Cloud-based simulation studies in R - A tutorial on using doRedis with Amazon spot fleets.

Simulation studies are helpful in testing novel statistical methods. From a computational perspective, they constitute embarrassingly parallel tasks. We describe parallelization techniques in the programming language R that can be used on Amazon's cloud-based infrastructure. After a short conceptual overview of the parallelization techniques in R, we provide a hands-on tutorial on how the doRedis package in conjunction with the Redis server can be used on Amazon Web Services, specifically running spot fleets. The tutorial proceeds in seven steps, ie, (1) starting up an EC2 instance, (2) installing a Redis server, (3) using doRedis with a local worker, (4) using doRedis with a remote worker, (5) setting up instances that automatically fetch tasks from a specific master, (6) using spot-fleets, and (7) shutting down the instances. As a basic example, we show how these techniques can be used to assess the effects of heteroscedasticity on the equal-variance t-test. Furthermore, we address several advanced issues, such as multiple conditions, cost-management, and chunking.

[Parental responses to children's pain : Analysis of the parental responses to their child's pain in the German general population]. / Elterliche Reaktionen auf kindlichen Schmerz : Analyse der elterlichen Reaktionen auf die Schmerzen ihres Kindes in der deutschen Allgemeinbevölkerung.

BACKGROUND: Parental reactions to their child's pain can comprise cognitive-affective and behavioral responses. Dysfunctional responses like parental catastrophizing may lead to an aggravation of the child's pain. OBJECTIVES: Aims of the online-based study were (1) to psychometrically evaluate existing questionnaires into cognitive-affective (Pain Catastrophizing Scale for Parents; PCS-P) and behavioral responses (Inventar zum schmerzbezogenen Elternverhalten; ISEV-E) within a sample of 105 healthy parents, and (2) to compare their responses to existing (inter)national clinical samples and to the reactions of 80 parents with self-reported chronic pain from the general population. METHODS: The assessment of parental pain-related reactions was online-based. RESULTS: While the factor structure of the ISEV-E could not be replicated, the three factors of the PCS-P could be replicated. Parental catastrophizing of the healthy parents was lower compared to clinical samples. Healthy parents did not differ from parents with chronic pain from the general population. CONCLUSION: The results offer a basis to grade parental catastrophizing, so that risk-groups can be identified.

Liebowitz Social Anxiety Scale (LSAS): Optimal cut points for remission and response in a German sample.

The Liebowitz Social Anxiety Scale (LSAS) is the most frequently used instrument to assess social anxiety disorder (SAD) in clinical research and practice. Both a self-reported (LSAS-SR) and a clinician-administered (LSAS-CA) version are available. The aim of the present study was to define optimal cut-off (OC) scores for remission and response to treatment for the LSAS in a German sample. Data of N = 311 patients with SAD were used who had completed psychotherapeutic treatment within a multicentre randomized controlled trial. Diagnosis of SAD and reduction in symptom severity according to the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, 4th edition, served as gold standard. OCs yielding the best balance between sensitivity and specificity were determined using receiver operating characteristics. The variability of the resulting OCs was estimated by nonparametric bootstrapping. Using diagnosis of SAD (present vs. absent) as a criterion, results for remission indicated cut-off values of 35 for the LSAS-SR and 30 for the LSAS-CA, with acceptable sensitivity (LSAS-SR: .83, LSAS-CA: .88) and specificity (LSAS-SR: .82, LSAS-CA: .87). For detection of response to treatment, assessed by a 1-point reduction in the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, 4th edition, rating, a reduction of 28% for the LSAS-SR and 29% for the LSAS-CA yielded the best balance between sensitivity (LSAS-SR: .75, LSAS-CA: .83) and specificity (LSAS-SR: .76, LSAS-CA: .80). To our knowledge, we are the first to define cut points for the LSAS in a German sample. Overall, the cut points for remission and response corroborate previously reported cut points, now building on a broader data basis.

Depressive Symptom Inventory Suicidality Subscale: Optimal Cut Points for Clinical and Non-Clinical Samples.

Suicide is a major cause of death in adulthood and specifically in patients suffering from mental illnesses. The Depressive Symptom Inventory Suicidality Subscale (DSI-SS) is widely used to detect and prevent suicidal ideation. The aim of the present study was to determine optimal cut points for the DSI-SS in different populations. We analysed the data of one population-based sample (n = 532), one outpatient sample (n = 180) and one inpatient sample (n = 244). Internal consistency, convergent validity and optimal cut points according to receiver operating characteristics were calculated. In all samples, we found excellent item-total correlations and internal consistencies for the DSI-SS. Zero-order correlations between the DSI-SS and theoretically related constructs showed positive correlation coefficients, ranging from 0.50 to 0.67. The DSI-SS differentiated well between patients with and without suicide attempts in the population-based sample, but less so in the inpatient sample and only marginally in the outpatient sample. A bootstrapping analysis showed some variability in the cut points that emerged as optimal, but there was no overlap between the different samples. The specific cut points that we identified may be used to improve the diagnostic utility of the DSI-SS and the chance to detect suicidal ideation. Copyright © 2016 John Wiley & Sons, Ltd. KEY PRACTITIONER MESSAGE: We developed cut points for the Depressive Symptom Inventory Suicidality Subscale, to improve the early and valid detection of suicidal ideation by this measure. The cut points that were identified as optimal varied between the samples. The cut points differentiated well in a non-clinical sample, but less well in outpatient and inpatient samples.

Difficulties in screening for peripheral neuropathies in children with diabetes.

AIMS: To assess the diagnostic utility of a novel abbreviated monofilament test in comparison with the tuning fork test to detect diabetic peripheral neuropathy in children. METHODS: A total of 88 children with Type 1 diabetes mellitus were screened for diabetic peripheral neuropathy using the monofilament test and the tuning fork. Nerve conduction studies were performed according to the 'gold standard' for neuropathy. We assessed the diagnostic utility and inter-rater agreement of the two screening methods. RESULTS: A total of 43 (49%) children (aged 6-18 years) had at least one abnormal nerve conduction study result. Diagnostic utility and inter-rater agreement were very low for both screening methods. The monofilament test yielded a sensitivity of 18% and a specificity of 80%. The tuning fork yielded a sensitivity of 0% and a specificity of 98%. CONCLUSION: The present study found that an abbreviated monofilament test has low diagnostic utility for the detection of early diabetic peripheral neuropathy because of its low reliability. The problem of reliability needs to be more thoroughly addressed in order to improve the screening procedures in diabetes management in childhood and adolescence.

Attitudes toward postmortem cornea donation in Germany: a multicenter survey.

PURPOSE: To analyse the willingness for postmortem cornea donation in Germany. METHODS: Employees in two cities (UKM, UKS), and university hospitals (STM, STE), members of the German Ophthalmological Society (DOG), and employees of an automobile company (BO) participated in a questionnaire about postmortem cornea donation attitudes. The questionnaire consisted of demographic items, motives concerning postmortem cornea donation, general attitudes toward donation, and questions concerning the perceived needs for information about donation. The statistical analyses included logistic regression with the target parameter of 'willingness to donate cornea postmortem'. RESULTS: Of the participants, 67.7 % (UKM, UKS), 70.9 % (STM, STE), 70.8 % (BO), and 79.4 % (DOG) declared their intention to donate their corneas postmortem. Younger age (p < 0.001), poorer general health (p < 0.05), faith in an eternal life (p < 0.05), disagreement with brain death diagnostics (p < 0.001), fear of receiving worse medical treatment (p < 0.001), and fear of the commercialization of organs (p < 0.001) were found to be risk factors for a negative attitude toward postmortem cornea. The majority of participants (57.4 %) indicated that additional information about donation would be appreciated, and the internet (69.9 %) was considered the most appropriate means for conveying this information. CONCLUSIONS: Emotional items were revealed to be the most relevant factors influencing the willingness to donate cornea postmortem, which may be counteracted by means of public education. The relatively low willingness among the medical staff contrasts with previous observations in a professional ophthalmologic society.

Influence of parental occupation on access to specialised treatment for paediatric chronic pain: a retrospective study.

BACKGROUND: The purpose of this study was to determine patients' travel distances to a tertiary paediatric pain clinic and to analyse the association between travel distance and the parents' occupational skill level and the patients' pain characteristics. PATIENTS AND METHODS: The retrospective study consisted of 2,248 children assessed at the first evaluation. All children (0-20 years) who visited the clinic during a 5-year period (2005-2010) were enrolled in this study. RESULTS: The mean travel distance was 81 km, and the 80 % catchment area was 109 km. Children of parents with a high occupational skill level had a 1.5-fold higher probability of travelling from outside the catchment area. The 80 % catchment area increased constantly with increasing occupational skill level. Additional significant factors for greater distance travelled were high impairment, musculoskeletal pain, long pain duration and a high number of previous physician contacts. CONCLUSION: The association between travel distance and parental occupational skill level suggests that there is social injustice due to access barriers based on socioeconomic deprivation and education. An increase in the number of health care facilities for chronic pain in children would be a first step in rectifying this injustice.

Childhood diabetic neuropathy: functional impairment and non-invasive screening assessment.

AIM: Sensory diabetic neuropathy, determined by nerve conduction studies, is common in children with Type 1 diabetes. Diabetic neuropathy diagnoses are rarely made in paediatric daily care because they are asymptomatic, vibration detection is mostly normal and nerve-conduction testing is impractical. The present study aims to: (1) describe somatosensory dysfunction in children with diabetes, (2) test whether diabetes duration and HbA(1c) are related to somatosensory dysfunction and (3) identify the best screening test for large-fibre dysfunction, as indicated by nerve conduction studies. METHODS: Forty-five children (age 13.2 ± 2.5 years) with Type 1 diabetes for 6.7 ± 2.5 years and matched control subjects were assessed by neurological examinations, nerve conduction tests and quantitative sensory testing on the feet using the protocol of the German Research Network on Neuropathic Pain. Abnormal nerve conduction was used as gold standard to define neuropathies. RESULTS: We found a high prevalence of mechanical (38%) and thermal (24%) hypoesthesia often associated with hyperalgesia (47%). Tactile hypoesthesia (33%) was more frequent than pallhypaesthesia (11%). Only cold detection and mechanical pain thresholds were related to HbA(1c). Tactile hypoesthesia had the highest sensitivity (75%), specificity (89%) and positive (75%) and negative (89%) predictive values for neuropathies defined by nerve conduction tests (31% abnormal). CONCLUSIONS: Almost half of the children with diabetes have subclinical large- and small-fibre neuropathies. Tactile detection was better than vibration for neuropathy assessment. Quantitative sensory testing is a valuable tool for assessment of neuropathy as well as a target of interventional studies in children with diabetes.

[Please don't hurt me!: a plea against invasive procedures in children and adolescents with complex regional pain syndrome (CRPS)]. / Bitte nicht noch mehr verletzen! : Plädoyer gegen eine invasive Schmerztherapie bei Kindern mit komplexem regionalem Schmerzsyndrom (CRPS).

BACKGROUND: Complex regional pain syndrome (CRPS; formerly known as Morbus Sudeck/reflex dystrophy) is diagnosed in children and adolescents, but the clinical presentation is often atypical. Unfortunately, potentially harmful, invasive treatments are used in pediatric patients. PATIENTS AND METHODS: A retrospective chart study of pediatric chronic pain patients with CRPS was performed. RESULTS: Over the course of 6 years, 37 (35 girls) children and adolescents took part in a multidisciplinary chronic pain inpatient program. At admission, patients took on average 4.4 (range 1-10) different medications and 29 different pharmaceuticals were used overall. Prior to admission, invasive pain treatments were performed without success in 16 of the children (43%). At least 13 children received two or more invasive treatments. Although sympathetic blocks were most prevalent, operations and regional anesthesia were also used. CONCLUSION: Despite a lack of evidence for invasive procedures, these continue to be used in children and adolescents with CRPS, who later respond positively to conventional treatment. The English full-text version of this article is available at SpringerLink (under "Supplemental").

Small-fiber neuropathy and pain sensitization in survivors of pediatric acute lymphoblastic leukemia.

BACKGROUND: Chemotherapy-induced Peripheral Neuropathy (CIPN) of large-fibers affects up to 20% of survivors of pediatric acute lymphoblastic leukemia (ALL). We aimed to describe small-fiber toxicity and pain sensitization in this group. METHODS: In a cross-sectional, bicentric study we assessed 46 survivors of pediatric ALL (Mean age: 5.7 ± 3.5 years at diagnosis, median 2.5 years after therapy; males: 28). INCLUSION CRITERIA: ≥6 years of age, ≥3 months after last administration of Vincristine, and cumulative dose of Vincristine 12 mg/m2. We used a reduced version of the Pediatric-modified Total Neuropathy Score (Ped-mTNS) as bedside test and Quantitative Sensory Testing (QST) for assessment of small- and large-fiber neuropathy as well as pain sensitization. We employed Nerve Conduction Studies (NCS) as the most accurate tool for detecting large-fiber neuropathy. RESULTS: Fifteen survivors (33%) had abnormal rPed-mTNS values (≥4 points) and 5 survivors (11%) reported pain. In QST, the survivor group showed significant (p < 0.001) inferior large-fiber function and pain sensitization when compared to healthy matched peers. We identified deficits of vibration in 33 (72%) and tactile hypoesthesia in 29 (63%), hyperalgesia to blunt pressure in 19 (41%), increased mechanical pain sensitivity in 12 (26%) and allodynia in 16 (35%) of 46 survivors. Only 7 survivors (15%) had pathologic NCS. CONCLUSION: QST is a sensitive tool that revealed signs of large-fiber neuropathy in two thirds, small-fiber neuropathy and pain sensitization in one third of survivors. Prospective studies using QST in pediatric oncology may help to elucidate the pathophysiology of small-fiber neuropathy and pain sensitization as well as their relevance for quality of survival.

Quantitative sensory testing profiles in children, adolescents and young adults (6-20 years) with cerebral palsy: Hints for a neuropathic genesis of pain syndromes.

INTRODUCTION: Many patients with cerebral palsy (CP) suffer chronic pain as one of the most limiting factors in their quality of life. In CP patients, pain mechanisms are not well understood, and pain therapy remains a challenge. Quantitative sensory testing (QST) might provide unique information about the functional status of the somatosensory system and therefore better guide pain treatment. OBJECTIVES: To understand better the underlying pain mechanisms in pediatric CP patients, we aimed to assess clinical and pain parameters, as well as QST profiles, which were matched to the patients' cerebral imaging pathology. PATIENTS AND METHODS: Thirty CP patients aged 6-20 years old (mean age 12 years) without intellectual impairment underwent standardized assessments of QST. Cerebral imaging was reassessed. QST results were compared to age- and sex-matched controls (multiple linear regression; Fisher's exact test; linear correlation analysis). RESULTS: CP patients were less sensitive to all mechanical and thermal stimuli than healthy controls but more sensitive to all mechanical pain stimuli (each p < 0.001). Fifty percent of CP patients showed a combination of mechanical hypoesthesia, thermal hypoesthesia and mechanical hyperalgesia; 67% of CP patients had periventricular leukomalacia (PVL), which was correlated with mechanic (r = 0.661; p < 0.001) and thermal (r = 0.624; p = 0.001) hypoesthesia. CONCLUSION: The combination of mechanical hypoesthesia, thermal hypoesthesia and mechanical hyperalgesia in our CP patients implicates lemniscal and extralemniscal neuron dysfunction in the thalamus region, likely due to PVL. We suspect that extralemniscal tracts are involved in the original of pain in our CP patients, as in adults.

Physician consultation in young children with recurrent pain-a population-based study.

Background. Recurrent pain is a common experience in childhood, but only few children with recurrent pain attend a physician. Previous studies yielded conflicting findings with regard to predictors of health care utilization in children with recurrent pain. Methods. The present study analyzes data from the German Health Interview and Examination Survey for Children and Adolescents (KiGGS) study comprising n = 2,149 children (3-10 years old) with recurrent pain to find robust predictors. We used multiple logistic regressions to investigate age, gender, socio-economic status (SES), migration background, pain intensity, pain frequency, pain-related disability, mental health problems, and health-related quality of life (HRQL) as predictors for visiting a doctor due to pain. Results. Overall, young girls with high pain-related disability, intensity, frequency, and migration background were more likely to attend a physician. Pain-related disability had the largest impact. Socioeconomic status, health-related quality of life and mental health problems were not systematically related to health care utilization. An analysis of the variability of these results indicated that several hundred participants are needed until the results stabilize. Conclusions. Our findings highlight the importance of pain-related disability and frequency in assessing the severity of recurrent pain. Generic predictors and demographic variables are of lesser relevance to children with recurrent pain. On a methodological level, our results show that large-scale studies are need to reliably identify predictors of health care utilization.

Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region.

The subcellular localization of the human Ca(2+)-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the DeltaN mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif.

Classifying the severity of paediatric chronic pain - an application of the chronic pain grading.

BACKGROUND: The chronic pain grading (CPG), a standard approach to classify the severity of pain conditions in adults, combines the characteristics of pain intensity and pain-related disability. However, in children and adolescents, the CPG has only been validated in a school sample, but not in the actual target population, i.e., clinical populations with pain. METHODS: In the present study, we applied the CPG to a tertiary sample of adolescents with chronic pain (n=1242). Construct validity, sensitivity to change and prognostic utility were examined. RESULTS: Results indicate that most adolescents were equally classified into the three higher severity grades. Higher CPG classification was associated with more pain locations, higher pain frequency, longer pain duration, extensive use of health care and more depressive symptoms. Adolescents with a high CPG received recommendations for inpatient treatment more often; however, the prognostic utility for therapy recommendation - as operationalized in this study - was low. Sensitivity to change was assessed via reassessment at follow-up for a subsample of 490 adolescents. The majority of adolescents improved to a less severe CPG; changes were more common in the high severity range. CONCLUSION: The CPG may be applied to adolescent tertiary care samples and to assess outcomes in clinical trials. However, in this study it was not appropriate to assign adolescent patients to different treatment options. Future work should focus on developing a comprehensive assessment tool for assigning patients to different treatments.

[Results of an internet-based survey amongst members of the German Ophthalmological Society concerning postmortem cornea donation]. / Ergebnisse einer internetbasierten Umfrage zur Hornhautspendebereitschaft bei DOG-Mitgliedern.

BACKGROUND: Analysis of willingness for postmortem cornea donation by professionals in ophthalmology and their motives in favor of or against donation. PATIENTS AND METHODS: 3887 members of the German Ophthalmological Society received an anonymous questionnaire concerning sociodemographic background, physical health, experiences with organ explantation and their former engagement and motives concerning organ and cornea donation. RESULTS: 722 of the questionnaires were partially and 533 completely answered with an average willingness for cornea donation of 79.4%. Significant parameters for cornea donation were gender, former experience with organ explantation, ophthalmological health and fear of false diagnosis of brain death, worse medical treatment or organ commercialization. Of the participants 53.9% suggested the internet as a favorite source of information in this matter. CONCLUSION: The factors which had a significant impact on cornea donation in this survey seem to be mainly a result of insufficient information. Detailed information regarding this topic should preferentially be presented on internet pages of professional societies and could probably increase donation approval of DOG members.

Developmental and sex differences in somatosensory perception--a systematic comparison of 7- versus 14-year-olds using quantitative sensory testing.

There are controversial discussions regarding developmental- and sex-related differences in somatosensory perception, which were found, eg, when comparing younger children (6-8 years), older children (9-12 years), and adolescents (13-16 years) using quantitative sensory testing (QST). The aim of our current study was to systematically assess the impact of age and sex using the QST protocol of the German Research Network on Neuropathic Pain (DFNS). QST, including thermal and mechanical detection and pain thresholds, was assessed in 86 healthy 7-year-old children (42 girls and 44 boys) and 87 healthy 14-year-old adolescents (43 girls and 44 boys). The sample size was calculated a priori to detect medium-sized effects as found in the previous studies with adequate power. Developmental and sex differences were tested using univariate analysis of variance. Children were more sensitive to most pain stimuli, except cold pain stimuli, compared with adolescents, but did not differ in mechanical and thermal detection thresholds except in regard to cold stimuli. Sex had an impact only on warm detection, with girls being more sensitive. There were no interactions between age and sex. In conclusion, developmental changes during the puberty appear to influence pain perception, whereas sex effects in childhood are negligible. At present, it is not clear what brings about the differences between adult men and women that are apparent in epidemiological studies. Our results contradict the hypothesis that differences in peripheral nerve-fiber functioning underlie sex effects.

[Transient global amnesia (TGA) during an oral provocation test]. / Transiente globale Amnesie (TGA) während oraler Arzneimittelexposition.

After the oral administration of the COX 2 inhibitor rofecoxib (Vioxx) as part of an oral provocation test, a 64-year-old woman developed acute anterograde and retrograde amnesia which lasted for several hours. The patient did not show symptoms of anaphylaxis. Neurological examination did not reveal focal symptoms or an epileptic seizure. Diffusion-weighted magnetic resonance imaging only showed a small region of increased signal intensity in the left hippocampus. The patient thus had no anaphylactic drug reaction, but a transient global amnesia (TGA) or amnestic episode. TGA is an acute, reversible, cerebral dysfunctional state which lasts from 1 h up to maximally 24 h and which is associated with anterograde and retrograde amnesia. While about half of the cases appear to occur spontaneously, other cases are preceded by physically or emotionally stressful events. In our patient it may have been the emotional distress caused by the oral provocation testing that has triggered the TGA. When TGA occurs during oral challenge of drugs, we recommend repeating the oral challenge of the tested drug.

Heterologous overexpression of human NEFA and studies on the two EF-hand calcium-binding sites.

Human NEFA is an EF-hand, leucine zipper protein containing a signal sequence. To confirm the calcium binding capacity of NEFA, recombinant NEFA analogous to the mature protein and mutants with deletions in the EF-hand domain were expressed in Pichia pastoris and secreted into the culture medium at high yield. The calcium binding activity of each purified protein was measured by a modified equilibrium dialysis using the fluorescent Ca2+ indicator FURA-2 and atomic absorption spectroscopy. A stoichiometry of 2 mol Ca2+/mol NEFA was determined. The Ca2+ binding constants were resolved by intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca2+ binding sites with Kd values of 0.08 microM and 0.2 microM. Circular dichroism (CD) spectroscopy showed an increase from 30 to 43% in the amount of alpha-helix in NEFA after addition of calcium ions. Limited proteolytic digestion indicated a Ca2+ dependent conformational change accompanied by an altered accessibility to the enzyme.

The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor.

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously been isolated from a KM3 cell line and immunolocalized on the plasma membrane, in the cytoplasma, and in the culture medium. Sequence analysis of a cDNA clone encoding NEFA identified a hydrophilic domain, two EF-hands, and a leucine zipper at the C-terminus. These characters are shared with nucleobindin (Nuc). In this paper we have further characterized NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra of recombinant NEFA indicated a helical content of 51% and showed that the EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and synthesized peptides revealed that the leucine zipper cannot form a homodimer. The leucine zipper may allow heterodimer formation of NEFA and an unknown protein. Phylogenetic analyses suggest that this protein is derived from a four-domain EF-hand ancestor with subsequent duplications and fusions. The leucine zipper and putative DNA-binding domains of NEFA have evolved secondarily from existing EF-hand sequences. These analyses provide insights into how complex proteins may originate and trace the precursor of NEFA to the common ancestor of eukaryotes.