Amyloid-β (Aβ) has been investigated as a diagnostic biomarker and therapeutic drug target. Recent studies found that cerebrospinal fluid (CSF) Aβ fluctuates over time, including as a diurnal pattern, and increases in absolute concentration with serial collection. It is currently unknown what effect differences in CSF collection methodology have on Aβ variability. In this study, we sought to determine the effect of different collection methodologies on the stability of CSF Aβ concentrations over time.Grouped analysis of CSF Aβ levels from multiple industry and academic groups collected by either lumbar puncture (n=83) or indwelling lumbar catheter (n=178). Participants were either placebo or untreated subjects from clinical drug trials or observational studies. Participants had CSF collected by lumbar puncture or lumbar catheter for quantitation of Aβ concentration by enzyme linked immunosorbent assay. Data from all sponsors was converted to percent of the mean for Aβ40 and Aβ42 for comparison. Repeated measures analysis of variance was performed to assess for factors affecting the linear rise of Aβ concentrations over time.Analysis of studies collecting CSF via lumbar catheter revealed tremendous inter-subject variability of Aβ40 and Aβ42 as well as an Aβ diurnal pattern in all of the sponsors’ studies. In contrast, Aβ concentrations from CSF samples collected at two time points by lumbar puncture showed no significant differences. Repeated measures analysis of variance found that only time and draw frequency were significantly associated with the slope of linear rise in Aβ40 and Aβ42 concentrations during the first 6 hours of collection.Based on our findings, we recommend minimizing the frequency of CSF draws in studies measuring Aβ levels and keeping the frequency standardized between experimental groups. The Aβ diurnal pattern was noted in all sponsors’ studies and was not an artifact of study design. Averaging Aβ concentrations at each time point is recommended to minimize the effect of individual variability. Indwelling lumbar catheters are an invaluable research tool for following changes in CSF Aβ over 24-48 hours, but factors affecting Aβ concentration such as linear rise and diurnal variation need to be accounted for in planning study designs.The online version of this article (doi:10.1186/s13195-015-0136-z) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by degeneration of neurons and their synapses leading to progressive cognitive impairment. In the United States, AD is estimated to be the third leading cause of death  and to hPrevious studies measured Aβ concentrations before and after amyloid deposition to understand potential changes in Aβ metabolism during AD pathogenesis. In these studies, CSF was collected from individuals infrequently via lumbar puncture, generally limited to the beginning and the end of the study. The importance of understanding Aβ metabolism for pharmacologic modeling during trials of investigational compounds led to clinical studies collecting CSF samples repeatedly over 24–48 hours via an indwelling lumbar catheter. A number of these studies have demonstrated considerable intra-subject variability in the levels of CSF Aβ  and haveUnderstanding how different collection methodologies affect the stability of CSF Aβ levels over time is of paramount importance for the design of clinical trials, where these biomarkers would be utilized to study pharmacodynamic activity, and ultimately may determine whether Aβ has utility in a diagnostic fashion. Several investigational compounds that target Aβ are currently in clinical trials . In manyn = 178). Our results suggest that the slope of the linear rise in CSF Aβ levels is dependent on the sampling frequency, but does not affect the hourly variability manifesting as an Aβ diurnal pattern. The results from this study will help guide clinical trial design, and provide insight into the relative stability of proteins associated with AD in CSF.To identify the most relevant factors that may contribute to intra-subject variability in CSF Aβ levels, and as part of the Alzheimer’s Disease Neuroimaging Initiative (ADNI) consortia, we shared data from multiple clinical studies on CSF Aβ measurements from placebo or untreated subjects and conducted a meta-analysis to identify factors that may result in changes in CSF Aβ levels. The combined dataset is the largest collection of participants with serial CSF sampling via a lumbar catheter , AbbVie Inc. , Eli Lilly and Company , Merck and Company , Radboud University Medical Center , and Washington University . All of the samples were obtained via either lumbar puncture or serial sampling through an indwelling lumbar catheter. Samples were obtained from subjects who participated in clinical research studies. Only participants who were given placebo treatment in pharmacological trials were included in this study. Table The clinical phase of the study was conducted from December 2007 to March 2008 at the California Clinical Trials Medical Group . Healthy male subjects between 20 and 45 years of age were enrolled in the study. This study was conducted using Good Clinical Practice, as defined by the International Conference on Harmonization, and in compliance with the Declaration of Helsinki, and was approved by the Institutional Review Board of the California Clinical Trials Medical Group where the study was conducted. All subjects provided written informed consent prior to study entry.For repeated CSF sampling, a 19G Dura-Flex PLUS epidural lumbar catheter  was placed at the L3/L4 interspace of each subject approximately 3 hours before dosing. Four CSF samples of approximately 7–10 ml each were collected in Falcon polypropylene tubes  within 2 hours before placebo administration starting at approximately 7:00 a.m., and then at hourly intervals for up to 41 hours after dosing. The start time of CSF collection for all participants was approximately between 6:30 and 7:30 a.m. Samples of CSF were immediately frozen and stored at or below –80 °C until shipment to the analytical laboratory  on dry ice. Aβ peptide concentrations were determined using MS6000 human (6E10) Aβ 3-plex kits . Samples were analyzed for concentrations of Aβ38, Aβ40, and Aβ42. The between-run and within-run coefficient of variation (CV) for the analytical quality control checks (QCs) for Aβ38 and Aβ40 concentrations was <25 %, whereas the between-run CV for the analytical QCs for Aβ42 concentration was >30 %. QCs from Aβ42 were therefore only evaluated within run and accepted when the within-run CV was <20 %. All samples for a subject were analyzed in the same plate or on the same day to minimize the variability of Aβ42 assessments. In addition, CSF Aβ42 levels were measured using high-performance liquid chromatography (HPLC) as described previously [The three AbbVie studies were all conducted at PAREXEL/California Clinical Trials . All three studies were conducted in compliance with the Declaration of Helsinki and the International Conference on Harmonization for Good Clinical Practice guidelines. Institutional review board approval was obtained from PAREXEL for all three studies and written informed consent was obtained from all subjects in all three studies. All subjects were screened to be in good general health and without neurologic disease. A lumbar catheter was placed immediately before CSF sample collection started. The subjects were encouraged to stay in bed, and were allowed free choice of when to sleep throughout the study. Each study was conducted on a different group of subjects and each with a different CSF sampling procedure. There were no subject drop-outs or refusals. In Study A1, six healthy young men (25–47 years old) participated, and 5 ml CSF and 4 ml blood were collected from each subject at 0 (6:00 a.m.), 1, 3, 5, 6, 7, 8, 10, 12, 14, 18, 22, and 26 hours. In Study A2, five healthy older men, two healthy older women (61–74 years old), and 15 subjects diagnosed with AD (58–83 years old) participated. Seven milliliters of CSF and 10 ml blood were collected from each subject at 0 (5:30 a.m.), 1, 4, 8, 12, 18, and 24 hours. In Study A3, eight healthy young men (24–45 years old) participated and were divided into two groups. In one group of four subjects, 6 ml CSF and 4 ml blood were collected from each subject at 18 time points over 24 hours (“higher frequency” period) at 0 (8:00 a.m.), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, and 24 hours. Ten days later, CSF was collected from the same four subjects at seven time points over 24 hours (“lower frequency” period) at 0 (8:00 a.m.), 1, 4, 8, 12, 18, and 24 hours. In another group of four subjects, CSF was first collected at lower frequency, followed by higher frequency CSF collection 10 days later from the same subjects. During each CSF collection in these three studies, the first 2 ml (corresponding to the tubing dead space) were voided. CSF aliquots of 250 μl were frozen at –80 °C immediately after collection in 1 ml siliconized polypropylene tubes.CSF Aβ38, Aβ40, and Aβ42 concentrations were measured using human Aβ38/Aβ40/Aβ42 multiplex assays (using 6E10 as detection antibody) following the manufacturer’s procedure . To avoid inter-plate variation, all samples from each subject were measured together in duplicates on the same plate, and all of the samples from the same study were measured on the same day. To avoid bias due to intra-plate variation, all samples from each subject were randomized on the plate. The means of the intra-plate CVs for the duplicate samples including a control human CSF sample were <10 %. The means of the inter-plate CVs for the control human CSF sample were <10 %.Eight clinical studies involving CSF sampling for analysis of Aβ peptides were sponsored by Eli Lilly and Company. Details of collections are presented in Tables Lilly Study 1 was conducted at Indiana University  in compliance with the Declaration of Helsinki and the International Conference on Harmonization for Good Clinical Practice guidelines. Institutional review board approval was obtained from Indiana University and written informed consent was obtained from all subjects. The study was designed to define within-subject variability of CSF Aβ isoforms from two lumbar punctures taken 1–2 weeks apart from 10 AD patients and nine healthy, age-matched volunteers 45 years of age or older (ages ranged from 21 to 84 years old) in the absence of any treatment . Subjectwas conducted at the Lilly Clinical Research Unit  in compliance with the Declaration of Helsinki and the International Conference on Harmonization for Good Clinical Practice guidelines. Institutional review board approval was obtained from Indiana University and written informed consent was obtained from all subjects. The study was a single-blind, placebo-controlled, multiple-dose, dose-escalation study of LY450139 conducted in a combined inpatient/outpatient fashion [Lilly Study 2  fashion . Ten heaLilly Study 3 was approved by the institutional review board for each participating site . The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. The study was a multisite randomized, double-blind, placebo-controlled trial also involving LY450139 . Thirty-Lilly Study 4 was approved by the institutional review board for each participating site , San Diego, CA, USA; University of Pennsylvania, Philadelphia, PA, USA; Oregon Health and Science University, Portland, OR, USA; Indiana University, Indianapolis, IN, USA; Washington University, St Louis, MO, USA; University of Washington, Seattle, WA, USA; Georgetown University, Washington, DC, USA). The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. The study was a multisite, randomized, parallel-arm, placebo-controlled, dose-escalation trial of LY450139 in mild to moderate AD patients 50 years of age or older (mean ± SD = 69 ± 10) with a treatment duration of approximately 14 weeks . ThirteeLilly Study 5-A and Study 5-B were conducted at the Lilly Research Laboratories Clinic . Institutional review board approval was obtained from Indiana University. The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. Study 5-A and Study 5-B were two parts of a double-blind, placebo-controlled, single ascending dose study of LY450139 . In partLilly Study 6 was conducted at the Lilly Clinical Research Unit. Institutional review board approval was obtained from Indiana University. The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. The study was a placebo-controlled, single-dose, pharmacokinetic/pharmacodynamic study of LY450139 in mild to moderate AD patients and healthy volunteers . Results13C6-leucine) as described previously [Lilly Study 7 was conducted at Washington University. Institutional review board approval was obtained from Washington University. The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. The study was a parallel, double-blind, randomized comparison of the effects of single oral doses of placebo and LY450139 on the rate of Aβ formation in seven healthy male volunteers aged 21–50 years (mean ± SD = 34 ± 7) . CSF waseviously , 29. TheLilly Study 8 was conducted at PAREXEL International Early Phase . Institutional review board approval was obtained from the California Institutional Review Board. The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. The study was an investigator-blind, placebo-controlled, randomized, single-dose design study of LY2811376 in healthy subjects . Ten of . Coating of wells with capture monoclonal antibodies provided specificity for the C-terminal antigenic determinants of Aβ42 (antibody 21F12) and Aβ40 (antibody 2G3). The Aβ species recovered from CSF in the two assays were detected using a biotin-labeled monoclonal antibody raised to an Aβ N-terminal antigenic determinant (antibody 3D6). Concentrations of Aβ species were determined by measurement of biotin with horseradish peroxidase-labeled streptavidin and a colorimetric TMB substrate. The CSF Aβ42 ELISA provided a dynamic range of 20–250 pg/ml with intra-assay precision ranging from 2.67 to 3.06 (%CV), and inter-assay precision ranging from 3.61 to 5.28 (%CV). The CSF Aβ40 ELISA provided a dynamic range of 20–250 pg/ml with intra-assay precision ranging from 2.97 to 7.53 (%CV), and inter-assay precision ranging from 3.32 to 7.53 (%CV). CSF ELISAs determined during analysis of specimens collected from the clinical study were comparable with those observed during formal validation of the assays as listed above. For Lilly Study 4, Study 5, and Study 8, this resulted in slightly shifted and broader dynamic ranges corresponding to 25–400 pg/ml for both analytes. For Lilly Study 7, CSF Aβ42 and Aβ40 were measured at Washington University using minor modifications of the proprietary ELISAs methods described above.For Lilly Study 1, Study 2, Study 3 and Study 6, CSF samples were analyzed at Eli Lilly and Company  using proprietary 96-well plate-format enzyme-linked immunosorbent assays (ELISAs) developed and validated for measurement of Aβ42 and Aβ40The seven Merck studies were conducted at three sites, including Vanderbilt University  PAREXEL/California Clinical Trials  SGS Clinical Research . Institutional review board approval was obtained at each study site: Vanderbilt University, California Clinical Trials Medical Group, and SGS Clinical Research. The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. All participants were in good general health and without neurologic disease. After overnight fast, intra-dermal lidocaine anesthesia was applied prior to insertion of the spinal needle. Intravenous infusion of heparinized 5 % dextrose in water was performed to maintain hydration. A spinal needle was inserted through either the L2/L3 or the L3/L4 vertebral interspace, between 7:00 and 8:00 a.m. After entry into the subarachnoid space, the intrathecal catheter was secured externally with adhesive tape. In Studies 1 and 2, the catheter was extended with silicon tubing attached to a peristaltic pump with a continuous pump rate of 0.1 ml/minute for 24 hours in Study 1 and 0.5 ml/minute for the following 15 hours in Study 2. In Studies 3–7, a 5 ml syringe was attached to the intrathecal catheter and CSF was collected by gentle aspiration. The sampling volume and intervals are presented in Table CSF samples from both Study 1 and Study 2 were analyzed for Aβ40 using an ELISA kit from Biosource International  and for Aβ42 using an ELISA kit from Innogenetics NV . CSF samples from Studies 3–7 were analyzed for Aβ peptides using ELISA kits from Meso Scale Discovery .The subject information and methods for serial CSF collection have been described previously . InstituInstitutional review board approval was obtained from Washington University. The protocol conformed to the International Conference on Harmonization guidelines and to the Declaration of Helsinki. Written informed consent was obtained from all subjects. Fifty-seven subjects ranging from age 19 to 84 years were enrolled in the study. An intrathecal lumbar catheter and an intravenous catheter were placed between 7:30 and 9:00 a.m. and sample collection started between 8:00 and 9:30 a.m. for all participants. Six milliliters of CSF were obtained each hour for 36 hours. The CSF aliquots were frozen at −80 °C immediately after collection in 1.7 ml Axygen maximum-recovery polypropylene tubes . Participants were encouraged to stay in bed and were allowed free choice of when to sleep, read, watch television, use their laptops, or talk throughout the study. Analysis methods for Aβ40 and Aβ42 are as described previously .The data were combined for an integrated analysis of the lumbar sample studies and from the indwelling catheter studies. Because each study and research group had different methods of sample collection, volume collected, and ELISAs, the data were transformed to a percentage of the mean in order to minimize the effects of the various extraneous factors. The data transformation was computed separately for Aβ40 and Aβ42.It is well known that CSF amyloid concentrations are log-normally distributed  and all All statistical programming and analysis was conducted in SAS version 9.1 or higher . The main statistical method used was the mixed procedure in SAS.To identify factors relevant to the CSF Aβ increase, CSF Aβ levels taken within the first 6 hours were analyzed for each individual. To identify differences in clinical trial design that may have affected Aβ levels, the data from Washington University was not included in this analysis because the same study design was employed for all of their studies and was the largest dataset . Owing to multiple observations from each subject, a linear mixed model was used to analyze the data only from the industry studies. The subject-specific random effects allow the model to account for the within-subject correlation. The response was log-transformed. The model included age group, number of draws within the first 6 hours, source/companies, and time as covariates. Except for time, all covariates were categorical in nature. There were three age groups defined as “young” (<30 years old), “medium” (between the ages of 30 and 50), and “old” (50 years and older). Similarly, the number of draws within the first 6 hours were also categorized into three levels : group 1 had three draws or less in the first 6 hours, group 2 had greater than three but less than seven draws in the first 6 hours, and group 3 subjects had seven draws or more within the first 6 hours. The data originated from four different sources. Additionally, an interaction effect between time and number of draws was considered in the model.SAS 9.2 was used to analyze the data from the model specified above. During the analysis we appropriately choose a covariance structure. The unequal size of the response vector led us to select the SAS option “KenwardRoger” as a method for denominator degree of freedom. The interaction effect and the corresponding main effects came out as highly significant for both Aβ isoforms. The normality assumption was also verified through residual plots.The data consisted of 392 observations from 57 subjects. The Aβ levels for the first 6 hours were used for the analysis. Both of the response variables (Aβ40 and Aβ42) were log-transformed to obtain sufficient symmetry in their distribution. Two age groups were created, based on whether a subject was under or over 65 years of age. Owing to the data being longitudinal I, a linear mixed model was most appropriate for analysis. The subjects were used as the block under repeated statement of mixed procedure of SAS to account for the correlation of the observations from the same subjects. The model included the age group and hours after first draw (including the 0 hour). The covariance structure was also chosen appropriately. The normality assumption was verified through residual plots.All participants from all sponsored studies using indwelling lumbar catheters were including for cosinor analysis. For each time point, Aβ concentrations for both Aβ40 and Aβ42 over time were expressed as the percent of the mean for each individual participant. Average percent of the mean for Aβ40 and Aβ42 was calculated at each time point for all of each sponsor’s participants. Owing to variability in study designs within individual sponsors, the number of samples collected at each time point is unequal. Cosinor analysis was performed similar to that described previously  except ty-intercept, b, is equal to the mesor in the above equation.The cosine transformation was applied to the time variable using 24 hours as the default circadian cycle. Graphpad Prism version 6.0b for Mac  was used to estimate the parameters of the circadian patterns for Aβ40 and Aβ42. The mesor (midline of the oscillation), amplitude (distance between the peak and mesor), and acrophase (the time corresponding to the peak of the curve) were calculated for each group. Note, the Variability in the concentration of Aβ40 and Aβ42 was found in the untreated and placebo-treated subjects enrolled in 16 separate serial CSF sampling studies . Further, the 95 % confidence intervals for amplitude do not cross zero for both sets of studies  data from three additional sponsors: AbbVie, BMS, and Lilly Fig. . DespitePlease see Additional file Statistical analysis was performed to identify factors that were significantly associated with the change in increasing linear trend for both Aβ40 and Aβ42 over time. We analyzed the slope of the Aβ increase over the first 6 hours relative to the number of CSF draws made during that time period. Since the studies differed in duration, only the first 6 hours were used in the analysis. The increase appeared to be the most dramatic during this time period. Factors such as age group, gender, ethnicity, frequency of draws, sponsor, and time were analyzed. For this analysis, subjects were divided into three age groups defined as “young” (<30 years old), “medium” (between the ages of 30 and 50), and “old” (50 years and older). Similarly, the number of draws within the first 6 hours were also categorized into three levels : group 1 had three draws or less in the first 6 hours, group 2 had greater than three but less than seven draws in the first 6 hours, and group 3 subjects had seven draws or more within the first 6 hours.Of these factors, only the frequency of the CSF draw, time, and the interaction of draw frequency and time were significant Table . The repp = 0.02); however, for Aβ40 there was no significant difference between the age groups in terms of the change in Aβ levels in the first 6 hours . Again, this difference between age groups and Aβ isoforms may be due to amyloid deposition, a factor not included in this analysis.Age was not a significant factor for the combined data. In the industry studies, however, only 15 out of the 107 subjects (minus the RUMC data) were older than 65 years. In contrast, 36 out of 57 subjects in the Washington University dataset were older than 65 years. Analysis of this dataset alone showed a trend towards an effect of age on the rise in Aβ levels in the first 6 hours. Younger subjects showed a greater change in Aβ42 levels over time relative to older subjects (p = 0.54 and p = 0.14 respectively) .For untreated or placebo-treated subjects enrolled in four separate clinical studies, intra-subject variability for CSF Aβ40 and Aβ42 collected by lumbar puncture were generally stable on comparison between baseline and endpoint sampling scheduled approximately 2 weeks apart. Occasionally, individuals exhibited notable shifts from baseline (increase or decrease); however, the majority of subjects exhibited stable results. Within individual studies, lumbar punctures were performed at approximately the same time of day. Overall, no significance difference between the endpoint and baseline for Aβ42 and Aβ40 was observed (ly) Fig. . In addiWe examined CSF Aβ40 and Aβ42 concentrations across multiple studies from four different companies and two academic institutions. While a tremendous amount of inter-individual variability existed, a general pattern of rapidly increasing concentrations of Aβ40 and Aβ42 in the first 12 hours of a study was found across all subjects in all indwelling lumbar catheter studies; in some cases, Aβ40 and Aβ42 reached levels as high as 200 % from the initial baseline draw. In most of the subjects, the slope of the linear rise decreased after the first 24 hours, but the levels did not return to baseline by 24 hours. Our results suggest that the use of indwelling catheters to collect multiple CSF samples results in an upward drift in the levels of both Aβ40 and Aβ42 relative to the initial draw. In contrast, the levels of Aβ40 and Aβ42 did not change significantly from baseline to second draw when samples were taken by lumbar puncture performed at a similar time of the day 2 weeks apart.The tremendous inter-subject variability of Aβ40 and Aβ42 seen over time could influence the results of AD prevention studies that target Aβ. Averaging Aβ concentrations at each time point is recommended to minimize the effect of individual variability and distinguish effects between experimental groups. We further recommend managing this variability by calculating the area under the curve using pharmacokinetic–pharmacodynamic models. For example, a previous study of a gamma-secretase inhibitor was able to show relatively small effects on Aβ in CSF collected via indwelling lumbar catheters using this technique .A circadian rhythm is a cyclic oscillation intrinsic to many biologic processes, such as body temperature and hormone levels, and is typically studied using cosinor analysis to fit a cosine transformation with a 24-hour period. Aβ concentrations have been found to fluctuate over time in several studies collecting CSF via a lumbar catheter , 9. Furtp = 0.06). This discrepancy is attributable to both the scatter of the data points in the first and last 6 hours of sampling due to CSF collection occurring at slightly different times of day across the AbbVie studies and a limited sampling time of 24 hours, the shortest period among all of the sponsors. Prior work analyzed a smaller group of subjects from the AbbVie studies  and reported that intra-subject variability was related to sampling frequency and/or sampling volume [n = 6–8/study) and not as a larger pooled analysis.Despite heterogeneity in the frequency of CSF collection in this study, we found evidence in data from all sponsors for a diurnal pattern of both Aβ40 and Aβ42. The only exception was the AbbVie data for Aβ42 which showed a mesor-to-peak amplitude of 4.29 % with 95 % confidence intervals that do not cross zero (0.71–7.87) suggesting a cosinor fit, but only a trend toward a difference with a straight line was noted . BMS, however, peaked earlier for both Aβ40 and Aβ42 at approximately 8 hours after collection started (14:00 p.m.). In contrast, Aβ40 and Aβ42 peaked at approximately 14 hours after collection started in the Lilly studies (16:00 p.m.) and at approximately 16–17 hours after collection started in the AbbVie studies (21:00–22:00 p.m.).These discrepancies in the amplitudes and peak time of CSF Aβ fluctuation are possibly due to differences in participant ages, sample collection schedules, analytic techniques used between sites, participant stress levels, participant amyloid status, and differences in sleep–wake activity between participants in the different groups. Our findings suggest that the Aβ diurnal pattern is not a timekeeper that directly interacts with the circadian clock, but is dependent on human processes with a diurnal pattern—such as changes in neuronal activity with the sleep–wake cycle as described previously , 33—and Since the studies enrolled subjects of different demographics and different protocols were employed for CSF collection across the studies, statistical analysis was conducted to identify factors that were associated with the rise in Aβ levels. Factors such as age, gender, clinical sponsor, and frequency of CSF draws were all analyzed. In the industry-sponsored studies, where considerable variability existed in the CSF collection designs, the frequency of CSF draws was significantly associated with the Aβ rise. The results indicated that performing more than seven CSF draws in the first 6 hours of the study had a large effect on the rise in CSF Aβ, while the rise in CSF Aβ was largely not evident in studies that collected six or less draws in the first 6 hours. This finding is similar to previous reports from AbbVie that sampling frequency was a factor in the Aβ linear rise . Severaln = 6; Table The Aβ linear rise is not a diurnal effect and is most probably a consequence of frequent CSF draws via an indwelling lumbar catheter leading to a shift in the rostrocaudal dynamics in CSF. A recent study showed that levels of Aβ were higher in CSF collected from the cisterna in patients suffering from trigeminal neuralgia relative to CSF from lumbar draws taken from patients suffering from tension-type headaches . PotentiThe studies conducted at Washington University in St Louis enrolled subjects in different age groups. In these studies, there was a trend towards age being a significant factor for the rise in CSF Aβ levels, with younger subjects showing significantly higher increases relative to older subjects for Aβ42. Previous studies examining age and the rise in CSF Aβ levels did not show an effect in amyloid-negative subjects . HoweverThe issue concerning whether or not significant fluctuations in CSF Aβ concentrations occur owing to CSF collection methodology is of paramount importance for the design of clinical trials where these biomarkers would be utilized to study pharmacodynamic activity, and ultimately may determine whether these biomarkers have utility in a diagnostic fashion. Further, the use of a filter system in the sampling setup employed by RUMC illustrates that the selection of sampling materials and the configuration of those materials on the recovery of Aβ species from CSF pulled through catheter systems needs to be carefully evaluated. Overall, our analysis has identified that frequency of CSF draws and age are factors that contribute to the linear rise in Aβ levels. The Aβ diurnal pattern, in contrast, is suggested to be an intrinsic process that occurs independent of CSF collection methodology. These findings are important for the design of studies of Aβ. For instance, a recent trial investigated whether sleep deprivation resulted in an increase in CSF Aβ concentration . The stuFrom our analysis, it is recommended that the frequency of CSF draws should kept to no more than 1/hour with a volume of 6–8 ml in order to minimize changes in CSF Aβ with frequent draws, particularly in the first 6 hours of collection; also, it is critical that a standard draw frequency be utilized across all study groups. Our data are not sufficient to determine whether these are the optimal CSF draw volume and frequency to control for the linear rise. Further, the linear rise needs to be accounted for during analysis of Aβ levels in CSF collected by indwelling lumbar catheters either by fitting both a cosine function and linear rise simultaneously as we have done, or by subtracting out the linear rise before cosinor analysis as reported previously .Indwelling lumbar catheters are an invaluable research tool for following changes in CSF Aβ over 24–48 hours, but factors affecting Aβ concentration such as linear rise and diurnal variation need to be accounted for in planning study designs.


Serum troponin is a useful laboratory study for the diagnosis of acute myocardial infarction. However, elevations can also be seen in a variety of other diseases processes. Falsely positive troponin values caused by interference with current troponin assays have been reported. We report a unique case that demonstrates the fluctuation of falsely elevated troponin correlating with hemoglobin, serving as a marker of heterophile antibody levels. A 74-year-old gentleman presented to our Emergency Department with a several-day history of increasing shortness of breath associated with a new-onset chest pain and a troponin I level of 77.28 ng/mL. Throughout his stay, fluctuations in measured troponin levels correlated strongly with fluctuations in hemoglobin levels. Several investigations confirmed false elevated troponin levels secondary to heterophile antibody interference. We conclude that hemoglobin trending in our patient represented a surrogate measure of his heterophile antibody titers with time and that fluctuations in these levels correlated with respective fluctuations in the falsely elevated troponin levels. Cardiac troponins T (cTnT) and I (cTnI) are regulatory proteins that control the calcium-mediated interaction between actin and myosin . The ske Relying on history, physical examination, and ECG abnormalities to diagnose acute myocardial infarction may often lead the clinician astray. Thus, the diagnosis of an acute myocardial infarction has become increasingly dependent upon the evaluation of cardiac enzymes, particularly cardiac troponins , 3. In addition to acute myocardial infarction, elevated serum troponins can also be seen in a variety of other diseases including sepsis or critical illness, tachycardia, LVH, heart failure, pulmonary embolism, myocarditis, myocardial trauma, and renal failure , 5. Alth Falsely elevated troponin values caused by interference with current troponin assays have been reported. We report a unique case that demonstrates the fluctuation of falsely elevated troponin correlating with hemoglobin, serving as a marker of heterophile antibody levels –17. A 74-year-old man, a retired railroad conductor, with a history of hypertension, hyperlipidemia, and noninsulin-dependent diabetes mellitus presented to our Emergency Department with a several-day history of increasing shortness of breath associated with a new-onset chest pain. ECG performed in the Emergency Department showed a right bundle branch block, left ventricular hypertrophy, and left atrial enlargement. Troponin I was elevated at 77.28 ng/mL.  The remainder of the cardiac enzymes were essentially normal: myoglobin (50 ng/mL), CK-MB (5.2 ng/mL), and creatine kinase (74 IU/L). D-Dimer was normal at 0.33 mcg/mL. BUN was 13 mg/dL and creatinine was normal at 0.65 mg/dL (reference range 0.64–1.27). Standard Acute Coronary Syndrome protocol was initiated, and the patient was admitted to the hospital. Transthoracic echocardiography showed left ventricular hypertrophy, mild diastolic dysfunction, and a normal ejection fraction with no evidence of wall motion abnormality. Troponin levels remained elevated throughout the entire hospitalization, in continued disproportion to the other cardiac enzymes,  which remained either normal or very mildly elevated throughout. His hospital course was complicated by bibasilar pneumonia and atelectasis requiring multiple bronchoscopies, intubation with ventilatory support, and ultimately a tracheostomy; bilateral lower extremity deep venous thromboses, for which he underwent successful inferior vena cava filter placement; a progressive thrombocytopenia which proved to be Heparin-Induced Thrombocytopenia-antibody positive; and finally, a life-threatening bleeding duodenal ulcer. A complete blood count trend analysis revealed a significant two-week down-trending of the patient's hemoglobin and hematocrit values; retrospectively a result of the slowly bleeding ulcer. Troponin levels was obtained serially throughout.  As the duodenal ulcer proved to be incendiary, and refractory to temporizing measures, the patient ultimately developed hemorrhagic shock and was taken emergently to the operating room where he underwent an exploratory laparotomy with pyloroplasty and successful suture ligation of the ulcer. In addition to extensive resuscitation with normal saline, the patient had required a total of 18 units of PRBC. 12 of these units were given within 24 hrs. Intraoperatively, the patient received 5 units of PRBC, 10 units of FFP, and 5 units of platelets. A patient's blood sample was sent out for a troponin I level on two separate occasions to a near-by hospital laboratory that used the Siemens ADVIA Centaur Immunoassay as opposed to the Beckman-Coulter Access AccuTnl Assay used by our hospital. Both times the result returned at <0.01 ng/mL. Additionally, a random troponin T level measured by Associated Regional and University Pathologists Laboratories using the Electrochemiluminescent Immunoassay was only 0.13 ng/mL. A heterophile antibody by latex agglutination was also sent out to ARUP Laboratories and returned negative. However, since this screen only tests for those heterophile antibodies known to develop in response to infection with Epstein-Barr Virus, this test was of limited value. Rheumatoid factor, which also may induce heterophilic antibodies , was als We contacted Beckman-Coulter and request a formal investigation for Heterophile antibodies for our patient. The following blocking reagents, produced by Scantibodies Laboratory, were introduced into the patient's serum and again run through the immunoassay: Goat IgG, Mouse IgG, Rabbit IgG, Bovine IgG, Poly Mak 33, Scavenger ALP, AP Mutein, HBR-1, HBR-Non Murine, and TRU Block . A blocking reagent is a preparation which, when added to immunoassay reagents, has the ability to block the binding of heterophile antibodies, effectively preventing heterophilic interference with another measured value. This is manifested by normalization or near-normalization of the measured value (in this case troponin I) after introduction of the blocking reagent. A blocking reagent can be either a specific blocking reagent, meaning that it has the ability to block only one specific heterophile antibody, or a nonspecific blocking reagent, meaning that it is capable of block heterophile antibodies in general, “nonspecifically” . The first 7 reagents listed previously are specific heterophile antibody blocking reagents. The last 3 are nonspecific heterophile antibody blocking reagents. 9 out of the 10 blockers had no effect on the resulting troponin I level. However, the addition of HBR-1, a nonspecific blocking reagent, reduced the troponin result by greater than 90% of the original value, indicating successful blockage of our patient's heterophile antibodies, drastically diminishing the spurious elevation, with near-normalization of the level. We suspected heterophile antibody interference in our patient for several reasons. The troponin elevation could not be explained by acute myocardial damage. There was no evidence of wall motion abnormalities, there was no clinical suggestion of myocarditis, the degree of LVH was moderate at best, and the degree of troponin elevation was inappropriate and inconsistent with what would be expected for any of these pathologies. Also, the troponin elevations were in extreme disproportion to the remainder of the cardiac enzymes, and the typical pattern of decay known of the troponin was not demonstrated. Finally, the presence of immune interference was suspected due to the dramatic fluctuations in troponin levels correlating with blood loss, blood transfusions (replacements), and overall volume status. The term “heterophile antibodies” has historically been used in reference to specific heterophile antibodies, produced by the human immune system in response to Epstein-Barr virus infection. These specific heterophile antibodies react to antigens from phylogenetically unrelated species. They agglutinate sheep erythrocytes, used in the classic Paul-Bunnell test, horse erythrocytes, used in the “Monospot” test, as well as ox and goat erythrocytes . Probably a better term, antianimal antibodies, encompasses the many different human heterophile antibodies that have the ability to bind to immunoglobulins of many different animal species. It is estimated that 10–40% of humans possess antianimal antibodies. “Anti-animal antibodies  arise as a result of iatrogenic and noniatrogenic causes and include human anti-mouse, -rabbit, -goat, -sheep, -cow, -pig, -rat, and -horse antibodies and antibodies with mixed specificity. Circulating antibodies can reach gram per liter concentrations and may persist for years” . Reagents used in contemporary troponin immunoassays are derived from immunoglobulins of other species. These antianimal antibodies, or “heterophile antibodies”, have the capability to interfere with the assays by simply binding these immunoglobulins, resulting in spurious troponin elevations. Even the newer ultrasensitive three-site sandwich troponin I immunoassays have been found to produce similar false positive results . The second troponin drop produced a glaring, abrupt dip on the troponin curve, as this drop was from 35.23 ng/mL to 11.20 ng/mL (a net of −24.03 ng/mL) over a period of only less than 32 hours. This highly precipitous decrease in troponin was the result of the patient's extensive upper gastrointestinal bleeding, copious fluid resuscitation, and massive blood transfusions described previously, which served to purge and dilute his heterophile antibodies, again mitigating interference with the troponin assay. Furthermore, according to our theory it follows that his troponin levels should gradually but steadily climb back up, once given an opportunity for his antibody levels to build back up to his baseline. Looking at the graph we can see that this trend too was demonstrated.Since the in vitro addition of HBR-1 to the patient's serum reduced the troponin result by greater than 90% of the original value, we know that the patient does indeed posses heterophile antibodies that interfere with the Beckman-Coulter Access AccuTnl Assay. Since HBR-1 is a nonspecific heterophile antibody blocker, it remains unknown what the specific heterophile antibody(ies) are. Several sources have been implicated as possible causes for inducing heterophile antibodies in humans, including exposure to animals, special diets, deliberate immunization, rheumatoid factors, blood transfusions, autoimmune diseases, dialysis, certain medications, and cardiac myopathy . The eli We conclude that hemoglobin trending in our patient represented a surrogate measure of his heterophile antibody titers with time and that fluctuations in these levels correlated very strongly with respective fluctuations in the falsely elevated troponin levels.


Populus sp.) and willows  are well known woody plants common throughout the northern hemisphere, both with fully sequenced genomes. They bear compact unisexual inflorescences known as “catkins.” Closely related genera of the “salicoid clade” within the family Salicaceae include the Asian genera Bennettiodendron, Idesia, Itoa, Poliothyrsis, and Carrierea and the Central American genera Olmediella and Macrohasseltia. Like willow and poplar, most of these genera are dioecious, but unlike willow and poplar they generally have loosely branched panicles rather than catkins, and less highly reduced flowers. However, the early developing inflorescences of Carrierea and Idesia show similarities to catkins which suggest possible pathways by which the salicoid catkin may have evolved.Poplars ( The catkin is a type of compact or string-like inflorescence characterized by a single relatively stout axis on which unisexual sessile or subsessile apetalous flowers are clustered in a spiral or whorled arrangement. It is an extremely striking characteristic of many common trees, particularly of northern temperate regions. Notable among these are members of the order Fagales  and the relatively distantly related family Salicaceae s. str. (willows and poplars). The similarities between the catkins of these two groups led to them being classified together for a century (see below). It is now accepted that the presence of catkins in the two groups is the result of convergent evolution.amentum, the Latin word for a thong or string, is less common. Its use in English dates from the late 18th century, sometimes anglicized as ament. The use of amentum in botanical Latin overlooks the Latin word for catkin, iulus, used as such by Pliny. However, apart from the occasional use of Juliflorae instead of Amentiflorae, this form never became established.In this paper ‘catkin’ will be used in preference to the alternative term ‘ament.’ According to the Oxford English Dictionary, the word catkin came into English in 1578 when Henry Lyte (1529-1607) coined it in his translation of Dodoens’ New Herbal as a translation of the Dutch “katteken” (kitten) used for the downy inflorescences of willows and other plants . The botLeitneria, Garryales . Remarkably, Rhoiptelea and Ticodendron, both unknown to Eichler.The striking amentaceous inflorescences of many trees quickly attracted the attention of botanists, some of whom thought that the catkin-bearing trees formed a natural group . This realization was confirmed by molecular phylogenetics and made clear in systems based on molecular phylogenetics such as the Angiosperm Phylogeny Group (APG) system  comprising only the genera rtiaceae . The hetrtiaceae , and it Salix and Populus are closely related sister genera which in turn are related to a group of seven other genera . Sister to that clade is a clade of the other four genera, Carrierea, Itoa, Macrohasseltia, and Poliothyrsis”  reduction events to n = 19.These nine genera have been referred to as the “salicoid clade” of the family Salicaceae . Salix atraploid  with a p . A reduction of inflorescence branching (from panicle to raceme) does occur as a sexual dimorphism in Itoa, in which the male flowers are said to be in racemes and the female flowers in panicles (Table 1). Furthermore, in Idesia the paniculate inflorescences are long, pendulous and fairly narrow, superficially resembling racemes .The genera of the salicoid clade have been described in various recent treatments . They arPseudosalix†  but somewhat paniculate inflorescences. Furthermore, the Eocene Populus tidwellii† Manchester, Judd & Handley  terminating the shoots that appear after bud-break of the terminal bud. This condition contrasts with Salix and Populus, in which the inflorescences are produced in lateral buds . Again, P. tidwellii†  and a vestigial ovary . The pistillate flowers have numerous small staminodes . The presence of vestigial sexual organs in most species but their complete absence in Salix and Populus indicates the extent of the process of floral reduction in those genera. However, it is not known whether the developmental pathways for vestigial pistils in males  has been completely removed as part of sex determination or merely reduced to the extent it no longer has anatomical consequence.All genera have a calyx of (3-)5(-6) sepals, except caducous . The staTable 1). These glands are generally assumed to be outgrowths of the disk. However, they are consistently associated with the stamens and staminodes, appearing interspersed among the stamens , or at the bases of the stamens in Olmediella  of incingly ,b that thomology .Populus. Fisher homologised this structure with perianth and with the disk glands of Salix: “The disk-shaped perianth of Populus, or its peripheral parts, is homologous with the nectary of Salix”  nectariferous glands, which occasionally are connate into a lobed glandular disk. These glands are obviously homologous to the cup-shaped disk in the poplars .” However, the cupular disk of Populus is vascularized, consistent with Fisher’s thesis that the disk glands of Salix have a perianth origin, assuming the two to have a common origin. However, it is also possible that as non-perianth disk glands evolved to increased complexity, vascularization was co-opted. Another possibility is that the cupular disk of Populus is indeed directly homologous with the calyx but not with the glands of Salix (which then have a non-perianth origin). Finally, the simplest explanation of all is that the cupular disk is merely an enlarged disk .Another puzzle is the “cupular disk” of f Salix” . SkvortsSalix and Populus differ, in reproductive morphology and phenology, from their close relatives. Knowledge of the inflorescence morphology and flowering behavior of related plants allows the formulation of scenarios by which catkins evolved in this clade.In this paper we seek to investigate whether the morphology of closely related non-catkin-bearing species can inform our understanding of the evolution of catkins in the Salicaceae. In particular we are interested in setting out the main ways in which Idesia, Carrierea, and Poliothyrsis, terminal resting buds were collected in spring before bud-break. In Salix and Populus lateral inflorescence buds were collected at the same time. A young inflorescence of Olmediella was collected at the same time from greenhouse-grown material. After budbreak young inflorescence shoots were also examined. A list of samples collected with accession numbers is given in Table 2.For Collections of inflorescence material were killed and fixed in formalin–acetic acid–alcohol (FAA) for approximately 1 week followed by storage in 70% ethanol. Some material was dehydrated through an ethanol series to 100% ethanol, transferred to Histoclear before embedding in Paraplast using standard protocols. The wax blocks were sectioned at 10 μm thickness on a rotary microtome (Leica RM2155) and the resulting sections were stained in 0.5% (w/v) solution of toluidine blue before mounting on microscope slides in DPX mountant. Images were captured using a Zeiss Axiocam HRc camera attached to a Leica DMLB microscope.Other material was dehydrated through an alcohol series into acetone and transferred to a critical-point drier (Tousimis Autosamdri 815B). Dried material was then sputter-coated with platinum in a sputter coater (Emitech K550). The material was examined on a Hitachi S-4700 II cold-field emission scanning electron microscope.Poliothyrsis, Idesia, and Carrierea set comparatively large terminal buds on all shoots of the previous year. The majority of these buds produce short shoots terminating in an inflorescence . Vegetative growth (and flowers of the following year) is therefore left to side shoots. This growth pattern corresponds with the “Modèle de Leeuwenberg” of Hallé and Oldeman  tending to contain catkins. In Poliothyrsis, nearly all growth is by terminal buds from side shoots of the previous year, these in turn terminate in inflorescences with between four and six leaves below then. In Carrierea there tend to be four to six leaves below each inflorescence and in Idesia four to five.In autumn  Oldeman . PopulusCarrierea  and Idesia  show very little development of the inflorescence when collected in February. In contrast, Populus and Salix  have fully formed flowers. The preformation and early development of inflorescences in Populus and Salix is well known, with inflorescences formed the previous year . In contrast, developmental timing in Idesia was found to be very different. No identifiable inflorescence meristems were found in buds sampled in February, although well-developed leaf primordia were present . Resampling in April revealed dramatic differences. A well-developed inflorescence meristem was found to be present, but no developed flowers . We conclude that inflorescence development in Idesia occurs in response to warming temperatures in the spring, although much of it is completed within the closed bud before bud break in May. At an early stage, the inflorescence meristem resembles a catkin in having numerous spirally arranged bracts and primordia on an axis. However, these primordia will develop into inflorescence branches and not individual flowers.ous year . FiguresSalix  is compared with its outgroup genera, for instance Idesia (Table 3), there are several traits that are shared. Our comparative investigation indicates that these apparently pre-existing shared traits  likely pre-date the evolution of catkins rather than being a consequence of the evolution of catkins. In contrast to the monoecious Fagales, where typically male and female catkins occur on the same tree, dioecy is universal in the catkin-bearing Salicaceae and their immediate relatives. Although bisexual teratomorphs are sometimes found, bisexual species in the catkin-bearing Salicaceae are exceedingly rare, although they do occur as a derived condition . Inflorescence development may start in the bud  but completes on the growing shoot . Preformation is obviously a necessary precondition for precocity (below), and precocity may be what is driving full preformation. Preformation in resting buds involves a paradox. In a normal bud there is no leaf production and shoot growth has ceased. The bud is in developmental stasis, at least vegetatively, in preparation for full dormancy. On the other hand with floral preformation there is much reproductive development in the bud with the formation of an inflorescence meristem and floral primordia and their development into flowers. The evolution of preformation implies increased developmental control in coupling two phase changes of the meristem: change from active growth to dormancy and change from vegetative to reproductive. In Idesia these phase changes seem to occur sequentially, first dormancy then inflorescence formation. In poplar and willow they appear to be coupled.This is the formation of structures a long time before they become visible or functional. In Salix as a whole shows a great deal of variation in this trait. Some are what salicologists call “coetaneous” (an archaic word for contemporaneous), meaning that the catkins are produced at the same time (contemporaneously) with the leaves. More precisely this means that the catkins are not sessile on the previous years growth but on, bud-break, a leafy shoot is produced which the catkin terminates. In this respect it is directly analogous with Idesia, in which the inflorescence terminates a short leafy shoot. A third type of reproductive behavior, “serotiny” (from the latin serotinus = coming late), also occurs in willows. This is an extreme form of the coetaneous habit. In botany serotiny is more commonly applied to delayed seed dispersal, but in willows it refers to delayed flowering. This occurs when catkins are poorly developed in bud and complete their development post-budbreak, thus apparently flowering with the current season’s growth. Although formal analyses are lacking , it is likely that coetaneous and serotinous willows are derived and may represent reversals. However, if the coetaneous habit is found to be primitive in Salix it provides a link to other genera of the salicoid clade.The term precocity is here applied to the breaking of reproductive buds before the vegetative buds to allow flowering before the development of a canopy. It should be noted that precocity as discussed here is seasonal precocity, not flowering as a juvenile . Seasonal precocity implies bud dimorphism (below). Although most willows are strictly precocious, the genus Salix and Populus. For instance Idesia bears its inflorescences on leafy shoots whereas the catkins of Populus are not associated with vegetative leaves. Evolutionary loss at the end of a shoot may simply be the consequence of growth ceasing early, while evolutionary gain at the tip may be the result of growth continuing for a longer period. Loss (in this case of leaves) at the base of a shoot is more problematic. It requires that a late developmental program (in this case inflorescence production) is brought forward to replace early developmental programs . The concepts of terminal and non-terminal deletion have been used in evolutionary analyses of other botanical systems, including of fossils  required for development. Bud dimorphism allows a marked “division of labor” between reproductive and vegetative meristems. In poplar and willow the catkin usually has no vegetative function whatsoever, and correspondingly the vegetative shoot has no reproductive function. In Idesia the distinction is blurred. Almost all shoots produce not only a terminal inflorescence but also numerous leaves below the inflorescence. Thus reproductive and vegetative functions are carried out by the same buds (repro-vegetative buds).In Idesia, with primordia and associated bract primordia  gives a possible scenario of how the catkin could have evolved. The primordia would normally develop into panicle branches and then into flowers. If the floral developmental pathway were to be brought forward in developmental time then it is possible to see how the result would be a series of bract associated flowers. This would be an example of heterochrony: a change in developmental timing  in willow. For nearly a century this question has been considered closed with the consensus that the disk glands of willow and the cupular disk of poplar represent the lost perianth. However, the recent identification of the relatives of Salix and Populus followed by the realization that they have both disk glands and a calyx have cast some doubt on this consensus . In the wind-pollinated Populus, large numbers of stamens have been retained, packed very tightly into the flowers in bud. This illustrates the constraint that the more pollen-wasteful process of wind pollination places on floral reduction in poplar.Small flowers are an obvious consequence of the evolution of the catkin as there is no space for elaborate flowers in a highly condensed inflorescence. Additionally some of the functions of individual flowers are, in the catkin, taken over by the inflorescence as a whole. An example is floral protection in bud which is done by the calyx in onsensus . In addiIdesia, the terminal buds on shoots produce inflorescences and the inflorescence terminates shoot growth. In poplar, terminal buds are never (or at least very rarely) reproductive. The catkin buds are all lateral (axillary) buds. The determinate growth of all terminal buds puts a constraint on the rate of height growth that can be attained by Idesia and its relatives which tend to be relatively small trees. Poplar, however, because its inflorescences are lateral, can maintain indeterminate growth, resulting in poplars being generally the fastest growing and tallest dicotyledonous trees in the northern hemisphere. Catkins are also lateral in willow, but in willow the terminal shoot tends to abort rather than form a resting bud for continued growth the following year, hence willows also tend to be smaller in stature. In the Juglandaceae, lateralization of the catkins is only partial, as while staminate catkins are general lateral, pistillate catkins are usually terminal  as it allows flowers to be pollinated before the emergence of the leafy canopy which may attenuate air movement among the branches. For insect-pollinated plants, precocity effectively removes competition for bees from other flowers. Willows  are generally insect pollinated (Andrena (Apis mellifera L.) early in the year when bees have few other food sources. However, a trade-off against the absence of competition for pollinators, is the fact that there may be fewer bees flying in early months of the year. This mechanism, of course, applies only to temperate regions with a pronounced cold season, and is not applicable to catkin-like inflorescences of tropical origin such as the related Lacistemataceae.The compactness of the catkin allows inflorescence development to be completed within the inflorescence bud. This in turn allows for precocious flowering. Precocity has an obvious consequence for wind-pollinated plants  genome was the third plant genome to be released  is a good example, in which a compact or lax infructescence (caused by variation of rachis length) is a characteristic of commercial importance  genes  genes .Salix and Populus will greatly facilitate the development of genomic resources for other genera in the salicoid clade. A complete genome of Idesia would be particularly valuable as an outgroup for Salix and Populus. Similarly, a member of the Salicaceae that is more distant (such as Azara) would be useful as an outgroup for the salicoid clade as a whole. Azara is outside the palaeopolyploidy event that has occurred in the salicoid clade, it would therefore be particularly useful to assess evolution of gene paralogues in Salix and Populus resulting from the whole genome duplication event.The rich genomic resources developing for The morphological richness of the Salicaceae coupled with the rapidly expanding genomic resources make this family, of all woody plant families, particularly promising for genome-enabled evolutionary developmental biology.QC and PR planned the study, collected material, supervised the anatomical work and wrote the paper. IN carried out the anatomical work and photomicrography and contributed to writing the paper.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.


N,N-dialkylsulfonamides, and reductive hydrodecyanation of malonitriles.A new ground-state organic electron donor has been prepared that features four strongly π-donating iminophosphorano substituents on a bispyridinylidene skeleton. Cyclic voltammetry reveals a record redox potential of −1.70 V vs. saturated calomel electrode (SCE) for the couple involving the neutral organic donor and its dication. This highly reducing organic compound can be isolated (44 %) or more conveniently generated in situ by a deprotonation reaction involving its readily prepared pyridinium ion precursor. This donor is able to reduce a variety of aryl halides, and, owing to its redox potential, was found to be the first organic donor to be effective in the thermally induced reductive S–N bond cleavage of  A (E1/2=−1.20 V vs. SCE) and Ba (E1/2=−1.24 V vs. SCE), have emerged as exciting new reagents in organic synthesis  and −1.51 V (Cc) vs. SCE -substituted bispyridinylidenes 1, we targeted bispyridinylidene 4 2 (KHMDS) cleanly produced the desired iminophosphorano-substituted donor 4, though it could only be isolated in low yield (12 %) owing to its poor solubility. Nevertheless, the isolated quantities were sufficient to allow for its chemical oxidation with hexachloroethane to 2+-2 Cl−4 and subsequent electrochemical analysis by cyclic voltammetry. In this way, redox potentials of −1.25 and −1.08 V vs SCE were determined for the +4/4 and 2+4/+4 couples, respectively. Though 4 should still be considered a relatively strong donor, these potentials are less reducing than for Bb ,Before attempting the preparation of 4 Scheme  derived 4, we then prepared tetrasubstituted donor 1 (Scheme 1). Initially, the 4-iminophosphorano functionality was introduced by the addition of 5 to a dichloromethane solution containing triethylamine and in situ generated dibromotriphenylphosphorane. The resulting chloropyridine 6 was isolated on a 60 g scale in 89 % yield, and was subsequently methylated at the pyridyl nitrogen to give chloropyridinium salt 7 . A combination of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and aminotriphenyl-phosphonium bromide was then used to generate nucleophilic Ph3P–NH, which in the presence of excess DBU, was able to substitute the chloride of 7 to give pyridinium salt 8 . The preparation of 1 was completed by the deprotonation of 8 with KHMDS in toluene. After three hours, donor 1, which is virtually insoluble in toluene, was isolated in a 44 % yield after being collected by filtration and extracted into benzene. 31P{1H} and 1H NMR spectra of the isolated solid show that 1 occurs as a 2:1 mixture of Z (31P: 0.4 and −7.8 ppm) and E (31P: −1.3 and −5.4 ppm) isomers. The preference for the Z isomer is supported by ROESY NMR experiments, and is in line with previous experimental1 should not be regarded as a major disadvantage, as the donor can be effectively used as a reductant when generated in situ. As for 4, donor 1 was oxidized to its more stable dichloride salt by its reaction with hexachloroethane, and analyzed by cyclic voltammetry. This electrochemical analysis revealed a half-wave potential of −1.70 V for the 2+1/1 couple, making 1 the strongest neutral organic electron donor by a substantial margin .Encouraged by the successful preparation of 1 over A and Ba, we were particularly keen to investigate the use of 1, generated in situ from an equimolar mixture of 8 and KHMDS, in the reductive S–N bond cleavage of sulfonamides , but was reduced to 10 in 65 % yield by Ba (6 equiv) after 72 h of photoexcitation.8 (equating to at most 4 equiv of 1), amine 10 was produced in good yield (75 %) within 24 h at 110 °C. Compound 11 a proved more challenging, but with eight equivalents of donor-precursor 8 (4 equiv of 1), yields of 12 (56 %) comparable to those achieved using 6 equiv of Ba under photolysis (59 %) were achieved.12 from the reduction of mesyl-substituted 11 b (6 %) were much lower than were achieved from tosyl-derived 11 a, owing to the absence of the relatively low-energy LUMO of the arene fragment in 11 b. Nevertheless, the outcomes are a testament to the strength of ground-state donor 1, which is the first ground-state organic electron donor able to effect the reduction of dialkylsulfonamides.Owing to the superior reducing power of s Scheme . In aren14 (92 %) and 16 (90 %) from compounds 13 and 15, respectively. The reduction of these substrates, which is facilitated by the formation of a resonance-stabilized nitrogen-containing leaving group, has been previously accomplished by donor A .12Moving to more activated sulfonamides, two equivalents of the in situ generated donor gave high yields of deprotected products 1 (2 equiv) reduces iodides 17 a and 19 a at room temperature, to products 18 and 20/21, respectively, where the formation of 21 suggests the involvement of aryl anion intermediates. Recent computational studies1. Importantly, under otherwise identical conditions, 17 a was quantitatively recovered in the absence of 8, demonstrating the necessity of donor 1 in effecting the reductions.In the case of aryl halides Scheme , donor 117 a and 19 a have been similarly effected by a number of organic donors,A, Ba, and Ca, so more challenging aryl halide substrates were also investigated. The related bromides 17 b and 19 b were reduced by donor 1 at higher temperatures and with longer reaction times , but the chloride 17 c remained inert to 1 under thermal conditions. In contrast, 1-chloroanthracene 22, with a lower energy LUMO owing to the extended π system, was easily reduced to anthracene 23 .The reduction of iodides 1 in the hydrodecyanation of malononitriles , 27 (91 %), and 29 (89 %). The lack of cyclized product in the case of 26 is in line with expectations; an initially formed radical intermediate 33, formed from generalized malononitrile substrate 32 should be easily reduced to the corresponding anion 34 under the heavily reducing reaction conditions.25, 27 and 29, in excellent yields. Neutral mononitrile 30 is also inert to reduction under these conditions, as was demonstrated in a separate reaction.These encouraging results prompted the investigation of the effectiveness of s Scheme . Such a 1 represents the most reducing organic neutral compound known, with its redox potential surpassing the previous record holder by 190 mV. It is the only organic electron donor with the ability to reduce dialkylarenesulfonamides as well as malononitriles without photoexcitation. Further reductions involving donor 1 are currently under investigation.In conclusion, tetra(iminophosphorano)-substituted bispyridinylidene


APOE ε4) on AD pathobiology. We evaluated cerebrospinal fluid (CSF) protein levels from 267 individuals clinically diagnosed as cognitively normal, mild cognitive impairment, and AD. Using linear models, we assessed the relationship between APOE ε4 genotype, CSF Complement 3 (C3), CSF amyloid-β (amyloid) and CSF hyperphosphorylated tau (ptau). We found a significant interaction between APOE ε4 and CSF C3 on both CSF amyloid and CSF ptau. We also found that CSF C3 is only associated with CSF ptau after accounting for CSF amyloid. Our results support a conceptual model of the AD pathogenic cascade where a synergistic relationship between the complement cascade (C3) and APOE ε4 results in elevated Alzheimer’s neurodegeneration and in turn, amyloid further regulates the effect of the complement cascade on downstream tau pathology.Inflammation is becoming increasingly recognized as an important contributor to Alzheimer’s disease (AD) pathogenesis. As a part of the innate immune system, the complement cascade enhances the body’s ability to destroy and remove pathogens and has recently been shown to influence Alzheimer’s associated amyloid and tau pathology. However, little is known in humans about the effects of the complement system and genetic modifiers of AD risk like the ε4 allele of apolioprotein E (The online version of this article (doi:10.1186/s40478-016-0339-y) contains supplementary material, which is available to authorized users. APOE ε4), the most common genetic risk factor for AD, has an important role in immune and inflammatory processes underlying Alzheimer’s neurodegeneration [Converging experimental and human biomarker evidence suggests that the immune system plays a critical role in Alzheimer’s disease (AD) pathobiology. In rodent models of AD, neuroinflammatory signals have been shown to correlate with amyloid and hyperphosphorylated tau (ptau) deposits as well as cognitive symptoms . Observaneration , 29. Furneration .APOE ε4 influences the relationship between inflammation  and amyloid and tau pathology.Recent work suggests an association between inflammatory pathways, amyloid, and tau pathology. In mouse models, Complement C1q has been shown to be necessary for amyloid-β associated synaptotoxicity . Using mAPOE ε4 and the complement system on amyloid and tau pathology in AD. We evaluated cerebrospinal fluid (CSF) measurements of the immune protein Complement 3 (C3) as well as amyloid β1-42 (amyloid) and ptau181p (ptau), which are sensitive in vivo markers of clinical diagnosis, decline, and pathology in AD [APOE ε4 and CSF C3 levels would be associated with low CSF amyloid  and elevated CSF ptau . Further, building on prior work [Here, we investigated the relationship between gy in AD , 24. Lowgy in AD . Low CSFgy in AD  and imprgy in AD . We hypoior work , 19, in n = 71), individuals diagnosed with amnestic MCI (n = 110), and probable AD (n = 56) from the Alzheimer’s Disease Neuroimaging Initiative (ADNI), which has been used in previously published studies [This study utilized samples from cognitively normal older adults  according to previously described methods [1-42 and ptau181p that are chemically bonded to color-coded beads along with analyte-specific detector antibodies. Baseline CSF C3 levels were measured using a separate multiplex immunoassay panel (Luminex technology), developed by Rules Based Medicine . CSF measurements in the immunoassay panel were processed and normalized according to previously described methods [10 transformed the data to achieve an approximately normal distribution. APOE genotype was previously generated by ADNI using DNA extracted by Cogenics  from a 3 mL aliquot of EDTA blood. APOE ε4 dosage was scored as count of ε4 allele  in all analyses.CSF amyloid β methods . This me methods , 26. BriWe used chi-squared and one-way ANOVA for demographic and biomarker summary statistics and univariate assessments. Multivariate linear models were used to assess all biomarker relationships, covarying for baseline age, baseline CDR-SB, and sex.APOE ε4 dosage on CSF amyloid and CSF ptau levels. Prior to our secondary analyses, we verified that there was an association between CSF amyloid and CSF ptau levels.We first tested for a statistical interaction between CSF C3 levels and Next, we evaluated two theoretical models by which CSF C3 levels might modify CSF ptau levels. In Model A, we tested whether CSF amyloid levels ‘mediate’ CSF ptau levels by adding it to a baseline model in which CSF C3 levels predict CSF ptau levels. In Model B, we test whether a statistical interaction between CSF C3 levels and CSF amyloid levels significantly predict CSF ptau levels.All analyses were performed using R. Plots were made using the ‘visreg’ package in R .APOE ε4 dosage, CSF amyloid, and CSF ptau.Our cohort was balanced with respect to age, sex, and CSF C3 levels as there were no significant differences by diagnostic group Table . There wAPOE ε4 dosage on CSF amyloid levels  = 23.8, p-value = 0.02) suggesting increased intracranial amyloid pathology among individuals with elevated intracranial C3 levels and increased number of APOE ε4 alleles  and APOE ε4 dosage .We found a significant statistical interaction between CSF C3 and les Fig. . With thAPOE ε4 dosage on CSF ptau levels  suggesting increased neurofibrillary pathology among individuals with elevated C3 levels and increased number of APOE ε4 alleles  but not CSF C3 .Similarly, we found a significant statistical interaction between CSF C3 and We assessed whether CSF amyloid influences the relationship between CSF C3 and CSF ptau by statistically testing two theoretical models . When CSF amyloid levels were added to the baseline model as a mediating variable, CSF C3 was significantly associated with CSF ptau (p = 0.04). Next we assessed Model B. In contrast to Model A, we found that there was no statistical interaction between CSF C3 and CSF amyloid on CSF ptau (p = 0.41).Full regression results from Models A and B  and APOE ε4 results in elevated Alzheimer’s associated pathology and in turn, amyloid further regulates the effect of the complement cascade on ptau pathology (Fig. Among individuals with elevated intracranial C3 and increased APOE and immune dysfunction, we found signs of elevated Alzheimer’s pathology in the presence of both APOE ε4 and elevated complement activation indicating that genetic propensity and inflammation may act synergistically to accelerate amyloid and tau pathology. In the presence of the statistical interaction, we found no effect of C3 on ptau suggesting that APOE may play an important role in influencing the relationship between inflammation and tau pathology. Interestingly, even with the interaction term in place, we found a significant effect of C3 on amyloid indicating that the complement cascade could influence amyloid pathology via both APOE-dependent and APOE-independent mechanisms. Additional experimental work will be required to delineate the precise mechanistic relationship between complement activation, APOE, amyloid and tau pathology.Building on prior genetic evidence implicating a relationship between Building on empirical research from prior studies , 19, ourOur findings suggest that a pro-inflammatory environmental milieu alters AD pathology in conjunction with genotype. This work further illustrates that inflammatory biomarkers along with genetic information may predict increased risk for AD  and otheAPOE ε4 influence ptau and amyloid remain to be determined. Our results will require follow-up in a larger, independent population-based cohort to determine the generalizability of this finding to diverse communities. Finally, our study highlights the need for future longitudinal studies that can directly test the causal effects suggested by our correlative analyses.Our study benefits from its use of a well-established, thoroughly characterized cohort of AD, MCI and cognitively normal healthy controls. In addition, our study benefits from using CSF, which is a biologically proximate measure of disease status and progression , 4. A liAPOE ε4 dosage on biomarkers indicative of elevated amyloid and tau pathology. Further, our mediation analyses indicate that C3 is associated with ptau only after adjusting for amyloid suggesting that amyloid may mediate the relationship between inflammation and tau pathology. Our findings underscore the importance of APOE ε4 in regulating the putative effects of C3 on both amyloid and ptau. More generally, this study highlights the importance of the innate immune system in modulating levels of the pathological proteins most associated with AD and its relevance as a biomarker and potential therapeutic target.In summary, we found a statistical interaction between elevated C3 and


The CREAM Study aimed to address the uncertainty around the effect of using antibiotics to treat clinically infected eczema on subjective eczema severity in children in primary care. Children were identified by GP practices and referred to the CREAM Study. A software package  that sits on primary care electronic medical record systems and can deliver pop-up reminders and templates to aid recruitment, was used within the study.Data on problems encountered during set-up and use were recorded systematically. Participating clinicians who had TT installed were surveyed and semi-structured interviews were conducted.Following a pilot study in 70 Scottish GP practices, TT was installed in practices participating in CREAM. Difficulties with installation, integration and operation of the software were encountered. A web-portal developed to counter these difficulties did not perform as required, therefore practices required installation of either an integrated or non-integrated (SATT) version of the software on all practice workstations potentially used by referring clinicians. Of 62 participating practices, integrated TT was installed in 26 (42%), SATT installed in 20 (32%) and 16 (26%) faxed referrals instead. Of the referrals made, only a very small proportion were sent using TT.Installation of TT required commitment at the surgery for the practice manager (or delegate) to allow remote access or install the software with telephone guidance. This resulted in delays in site activation, and unnecessary / inappropriate pop ups irritated GPs, and contributed to loss of interest in the study which impacted on recruitment rates.


For more than 70 years unusual sauropod trackways have played a pivotal role in debates about the swimming ability of sauropods. Most claims that sauropods could swim have been based on manus-only or manus-dominated trackways. However none of these incomplete trackways has been entirely convincing, and most have proved to be taphonomic artifacts, either undertracks or the result of differential depth of penetration of manus and pes tracks, but otherwise showed the typical pattern of normal walking trackways. Here we report an assemblage of unusual sauropod tracks from the Lower Cretaceous Hekou Group of Gansu Province, northern China, characterized by the preservation of only the pes claw traces, that we interpret as having been left by walking, not buoyant or swimming, individuals. They are interpreted as the result of animals moving on a soft mud-silt substrate, projecting their claws deeply to register their traces on an underlying sand layer where they gained more grip during progression. Other sauropod walking trackways on the same surface with both pes and manus traces preserved, were probably left earlier on relatively firm substrates that predated the deposition of soft mud and silt . Presently, there is no convincing evidence of swimming sauropods from their trackways, which is not to say that sauropods did not swim at all. However, the notion of swimming sauropods has even older roots originating with the archaic, and now largely discredited idea that sauropods were aquatic either to support their massive weight or to escape predators. So were there any sauropods possibly performing an aquatic or semi-aquatic lifestyle, and is there evidence that they were able to swim ? The track literature still continues to raise this question periodically. In 1944, Roland T. Bird suggested the swimming sauropod hypothesis based on a trackway composed almost exclusively of manus prints from the Cretaceous of Texas. Bird suggested that the trackway could have been produced by a sauropod ‘punting’ off the bottom with its forelimbs, while the hindlimbs floated clear of the substrateet al.et al.20In 2000, workers from the Research Center of Paleontology of the Bureau of Geology and Resource Exploration of Gansu Province discovered ten dinosaur tracksites in a small area representing the Hekou Group in Yanguoxia, Gansu Province16et al.et al.Unusual sauropod tracks were found at No. 2 site of Liujiaxia Dinosaur National Geopark (YSII), which preserve only clear claw traces without the impressions of the whole pes or any trace of the associated manus. These occur in a more or less regular trackway pattern, but predominantly show only large pes claw traces . In a prLiujiaxia Dinosaur National Geopark lies 54 km west of the city of Lanzhou, on the north shore of Taiji Lake (Yanguoxia Reservoir), Yongjing County, Linxia Hui Autonomous Prefecture, Gansu Province. The track locality is positioned at the southeastern edge of the Lanzhou−Minhe Basin.et al.et al.The Lower Cretaceous Hekou Group at Lanzhou-Minhe Basin is a continental clastic deposit of great thickness18In 1997, the Hekou Group was divided into eight informal formation-level units282925Deposits producing tracks in the Yanguoxia Region consist of purple mudstone and silty mudstone interbedded with fine sandstone. Blue grey silty mudstone beds are commonly seen in upper parts of these deposits. A number of ripple beds, parallel beds and asymmetric ripple marks are seen in the sandstone with rain prints, mud cracks and invertebrate traces on the surface, suggesting a depositional environment typical of shallow shore lacustrine facies2 , which were converted into scaled 3D textured mesh models using Agisoft Photoscan35The Yanguoxia No. 2 tracksite preserves at least nine large-sized digit-only pes trackways, catalogued as YSII-SS1−SS9  . TrackwaAmong all the pes-only trackways, the best preserved is YSII-SS1. In YSIILingulate sand mounds are preserved at the posterior end of the all the pes traces in YSII-SS1 (except RP1), showing that the substrate was pushed up posteriorly behind the deeply impressed digits. This configuration is a common feature of swim tracks209The other trackways are similar to YSII-SS1 in their morphology. Digit I impression of SS2-LP2 has sand mounds projecting distally while SS5-LP1 shows evident dragging marks extending anteriorly.Brontopodus39404142Brontopodus-type: wide−gauge, pes tracks longer than broad, with large, outwardly directed claw marks of digits I–III, a small digit IV trace and small callosity or pad mark representing digit V. Manus tracks are semicircular to U−shaped with rounded posteromedial/posterolateral traces of digits I and V, respectively44Most sauropod trackways in China are wide- (or medium-) gauge and are therefore referred to the ichnogenus Brontopodus-type trackways  present in titanosauriform sauropods. So, a final attribution and interpretation of the trackmaker cannot be given here.The wide gauge pattern of the The Yanguoxia No. 2 tracksite yields rich theropod, sauropod and ornithopod trackwayset al.sensu Marty 7Although Li et al.Based on the incompletely exposed Yanguoxia No. 2 tracksite, Zhang et al.Li Diplodocus and Apatosaurus, which possessed posterior centers of mass (CoM) could have produced pes-only tracks. But Brachiosaurus belonging to Titanosauriformes and hypothesized to have had a more anterior CoM, would have ‘tipped’ forwards, meaning that when partially buoyant it would have left manus-only tracks: see Lockley5354HendersonKyuangyuanpus a possible crocodilian trackHere it should be noted that the displaced wedge of sand is a reflection of foot registration dynamics and substrate conditions, especially posteriorly directed force during kick off. Such displaced sand wedges, referred to as “sand crescents” occur behind mammaloid tracks in Permian through Mesozoic sand dune facies57et al.18 stressed that “ the track maker was walking on a higher or overlying stratigraphic layer, through which only the pes claws penetrated” and further inferred (op. cit. p. 53–54) that “the irregular pes-only sauropod trackway is … evidence of an animal walking on an overlying surface, now removed. The track maker dug its longer claws (pes digits I–III) into the soft underlayers. Assuming the overlying layer to have been soft, as indicated by the depth of the claw impressions in the layer beneath, the irregular trackway may have been the result of the large animal varying its gait to negotiate a difficult and potentially dangerous surface where it may have run the risk of slipping or getting stuck. The irregular trackway, with traces of spread or projecting claws could be interpreted as evidence of such a reaction to an unstable substrate” These inferences require further evaluation in the light of the newly exposed evidence available for the present study.Zhang et al.et al.et al. At the time when Zhang It is important to note that in this interpretation the pes-only sauropod tracks are not undertracks transmitted through layers of overlying sediment, but they are actually true tracks, albeit only representing the ungual (claw) portions of the foot, that penetrated the overlying mud-silt sediment and directly registered in the sand: see ThulbornGiven the speed estimates derived from the trackways and the fact that sauropods were graviportal, they, unlike our human subject, probably could not run. On the other hand, recent elephants of similar proportions are able to run, and the same may have been true also for very small juvenile sauropods for example. The latter, however, cannot be proved by trackways presently. In the scenario where their main purchase was on a buried substrate layer with their toe tips, the posterior force exerted would be similar to that made by an individual that was running and exerting force in the kick-off phase.We note that despite proposing the above arguments other scenerios might be possible. The difficulties with proposing an unambiguous interpretation are that the sedimentological history of the deposition of the sandstone and overlying silts and muds cannot be known with certainty. Deposition rates, levels of sediment saturation and duration of substrate exposure to subaerial or subaqueous conditions cannot be determined precisely even with detailed sedimentological analysis.As noted above, there is, as yet, no convincing trackway evidence for swimming sauropods, despite many attempts to promote this scenario based on manus-only or manus-dominated trackways. Given the arguments above, the rare occurrence of pes-only trackways also fails to provide a convincing argument for a swimming scenario. As noted below, the speed estimates derived from these trackways also gives no support for a swimming sauropod scenario.The typical sauropod tracks on the same surface as the pes-only sauropod tracks have a typical L/W (length/width) ratio of 1.3 (as YSII-S3). The width of pes-only sauropod tracks YSII-SS1 (average 64.7 cm) is nearly equal to that of the walking sauropod tracks from the same surface. Track length of YSII-SS1 (84.1 cm) can be roughly figured out by using the L/W ratio of the latter. A foot length/hip height ratio for a sauropod is 4.0The digit-only ornithopod trackways that are also found at the No. 1 and No. 2 tracksites, as well as small-sized sauropod trackways, indicate similar preservation pattern. The digit-only ornithopod trackways include at least five trackways at No. 1 tracksite of Liujiaxia Dinosaur National Geopark and 49 trackways from No. 2 tracksite. There are only two small digit-only sauropod trackways from the No. 2 tracksite.et al.Fujita et al.Moreover, Fujita Compared with the 54 narrow-gauge ornithopod trackways, the two wide-gauge small-sized sauropod trackways at No. 2 tracksite only preserve digit impressions (YSI-SW [No. w sauropod trackway] and YSI-SV [No. v sauropod trackway]). They are morphologically almost the same as the anterior parts of the small-sized sauropod pes track from the Litan site, YanguoxiaWe report an assemblage of nine sauropod trackways consisting of digit-only pes tracks from the Lower Cretaceous Hekou Group of Gansu.To date this is the only site preserving this type of trackway.The trackway pattern is similar to that found in trackways of typical walking sauropods except that the toe impressions are much deeper.The tracks also show a displaced wedge of sand similar to that commonly found in other tracks made in sandy substrates.The only other well-known category of unusual incomplete sauropod trackway, that of manus-only or manus-dominated trackways, has been the subject of much debate. Previously many authors have interpreted these as evidence of swimming. However, others have disputed these claims and interpreted manus-only trackways as undertracks or examples of unusual preservation that cannot be used to support the swimming sauropod scenario.The digit-only pes sauropod trackways are here interpreted as evidence of animals walking on a silt and mud substrate above the sandstone layer into which the pes claws penetrated from above.Thus the digit-only pes trackways have a preservational explanation and are not evidence of swimming or unusual behavior.How to cite this article: Xing, L. et al. Digit-only sauropod pes trackways from China - evidence of swimming or a preservational phenomenon?. Sci. Rep.6, 21138; doi: 10.1038/srep21138 (2016).


To investigate the role of homocysteine in neuronal injury in HIV infection.12, serum folate, CSF, and plasma HIV RNA, the immune activation marker neopterin in CSF and serum, and albumin ratio as a marker of blood-brain barrier integrity. Twenty-two subjects provided a second sample median of 12.5 months after antiretroviral treatment initiation.Using a cross-sectional design and archived samples, we compared concentrations of plasma homocysteine and cerebrospinal fluid (CSF) neurofilament light protein (NFL), a sensitive marker of neuronal injury, in 83 HIV-1-infected subjects without antiretroviral treatment. We also analyzed plasma vitamin Bp < 0.0001). As expected, there was a significant inverse correlation between homocysteine and B12  and folate  levels. In a multiple linear regression analysis homocysteine stood out as an independent predictor of CSF NFL in HIV-1-infected individuals. The correlation of plasma homocysteine and CSF NFL was also present in the group receiving antiretroviral therapy .A significant correlation was found between plasma homocysteine and CSF NFL concentrations in untreated individuals (r = 0.52, 12/folate deficiency appear to play a role in neural injury in HIV-infected individuals.A correlation between plasma homocysteine and axonal injury, as measured by CSF NFL, was found in both untreated and treated HIV. While this study is not able to prove a causal link, homocysteine and functional B Prior to the discovery of effective antiretroviral therapy (ART), HIV-infected individuals had about a 30% overall risk of developing HIV-related dementia (HAD) . Althoug12 and B6, and therefore elevated levels of homocysteine are an indicator of B12 and/or folate deficiency : CD4+ > 350 (n = 21); CD4+ 200–349 (n = 20); CD4+ 50–199 (n = 22); and CD4+ < 50 (n = 17), based on earlier findings of higher CSF-NFL levels in untreated subjects with lower CD4 cell counts [The untreated neuroasymptomatic individuals were divided into four subgroups according to CD4l counts .+ count, CSF, and blood albumin levels were analyzed according to local laboratory standards. CSF:blood albumin ratio was calculated as a measure of blood-brain barrier dysfunction.All samples were frozen to –70°C within an hour of sampling and stored until analysis. CSF samples were centrifuged to remove cells and aliquoted before freezing. Levels of NFL in CSF were determined using a commercial ELISA, according to the manufacturer’s instructions . For neopterin a commercially available immunoassay  was used. CD412 and serum folate were quantified using an electrochemiluminescence immunoassay on a cobas e analyzer . Total homocysteine was determined in EDTA plasma by stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) using a Quattro micro instrument , essentially as described by Magera et al. [Plasma vitamin Ba et al. . Levels P values were adjusted for multiple comparisons using the Tukey’s Range test. Predictors of log10 plasma homocysteine and CSF NFL were analyzed by multiple linear regression analysis with forward selection. In the analyses CD4+ count, CSF:blood albumin ratio, plasma and CSF HIV RNA, serum and CSF neopterin, CSF NFL, plasma homocysteine, plasma vitamin B12, and serum folate were log transformed to reduce skewness. P < 0.05 was considered significant.Correlations were analyzed by Pearson correlation analysis. Paired T-test was used to compare the treatment group before and during treatment. Differences between groups were analyzed with ordinary one-way ANOVA and All statistical tests were performed with SPSS version 21  or Prism version 6.0 .The entire study and the procedure used to assure the anonymity of the data was approved by the Research Ethics Committee of the University of Gothenburg, in accordance with the Helsinki Declaration of 1975, as revised in 2000. All participants gave their informed consent in writing. All patient information was anonymized prior to analysis by study Principal investigator (MG) at the Department of Infectious Diseases, at Sahlgrenska University Hospital.12 levels were below 140 pmol/L in 2 subjects, and below 250 pmol/L in 32. One individual had a high serum folate concentration. Of the three individuals with HAD, two had hyperhomocysteinemia and two had CD4+ counts < 200. All three had very high levels of CSF HIV RNA and CSF NFL.Among the 80 untreated neuroasymptomatic subjects, 20 had moderate hyperhomocysteinemia (> 15 μmol/L). Twenty had elevated levels of CSF NFL, compared to age-dependent laboratory norms. Bp = 0.0002). The group with CD4+ count < 50 had homocysteine levels significantly higher than the other groups (p = 0.0001). The CD4+ < 50 group had significantly higher levels than the groups with CD4+ > 350 and CD4+ 200–349. The CD4+ 50–199 group had higher levels compared to the CD4+ > 350 group (12 or serum folate levels (Comparison of the subgroups of untreated neuroasymptomatic subjects showed a significant difference in homocysteine levels between the groups (r groups . Differe50 group . No signe levels .p < 0.0001) between the plasma level of homocysteine and CSF NFL , and of homocysteine and folate . Homocysteine was also found to correlate significantly to the CSF:blood albumin ratio , CD4+ count , age , and treatment with trimethoprim-sulfamethoxazole . Plasma homocysteine correlated to serum neopterin , but no correlation was found between homocysteine and CSF neopterin. No significant correlation was found between CSF NFL and B12 concentrations. A weak correlation was found between serum folate and CSF NFL .We found a significant correlation Click here for additional data file.