the study. It was registered at In total, 116 subjects including 44 MZ and 14 same-sex DZ twin pairs were included in the study. One MZ twin pair was excluded from the analyses of the intravenous midazolam administration because of erroneous blood sampling. All subjects were healthy according to their medical history, medical examination, electrocardiogram, urine status, and clinical chemistry analyses. The latter included sodium, potassium, calcium, aspartate aminotransferase, creatinine, total bilirubin, hemoglobin, erythrocyte, thrombocyte, and leucocyte counts. Written informed consent was obtained from each subject before participation in the study. Except for hormonal contraceptives, no other drugs or potentially interacting nutrients  were allowed for 1 week prior to the study days and until the last blood and urine sampling was completed. Smoking was allowed if both siblings of each pair smoked to a similar extent. Subjects with history of alcohol or drug addiction were excluded prior to any study related procedures. Additionally, subjects were not allowed to drink alcohol from 48 h prior to 72 h after midazolam application and alcohol breath test was absolved in the morning of each study day. Other dietary habits were recorded in standardized nutrition questionnaires . Lean boMidazolam was administered as a 2 min intravenous infusion with a total dose of 0.2 mg. The phenotyping test with midazolam was performed on three different occasions with a time interval of at least 1 week and maximally 3 months. Blood samples were drawn at 0 (before infusion), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 24 h after the midazolam infusion.Cholesterol and 4ß-OH cholesterol were measured in the baseline blood samples before drug administration. All samples were centrifuged for 10 min (4000 rpm) at 4°C and the plasma was stored at −20°C. To avoid possible circadian variation of the cortisol to 6ß-OH-cortisol metabolism , a 24 h The concentrations of midazolam, 1-OH-midazolam, 6-ß-hydroxycortisol and cortisol were determined by LC-MS-MS analysis on an Agilent 6460 triple quadrupole mass spectrometer . Sample work-up and LC-MS-MS analysis of midazolam was performed as described previously  using miTM 4 μm Polar RP 80Å, 150 × 2 mm, Phenomenex, Aschaffenburg, Germany) with a gradient of acetonitrile and water with 0.1% formic acid.Urine samples were centrifuged after thawing, and 10 μl were diluted with 90 μl of water for determination of cortisol and 6ß-OH-cortisol. After addition of internal standard mixture (10 pmol cortisol-d4 and 100 pmol 6ß-OH-cortisol-d4) the samples were centrifuged, and 10 μl of the supernatant were used for LC-MS-MS analysis. HPLC separation was achieved on a reversed phase column trifluoroacetamide (BSTFA) for 30 min at room temperature.Cholesterol and 4ß-hydroxycholesterol were determined by GC-MS in EI mode, on a 5975 XL MSD coupled to a 7890A GC . Cholesterol quantification was performed as described previously  with minSample preparation for 4ß-hydroxycholesterol was performed according to a previously published method  with minCalibration samples for cholesterol were prepared in isooctane with 10% 2-propanol in the concentration range from 1 to 30 μg. Calibration samples for 4ß-hydroxycholesterol were prepared in isooctane with 0.8% 2-propanol from 5 to 100 ng. Calibration samples for cortisol and 6ß-hydroxycortisol were prepared in water from 0.025 to 10 pmol, or 0.25 to 100 pmol, respectively. Calibration samples were worked up as the samples, and analyzed together with the unknown samples. Calibration curves based on internal standard calibration were obtained by weighted (1/x) linear regression for the peak area ratio of the analyte to the respective internal standard against the amount of the analyte. The concentration in unknown samples was obtained from the regression line.TM DNA Blood 350 μl Kit using the Bio-robot EZ1TM . The ∗1bCYP3A4 and ∗22 alleles as well as the ∗1/∗3CYP3A5 polymorphism were genotyped by DNA sequencing using fluorescence-labeled dideoxynucleotides and detection via Gene Mapper v3.7 Software® . Zygosity was assessed by analyzing variants in 23 polymorphic genes. Monozygosity was concluded when there were no differences regarding the polymorphisms in both siblings of one twin pair.DNA was isolated from whole venous blood by automated solid phase extraction with the EZ1420 was calculated from time of dose to 420 min after dosage. Total plasma clearance (Cl) after administration of midazolam was calculated as the ratio dose over AUC. Time (tmax) and amount of maximum plasma concentration (Cmax) were given as measured and the terminal half-life (t1/2) was calculated as t1/2 = ln(2)/lambda z.Pharmacokinetic parameters were estimated by non-compartmental analyses using the WinNonlin software . The AUC from zero to infinity (AUC), was calculated by the linear/log trapezoidal rule and using the terminal elimination rate constant (lambda z). AUCW) and inter-individual (SDB) standard deviation was calculated as described by Predefined primary endpoint was midazolam AUC. All concentrations and pharmacokinetic parameters were determined on three independent occasions to compare intra- versus interindividual variation. The genetic component (rGC) concerning intra- (SDWe studied 44 monozygotic and 14 dizygotic twin pairs. There were no significant differences in demographic data between MZ and DZ twin pairs . No seri∗min/L) and a 10.4-fold variation of 1-OH-midazolam AUC (30.95 – 322.8). There were no statistically significant differences in the pharmacokinetic parameters between the monozygotic and dizygotic twins (Midazolam was administered intravenously on three independent occasions under supine and resting conditions. As illustrated in ic twins . Intra-iAs illustrated in the lower part of p = 0.03) and 4ß-hydroxy-cholesterol (p < 0.01)  versus 432.1 μg ∗ min/L (287.8 – 897.4 μg ∗ min/L)]. Higher lean body mass was associated with low AUC according to linear regression analysis. The median midazolam AUC in heterozygous carriers of the ∗22CYP3A4 polymorphism (n = 4) was 35% higher than in subjects with the ∗1/∗1 and ∗1/∗1B genotypes (n = 110) (595.1 μg ∗ min/L versus 439.1 μg ∗ min/L).Midazolam pharmacokinetics were significantly influenced by sex, lean body weight, type of nutrition, usage of hormonal contraceptives, and by the morphism . Median p = 0.012), but there were no statistically significant effects of the CYP3A genotypes , with a median 4ß-OH-cholesterol level of 23.01 ng/ml in homozygous carriers of the ∗3CYP3A5 allele and 30.69 ng/ml in heterozygous carriers of the active ∗1CYP3A5 allele. Our study included 16 carriers of the ∗1CYP3A5 genotype, and from the genotype-related differences  and constantly acting environmental (including epigenetic) effects (C) was not very precise as given in A high intra-individual constancy  and a hiDue to its high sensitivity to inhibitors and inducers, midazolam is currently a preferred CYP3A probe drug in drug research, but data shown here indicate that midazolam may also reflect other processes and activities besides CYP3A. With a clearance of about 0.5 L/min , midazolIn vitro, one often prefers measuring enzyme activity based on the metabolite formation. In vivo, in our study, there was a high intra-individual constancy and significant heritability of the main midazolam metabolite 1-OH-midazolam  in 50 healthy volunteers (Our data on the poor correlation between 4ß-OH-cholesterol and midazolam AUC is in line with results published by others: For instance, the correlation between midazolam total clearance and 4ß-OH-cholesterol plasma concentration was only 0.24 (lunteers . This islunteers . One explunteers , the hallunteers  but othelunteers . Thus, 4in vivo markers of CYP3A activity used here most likely would have correlated much stronger than found here. Heritability of induced CYP3A activity was indeed found to be about 66% after a 14 days treatment with St. John’s wort (The cortisol metabolite 6ß-OH-cortisol has been in use in clinical pharmacology for more than 50 years . Under bn’s wort , but thein vivo biomarker of CYP3A activity does not exist. The multivariate twin study design allowed searching for the common hidden factor using structural equation modeling, but this did not identify a strong genetic correlation between all four presumed CYP3A biomarkers. The relatively poor correlation between different biomarkers of CYP3A activity may not generally devalue them, but we should keep in mind that CYP3A biomarkers may reflect to a significant extent more of other processes than commonly presumed. The fraction of variation in CYP3A4 activity currently explained by defined genetic polymorphisms is relatively small and according to our study, search for further genomic biomarkers to predict reliably the individual CYP3A activity may not be very promising. Instead, functional CYP3A biomarkers may be more suitable for therapy individualization and further search for epigenetic factors modulating intermediate- and long-term CYP3A activity may be promising. However, the high heritability of 1-OH-midazolam might stimulate further research on the underlying genetic polymorphisms relevant for this metabolite.In a population not exposed to known inducers or inhibitors, heritability of CYP3A activity was found to be relatively small, particularly when using midazolam as biomarker. Considering the known very strong susceptibility of CYP3A4 and CYP3A5 to enzyme induction and enzyme inhibition, this finding may not appear unexpected. In our study, the estimates of CYP3A heritability varied depending on the biomarker used, which could partially be explained by the specific features of each probe substance, but it clearly indicates that the one and only correct The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethikkommission der Universitätsmedizin Göttingen, Georg-August-Universität Göttingen. The patients/participants provided their written informed consent to participate in this study.JM, WB, and JB wrote the manuscript. JB, DS, MS, and RK designed the research. JM, JB, MT, JS, DS, CS-S, and UH performed the research. WB, JB, JM, and JB analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eorubeta nevadensis in deposits of the Sheep Pass Formation, ranging from Late Cretaceous to Eocene, in the south Egan Range, Nevada. This formation represents a lacustrine basin within the Sevier retroarc hinterland. The formation is subdivided into six members (A–F); of interest here are members B and C. The base of member B is ?uppermost Cretaceous-Paleocene, while member C is Paleocene. Member B frogs are preserved in three taphonomic modes. Mode 1 frogs are nearly complete and accumulated under attritional processes, with frogs settling on microbial mats, as evidenced by crenulated fabric of entombing limestone. Mode 2 involves accumulation of frogs as a result of attritional processes. These frogs are mostly complete with some showing evidence of invertebrate scavenging. Possible scavengers are gastropods, ostracods, and decapods. Mode 3 is represented by isolated, reworked remains of frogs as a result of storm activity, supported by the association of elements with disarticulated bivalves and mud rip-up clasts. Member C preserves frogs in two taphonomic modes. Mode 4 are nearly complete frogs that accumulated during discrete mass mortality events. Numerous individuals are preserved along bedding planes in identical preservational states. Mode 5 is beds of frog bone hash, which represent increased energy to the depositional system (likely tempestites) and reworking of previously buried frog remains. Taphonomy of the frogs, along with the associated fauna and flora, are consistent with preservation in a cool, temperate lake basin, supporting previous interpretations that the Nevadaplano was an elevated plateau during the late Cretaceous through the Eocene. This is a period of time coincident with a climatic thermal optimum, thus the most parsimonious explanation for a temperate lake at the latitude of east-central Nevada is to invoke high elevation, which is consistent with independent structural and clumped stable isotope studies.Here we report on exceptional preservation of remains of the frog  Eorubeta nevadensis. Ancient high-elevation sedimentary packages are rarely preserved in the stratigraphic record due to intense erosional processes at high elevations over extended periods of time. In addition to the processes of erosion, the Sevier hinterland has been subjected to several episodes of Paleogene and Neogene extension and volcanism that have further disrupted the original continuity of synorogenic deposits of the Sheep Pass Formation  from the Sheep Pass Formation type section, many of which are mostly complete, as well as a number of laterally extensive frog bonebeds. Exceptional preservation in this case refers to common preservation of nearly complete individuals as well as the abundance of elements; no soft tissue preservation has been found as yet. Frogs are the only vertebrates identified within the Sheep Pass Formation type section to date . We have also documented potential decapods, trace fossils, and scattered plant remains, in association with previously documented ostracod and mollusk faunas. These discoveries present an interesting pattern of community structure and preservation within the Sevier hinterland during the latest Cretaceous and Paleogene. In this article we describe the taphonomic parameters of fossil material collected from the Sheep Pass Formation type section and interpret the preservational modes of the fossils. We also consider what this fossil material may reveal about the ecology and evolution of this long-lived, high-altitude lake basin.The Sevier orogen is typified by thin-skinned thrust faulting and resultant crustal thickening in the Sierra Nevada retroarc region, resulting from prolonged Jurassic to Paleogene eastward subduction of the oceanic Farallon plate beneath the western margin of North America . SynconvThe Sheep Pass Formation, first described by Nyctitherium within Paleocene to Eocene lacustrine carbonates of the Grant Range /He cooling ages of detrital zircons, with the upper portion of Member A also yielding two euhedral detrital zircons with respective U-Pb ages of 68 and 70 ± 1 Ma . The basFossil localities were discovered by prospecting exposures of the Sheep Pass Formation type section within the Sheep Pass Canyon area. The positions of fossil sites, including isolated elements, were plotted on a topographic map, their GPS coordinates recorded (these coordinates are on file at the respective repositories of the specimens), then specimens were taphonomically assessed before being collected  or later in the laboratory .in situ. In situ material was discovered by splitting the exposed mudstones with a rock hammer. Collected specimens are reposited at:Sedimentological data were recorded at each fossil locality to determine their depositional environments. Only surface collections were made; no excavations were conducted. Fossil material was noted as float or as Sierra College Natural History Museum, Rocklin, CA (SCNHM)Carnegie Museum of Natural History, Pittsburgh, PA (CM)Las Vegas Natural History Museum, Las Vegas, NV (LVNHM)Each fossil locality was placed within the stratigraphic framework of Taphonomic observations recorded included spatial distributions of specimens, degree of articulation and/or close association of disarticulated elements, presence of bone-on-bone contacts, types of bone breakages, and alteration halos. These taphonomic modifiers were revisited in the lab as well, and under light microscope. We obtained data on surface modification in the lab, after preparation of elements, because such features are often obscured by matrix . SurfaceMember B of the Sheep Pass Formation type section has produced a diverse assemblage of plants, invertebrates, and vertebrates. Ostracods are the most numerous invertebrates, and frogs are the only vertebrates recovered to date, with the exception of a toothless mammal jaw. In addition to body fossils, there are also some invertebrate traces preserved within Member B. Plant body fossils are present in isolated horizons throughout Member B. Fossils are found in at least three different lithofacies: dolomitic clayshales, dolomitic microbialites, and tempestites.There are at least four types of plant body impressions. The first type consists of portions or bracts of a larger organ that is 1 cm long and up to 1.5 mm in diameter. Perpendicular to the long axis are shorter appendages. The second type consists of long (up to 20 cm), 2–3 cm wide impressions, with parallel structures along the long axis. These impressions maintain their width from the base until they finally taper to a tip at their distal end . These aType 2 impressions are likely autochthonous and represent a period of time when the lake level was low enough for these plants to subsist in this portion of the basin. Lower and higher in the stratigraphic section the water was probably too deep for such plants. This interpretation is supported by the absence of fossil gymnosperms higher or lower in the section. Allochthonous macrofloral material in lakes rarely travels farther than 50 m from the source plant ; thus weThe scarcity of plant fossils does not allow for a more comprehensive picture of the plant life within the basin in which the Sheep Pass Formation type section was deposited. Little can be said about the plant record, as to what the surrounding foliage was like, nor can these fossils lend themselves to rigorous paleoclimatic analysis. The previous palynological study by Clinocypris?, sp.Paracypridopsis? sp., and Cypridea bicostata . Previous workers concluded that ostracods  and mollValvata, Hydrobia-Sphaeriidae association; along with the ostracod assemblage, these mollusk taxa support the interpretation of an open, alkaline-lacustrine setting (Other invertebrates preserved in Member B include occasional gastropods and numerous bivalves.  setting . Analogo setting .Bivalves are preserved in two taphonomic modes. The less common mode of preservation is as isolated, articulated specimens in planar-laminated, dolomitic limestone. These specimens are interpreted to be the result of attritional accumulation over time. The second, and more common, preservational mode is articulated and disarticulated valves found amongst irregular clasts of underlying clayshale strata in an otherwise silty, irregularly-bedded, dolomitic limestone. We interpret this preservational style to represent tempestite beds, with mudrock clasts being mud rip-up clasts. That, coupled with the dense accumulation of articulated and disarticulated specimens, suggests that at least some of the valves were remobilized. The irregular bedding, disarticulated valves, and mud rip-up clasts indicate that these beds represent periods of increased wave energy in the system rather than fluvial influxes, because there are no signs of sedimentary structures indicative of fluvial processes, such as clast imbrications or cross-laminae.Numerous frog specimens have been recovered from Member B of the Sheep Pass Formation within the Sheep Pass type section. These specimens are found stratigraphically throughout this member . All speFrogs are preserved in three taphonomic modes in Member B. Mode 1 includes individuals preserved as nearly complete skeletons only within those dolomitic mudstones with a crenulated-fabric (i.e., SCNHM VAF 3, Taphonomic Mode 2 is similar to the first in that specimens are typically nearly complete; the difference is that there are no signs of microbial mats, as indicated by the lack of the crenulated fabric sedimentary structures. This mode is hosted in a planar-laminated, dolomitic limestone and, unlike the first vertebrate taphonomic mode, these are the horizons that contain abundant ostracods (cf. SCNHM VAF 26 A, The final taphonomic mode of frog elements in Member B, Taphonomic Mode 3, is related to the taphonomic mode described for bivalves in the beds interpreted as tempestite horizons. These frog elements are typically found isolated and associated with disarticulated bivalve valves and mud rip-up clasts. The frog bones are interpreted as having been reworked from either the lake bottom or underlying sediments. There are numerous burrows preserved within the dolomitic mudrocks of Member B. One type consists of randomly sinuous, continuous trails , roughlyA number of frog specimens have been recovered near the top of Member C . In addiMember C differs from Member B in that invertebrate remains are found within the same lithofacies and with similar preservation as the vertebrates. Two taphonomic modes are observed. Taphonomic Mode 4 consists of frogs, ostracods, and bivalves occurring together within horizontally laminated, calcareous siltstones (cf. SCNHM VAF 4, The accumulation mechanism may be a result of either biotic or abiotic factors. If these were terrestrial frogs, then the accumulation could be attributed to the congregation of frogs in the lake to breed. Abiotic accumulation mechanisms could have included carcasses being blown by lake fetch e.g., , low-denTaphonomic Mode 5 is odd, in that frogs are the only preserved organisms and they are completely disarticulated; invertebrates are completely lacking (cf. SCNHM VAF 11, Actualistic studies of frog and toad carcasses in pond water show that frogs can begin disarticulating within 21 days, but they remain mostly articulated for up to 45 days . TherefoValvata, Hydrobia, Sphaeriridae in a tropical to subtropical realm does not exist. It was when Results of the taphonomic analysis complements previous studies , which iAlthough the frogs of taphonomic modes 1–4 are mostly complete, they show no signs of soft-tissue preservation, in contrast to other examples of frog-bearing lagerstatten. Although soft-tissue can be conserved for a number of years via microbial mat action , geologiA puzzling aspect of the paleontology of the type section of the Sheep Pass Formation is that no identifiable remains of any vertebrates other than frogs, with the exception of the partial mammal jaw, have been found, including aquatic and semi-aquatic groups such as fish, turtles, crocodilians, or waterfowl. The invertebrate fauna is considerably more diverse. One locality within the Sheep Pass Formation at Elderberry Canyon, which lies north of the type section, does produce a large and varied fauna of frogs, reptiles, birds and at least 30 different types of mammals . The mamEorubeta nevadensis. The majority of frog carcasses were preserved in an environment conducive to rapid settling to the lake bed without going through the bloat-and-float process. Accumulation processes differ between the two members in that frogs found in Member B are a result of attritional processes, whereas those in Member C were subjected to punctuated mortality events. These taphonomic modes (The Sheep Pass Formation type section, spanning the Maastrichtian to the Eocene, represents a highland, cool water, alkaline, lacustrine setting on the Nevadaplano of east-central Nevada. This section preserves an abundant invertebrate fauna of gastropods, bivalves, and crustaceans. It also preserves exceptionally abundant, well preserved, nearly complete, semi-articulated, and close associations of elements of numerous frog skeletons of the species ic modes  should b10.7717/peerj.9455/supp-1Data S1Click here for additional data file.