Positive cooperativity during Azotobacter vinelandii nitrogenase-catalyzed acetylene reduction.

The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (Fe8S7) and the FeMoco (MoFe7S9-C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF4]- transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H+ to H2 was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H+ reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H+ reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αß halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H+ reduction to H2. For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis.

Identification of a single MPV17 nonsense-associated altered splice variant in 24 South African infants with mitochondrial neurohepatopathy.

MPV17-related mitochondrial neurohepatopathy is a rare genetic disorder worldwide. We report on a novel pathogenic variant in the MPV17 gene in 24 unrelated neurohepatopathic infants of non-consanguineous Black South African heritage. Exome sequencing identified homozygosity for a c.106C>T nonsense variant in exon 3 of the human MPV17 gene in 2 unrelated index patients. mRNA analysis revealed transcripts both with and without exon 3, indicating both reduced splice efficiency and premature termination as mechanisms for disease. Carrier frequency in this population was found to be 1 in 68 (95% CI; 1/122-1/38) with an estimated newborn incidence of 1 in 18 496 (95% CI; 1/59 536-1/5776). Affected infants all presented with infantile onset neurohepatopathy with none surviving beyond infancy. This description of a relatively common pathogenic variant underlying a previously uncharacterized severe neurohepatopathy in South Africa will engender increased awareness, earlier diagnosis and possibly improve outcome if preventative or specific therapeutic options can be found.

Extensively hydrolysed infant formulas: Need for aligned definition of peptide size characteristics and standardisation of analytical methods.

Letter by Nutten et al. on article by Levin et al. (Levin ME, Blackhurst DM, Kirstein F, Kok D, van der Watt GF, Marais AD. Residual allergenicity of amino acid-based and extensively hydrolysed cow's milk formulas. S Afr Med J 2017;107(9):763-767. S Afr Med J 2017;107(3):258-263. https://doi.org/10.7196/SAMJ.2017.v107i9.12137); and response by Levin et al.

Residual allergenicity of amino acid-based and extensively hydrolysed cow's milk formulas.

BACKGROUND: Criteria for labelling infant feeds as suitable for the dietary management of cow's milk protein allergy (CMPA) rely on proving the hypoallergenicity of such feeds or clinical studies showing that the feeds are tolerated by 90% of children with proven CMPA. South African (SA) labelling legislation does not indicate what testing is necessary to prove hypoallergenicity. OBJECTIVES: To evaluate all extensively hydrolysed cow's milk formulas and amino acid-based formulas available in SA for residual allergen content, protein size and amino-acid content. RESULTS: All amino-acid and extensively hydrolysed formulas were found to be similar in composition, with no residual cow's milk allergens detectable by enzyme-linked immunosorbent assay. Furthermore, proteins were absent and only small molecules in the size range of amino acids and possibly of very small oligopeptides were detected. CONCLUSIONS: These findings indicate that the formulas are extremely likely to be compliant with the definition of hypoallergenicity as tolerance in 90% of proven sufferers from cow's milk allergy. The formulas may therefore be labelled as suitable for the dietary management of infants with CMPA.

After 50 years and 200 papers, what can the Midspan cohort studies tell us about our mortality?

OBJECTIVE: To distil the main findings from published papers on mortality in three cohorts involving over 27,000 adults, recruited in Scotland between 1965 and 1976 and followed up ever since. METHOD: We read and summarized 48 peer-reviewed papers about all-cause and cause-specific mortality in these cohorts, published between 1978 and 2013. RESULTS: Mortality rates were substantially higher among cigarette smokers in all social classes and both genders. Exposure to second-hand smoke was also damaging. Exposure to higher levels of black smoke pollution was associated with higher mortality. After smoking, diminished lung function was the risk factor most strongly related to higher mortality, even among never-smokers. On average, female mortality rates were much lower than male but the same risk factors were predictors of mortality. Mortality rates were highest among men whose paternal, own first and most recent jobs were manual. Specific causes of death were associated with different life stages. Upward and downward social mobility conferred intermediate mortality rates. Low childhood cognitive ability was strongly associated with low social class in adulthood and higher mortality before age 65 years. There was no evidence that daily stress contributed to higher mortality among people in lower social positions. Men in manual occupations with fathers in manual occupations, who smoked and drank >14 units of alcohol a week had cardiovascular disease mortality rates 4.5 times higher than non-manual men with non-manual fathers, who neither smoked nor drank >14 units. Men who were obese and drank >14 units of alcohol per day had a mortality rate due to liver disease 19 times that of normal or underweight non-drinkers. Among women who never smoked, mortality rates were highest in severely obese women in the lowest occupational classes. CONCLUSION: These studies highlight the cumulative effect of adverse exposures throughout life, the complex interplay between social circumstances, culture and individual capabilities, and the damaging effects of smoking, air pollution, alcohol and obesity.

A cardiovascular disease policy model: part 2-preparing for economic evaluation and to assess health inequalities.

OBJECTIVES: This is the second of the two papers introducing a cardiovascular disease (CVD) policy model. The first paper described the structure and statistical underpinning of the state-transition model, demonstrating how life expectancy estimates are generated for individuals defined by ASSIGN risk factors. This second paper describes how the model is prepared to undertake economic evaluation. DESIGN: To generate quality-adjusted life expectancy (QALE), the Scottish Health Survey was used to estimate background morbidity (health utilities) and the impact of CVD events (utility decrements). The SF-6D algorithm generated utilities and decrements were modelled using ordinary least squares (OLS). To generate lifetime hospital costs, the Scottish Heart Health Extended Cohort (SHHEC) was linked to the Scottish morbidity and death records (SMR) to cost each continuous inpatient stay (CIS). OLS and restricted cubic splines estimated annual costs before and after each of the first four events. A Kaplan-Meier sample average (KMSA) estimator was then used to weight expected health-related quality of life and costs by the probability of survival. RESULTS: The policy model predicts the change in QALE and lifetime hospital costs as a result of an intervention(s) modifying risk factors. Cost-effectiveness analysis and a full uncertainty analysis can be undertaken, including probabilistic sensitivity analysis. Notably, the impacts according to socioeconomic deprivation status can be made. CONCLUSIONS: The policy model can conduct cost-effectiveness analysis and decision analysis to inform approaches to primary prevention, including individually targeted and population interventions, and to assess impacts on health inequalities.

Hepatitis C virus in Mexican Americans: a population-based study reveals relatively high prevalence and negative association with diabetes.

This study aimed to estimate the prevalence and risk factors for hepatitis C virus (HCV) infection in Mexican Americans living in South Texas. We tested plasma for the presence of HCV antibody from the Cameron County Hispanic Cohort (CCHC), a randomized, population-based cohort in an economically disadvantaged Mexican American community on the United States/Mexico border with high rates of chronic disease. A weighted prevalence of HCV antibody of 2·3% [n = 1131, 95% confidence interval (CI) 1·2-3·4] was found. Participants with diabetes had low rates of HCV antibody (0·4%, 95% CI 0·0-0·9) and logistic regression revealed a statistically significant negative association between HCV and diabetes (OR 0·20, 95% CI 0·05-0·77) after adjusting for sociodemographic and clinical factors. This conflicts with reported positive associations of diabetes and HCV infection. No classic risk factors were identified, but important differences between genders emerged in analysis. This population-based study of HCV in Mexican Americans suggests that national studies do not adequately describe the epidemiology of HCV in this border community and that unique risk factors may be involved.

Contributions of maternal and paternal adiposity and smoking to adult offspring adiposity and cardiovascular risk: the Midspan Family Study.

OBJECTIVE: Obesity has some genetic basis but requires interaction with environmental factors for phenotypic expression. We examined contributions of gender-specific parental adiposity and smoking to adiposity and related cardiovascular risk in adult offspring. DESIGN: Cross-sectional general population survey. SETTING: Scotland. PARTICIPANTS: 1456 of the 1477 first generation families in the Midspan Family Study: 2912 parents (aged 45-64 years surveyed between 1972 and 1976) who had 1025 sons and 1283 daughters, aged 30-59 years surveyed in 1996. MAIN MEASURES: Offspring body mass index (BMI), waist circumference (WC), cardiometabolic risk (lipids, blood pressure and glucose) and cardiovascular disease as outcome measures, and parental BMI and smoking as determinants. All analyses adjusted for age, socioeconomic status and family clustering and offspring birth weight. RESULTS: Regression coefficients for BMI associations between father-son (0.30) and mother-daughter (0.33) were greater than father-daughter (0.23) or mother-son (0.22). Regression coefficient for the non-genetic, shared-environment or assortative-mating relationship between BMIs of fathers and mothers was 0.19. Heritability estimates for BMI were greatest among women with mothers who had BMI either <25 or ≥30 kg/m(2). Compared with offspring without obese parents, offspring with two obese parents had adjusted OR of 10.25 (95% CI 6.56 to 13.93) for having WC ≥102 cm for men, ≥88 cm women, 2.46 (95% CI 1.33 to 4.57) for metabolic syndrome and 3.03 (95% CI 1.55 to 5.91) for angina and/or myocardial infarct (p<0.001). Neither parental adiposity nor smoking history determined adjusted offspring individual cardiometabolic risk factors, diabetes or stroke. Maternal, but not paternal, smoking had significant effects on WC in sons (OR=1.50; 95% CI 1.13 to 2.01) and daughters (OR=1.42; 95% CI 1.10 to 1.84) and metabolic syndrome OR=1.68; 95% CI 1.17 to 2.40) in sons. CONCLUSIONS: There are modest genetic/epigenetic influences on the environmental factors behind adverse adiposity. Maternal smoking appears a specific hazard on obesity and metabolic syndrome. A possible epigenetic mechanism linking maternal smoking to obesity and metabolic syndrome in offspring is proposed. Individuals with family histories of obesity should be targeted from an early age to prevent obesity and complications.

A cardiovascular disease policy model that predicts life expectancy taking into account socioeconomic deprivation.

OBJECTIVES: A policy model is a model that can evaluate the effectiveness and cost-effectiveness of interventions and inform policy decisions. In this study, we introduce a cardiovascular disease (CVD) policy model which can be used to model remaining life expectancy including a measure of socioeconomic deprivation as an independent risk factor for CVD. DESIGN: A state transition model was developed using the Scottish Heart Health Extended Cohort (SHHEC) linked to Scottish morbidity and death records. Individuals start in a CVD-free state and can transit to three CVD event states plus a non-CVD death state. Individuals who have a non-fatal first event are then followed up until death. Taking a competing risk approach, the cause-specific hazards of a first event are modelled using parametric survival analysis. Survival following a first non-fatal event is also modelled parametrically. We assessed discrimination, validation and calibration of our model. RESULTS: Our model achieved a good level of discrimination in each component (c-statistics for men (women)-non-fatal coronary heart disease (CHD): 0.70 (0.74), non-fatal cerebrovascular disease (CBVD): 0.73 (0.76), fatal CVD: 0.77 (0.80), fatal non-CVD: 0.74 (0.72), survival after non-fatal CHD: 0.68 (0.67) and survival after non-fatal CBVD: 0.65 (0.66)). In general, our model predictions were comparable with observed event rates for a Scottish randomised statin trial population which has an overlapping follow-up period with SHHEC. After applying a calibration factor, our predictions of life expectancy closely match those published in recent national life tables. CONCLUSIONS: Our model can be used to estimate the impact of primary prevention interventions on life expectancy and can assess the impact of interventions on inequalities.

Update on blood culture-negative endocarditis.

Blood culture-negative endocarditis is often severe, and difficult to diagnose. The rate of non-documented infective endocarditis has decreased with the advent of molecular biology - improved performance for the diagnosis of bacterial endocarditis with blood cultures sterilized by previous antibacterial treatment - and cardiac surgery - access to the main infected focus, the endocardium, for half of the patients. Blood culture-negative endocarditis are classified in 3 main categories: (i) bacterial endocarditis with blood cultures sterilized by previous antibacterial treatment (usually due to usual endocarditis-causing bacteria, i.e. streptococci, more rarely staphylococci, or enterococci); (ii) endocarditis related to fastidious microorganisms (e.g. HACEK bacteria; defective streptococci - Gemella, Granulicatella, and Abiotrophia sp. - Propionibacterium acnes, Candida sp.): in these cases, prolonged incubation will allow identifying the causative pathogen in a few days; (iii) and the "true" blood culture-negative endocarditis, due to intra-cellular bacteria that cannot be routinely cultured in blood with currently available techniques: in France, these are most frequently Bartonella sp., Coxiella burnetti (both easily diagnosed by ad hoc serological tests), and Tropheryma whipplei (usually diagnosed by PCR on excised cardiac valve tissue). Non-infective endocarditis is rare, mostly limited to marantic endocarditis, and the rare endocarditis related to systemic diseases (lupus, Behçet).

Plasma sarcosine does not distinguish early and advanced stages of prostate cancer.

INTRODUCTION: Diagnosis of prostate cancer by prostate specific antigen (PSA) is error-prone and cannot distinguish benign prostatic hyperplasia (BPH) from malignant disease, nor identify aggressive and indolent types. METHODS: We determined serum sarcosine (N-methylglycine) in 328 cancer patients by gas chromatography (GC)/mass spectroscopy (MS) and searched for correlations with early (stage T1/T2) and advanced (stage T3/T4) disease. RESULTS: Serum sarcosine of male control patients ranged from 1.7 µmol/l to 4.8 µmol/l. In prostate cancer patients, sarcosine ranged from 2.8 µmol/l to 20.1 µmol/l. Expressed as the sarcosine/alanine ratio, serum control values were 9.4 ± 5.5 x 10(-3) (mean ± SD) compared with 21.6 ± 9.0; 28.5 ± 16.6; 22.7 ± 7.7 and 22.2 ± 11.0 for patients diagnosed with T1, T2, T3 and T4 prostate tumours, respectively. The small differences between T1, T2, T3 and T4 patients were not statistically significant (p=0.51). However, the conventional PSA marker significantly correlated with T stage in these patients (r=0.63; p<0.009). CONCLUSIONS: The median sarcosine/alanine ratios among patients with early and advanced prostatic cancer ranged from 21.6 ± 9.0 to 28.5 ± 16.6 and were fairly constant, showing no statistically significant differences between T-stages. The results are consistent with published data in urine and serum which find differences between controls and patients with metastatic prostate cancer to be small and sarcosine to be uninformative regarding prostate cancer progression. By multi-comparison of PSA with T-stages in the same group of patients, we found significant correlations confirming the well-known merits and limitations of this marker.

Development of a surface scanning soil analysis instrument.

ANSTO is developing a nuclear field instrument for measurement of soil composition; particularly carbon. The instrument utilises the neutron activation approach with clear advantages over existing soil sampling and laboratory analysis. A field portable compact pulsed neutron generator and γ-ray detector are used for PGNAA and INS techniques simultaneously. Many elements can be quantified from a homogenised soil volume equivalent to the top soil layers. Results from first test experiments and current developments are reported.

Plasma free fatty acid reference interval in South African neonates in the first week of life.

BACKGROUND: Non-esterified fatty acid (NEFA) levels are an important diagnostic tool in the investigation of neonatal hypoglycaemia. NEFA reference intervals have not been reported for neonates previously. METHODS: The objective of this study was to determine an NEFA reference interval for neonates. RESULTS: Heparinized plasma obtained from 106 healthy neonates in the first week of life was analysed using the Roche "Free fatty acid, Half-micro test" kit. Results were then analysed statistically for normality (Shapiro-Wilk test) and reference interval determined non-parametrically (bootstrap method). CONCLUSIONS: NEFA levels displayed a non-Gaussian distribution and the reference interval (2.5th and 97.5th percentiles) was 0.2-1.5 mmol/L (90% confidence intervals 0.1-0.3 and 1.4-2.0 mmol/L, respectively). The NEFA reference interval in South African neonates less than a week old is similar to that described in infants (1-12 months), indicating that this reference range can be used over the entire neonatal period.

A survey of foot problems in juvenile idiopathic arthritis.

BACKGROUND: Evidence suggests that foot problems are common in juvenile idiopathic arthritis (JIA), with prevalence estimates over 90%. The aim of this survey was to describe foot-related impairment and disability associated with JIA and foot-care provision in patients managed under modern treatment paradigms, including disease-modifying anti-rheumatic drugs (DMARDs) and biologic therapies. METHODS: The Juvenile Arthritis Foot Disability Index (JAFI), Child Health Assessment Questionnaire (CHAQ), and pain visual analogue scale (VAS) were recorded in 30 consecutive established JIA patients attending routine outpatient clinics. Foot deformity score, active/limited joint counts, walking speed, double-support time (s) (DS) and step length symmetry index % (SI) were also measured. Foot-care provision in the preceding 12 months was determined from medical records. RESULTS: Sixty-three per cent of children reported some foot impairment, with a median (range) JAFI subscale score of 1 (0-3); 53% reported foot-related activity limitation, with a JAFI subscale score of 1 (0-4); and 60% reported participation restriction, with a JAFI subscale score of 1 (0-3). Other reported variables were CHAQ 0.38 (0-2), VAS pain 22 (0-79), foot deformity 6 (0-20), active joints 0 (0-7), limited joints 0 (0-31), walking speed 1.09 m/s (0.84-1.38 m/s), DS 0.22 s (0.08-0.26 s) and SI +/-4.0% (+/-0.2-+/-31.0%). A total of 23/30 medical records were reviewed and 15/23 children had received DMARDS, 8/23 biologic agents and 20/23 multiple intra-articular corticosteroid injections. Ten children received specialist podiatry care comprising footwear advice, orthotic therapy and silicone digital splints together with intrinsic muscle strengthening exercises. CONCLUSION: Despite frequent use of DMARD/biologic therapy and specialist podiatry-led foot care, foot-related impairment and disability persists in some children with JIA.

Impact of deprivation on cardiovascular risk factors in people with diabetes: an observational study.

AIM: To examine the influence of deprivation on prevalence of diabetes and of cardiovascular disease risk factors in people with diabetes. METHODS: Cross-sectional study of 52 280 people in diabetes registers of Greater Glasgow and Lothian NHS Board areas linked to hospital admission data. Results Age- and sex-adjusted prevalence of diabetes increased from 2.3% in the least deprived quintile (Q1) to 3.3% in the most deprived quintile (Q5; P < 0.001), as did prevalence of vascular disease (Q1 20%, Q5 27%; P < 0.001). Prevalence of current smoking (Q1 13%, Q5 32%; P < 0.001), obesity (Q1 38%, Q5 51%; P < 0.001) and above-target glycated haemoglobin (HbA(1c); > or = 7.5%: Q1 46% vs. Q5 47%; P = 0.01) were higher in the most deprived quintile. In contrast, the proportion of people with above-target cholesterol were similar (proportion > or = 5.0 mmol/l: Q1 26%, Q5 24%; P = 0.07) and the proportion of people with above-target systolic blood pressure (SBP) was lower (SBP > or = 140 mmHg: Q1 44%, Q5 37%; P = 0.02) in the most deprived quintile. In people with diabetes and prevalent vascular disease, deprivation was associated with failure to reach cholesterol target [odds ratio cholesterol > or = 5.0 mmol/l: Q5 vs. Q1 1.23 (1.04-1.45) P = 0.013]. SBP and cholesterol were markedly lower compared with previous population surveys. CONCLUSIONS: The burden of diabetes and vascular disease is greater in more deprived populations. Our data confirm a major advance in management of cholesterol and blood pressure management. Deprivation is still associated with failure to reach cholesterol targets in secondary prevention as well as higher prevalence of obesity and smoking.

Living and health conditions of Palestinian refugees in an unofficial camp in the Lebanon: a cross-sectional survey.

OBJECTIVE: To determine the living conditions and self-reported health of Palestinian refugees living in an unofficial camp in Lebanon. DESIGN: Cross-sectional survey. SETTING: Gaza displacement centre, Beirut, Lebanon. PARTICIPANTS: 97 Households and 437 residents. MAIN OUTCOME MEASURES: Household characteristics, including the number of rooms per household; access to outside air; the presence of mould and dampness. Resident characteristics, including age; educational attainment; and chronic conditions. RESULTS: Half of the households surveyed had only one room; 44% had three or more people per room; 11% had no external ventilation; 49% had no heating; 54% had mould and dampness. The use of wood or charcoal for heating was associated with an increase in mould and dampness (p = 0.015). 135 Members of the population (31%) were aged under 15 years; 130 (30%) had a chronic condition. Logistic regression results showed that overcrowding (odds ratio (OR) 3.26) and a member of the household living in Gaza buildings for more than 15 years (OR 0.48) were significantly associated with children under 15 years. Age over 45 years (OR 5.32), a member of the household in full-time employment (OR 0.58) and a member of the household living in Gaza buildings for more than 15 years (OR 1.71) were significantly associated with chronic disease. CONCLUSION: This study demonstrates the poor conditions under which Palestinian refugees in unofficial camps live, resembling the slum housing of the United Kingdom in the last century. In the absence of routine data collection, research may be the only way to obtain such data for future public and environmental health planning.

Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI-74330, using a murine CXCR3 internalization assay.

BACKGROUND AND PURPOSE: Pharmacokinetic/pharmacodynamic (PK/PD) models are necessary to relate the degree of drug exposure in vivo to target blockade and pharmacological efficacy. This manuscript describes a murine agonist-induced CXCR3 receptor internalization assay and demonstrates its utility for PK/PD analyses. EXPERIMENTAL APPROACH: Activated murine DO11.10 cells were incubated with agonist in the presence or absence of a CXCR3 antagonist and changes in surface CXCR3 expression were detected by flow cytometry. For PK/PD analysis, mice were dosed with a small molecule CXCR3 antagonist, NBI-74330, (100 mg kg(-1)) orally or subcutaneously and plasma samples taken at specified timepoints for the CXCR3 internalization assay. KEY RESULTS: Surface CXCR3 expression was specifically decreased in response to CXCL9, CXCL10 and CXCL11. CXCL11 was the most potent CXCR3 agonist in buffer (pA50=9.23+/-0.26) and the pA50 for CXCL11 was unaltered in murine plasma (pA50=9.17+/-0.15). The affinity of a small molecule CXCR3 antagonist, NBI-74330, was obtained in the absence or presence of plasma (buffer pA2 value: 7.84+/-0.14; plasma pKB) value 6.36+/-0.01). Administration of NBI-74330 to mice resulted in the formation of an N-oxide metabolite, also an antagonist of CXCR3. Both antagonists were detectable up to 7 h post oral dose and 24 h post subcutaneous dose. Measurement of CXCR3 internalization demonstrated significant antagonism of this response ex vivo, 24 h following subcutaneous administration of NBI-74330. CONCLUSIONS AND IMPLICATIONS: The CXCR3 receptor internalization assay provides a robust method for determining agonist potency orders, antagonist affinity estimates and PK/PD analyses, which discriminate between dosing regimens for the CXCR3 antagonist NBI-74330.

Neuropsychological abnormalities in patients with dementia in CRF 01_AE HIV-1 infection.

HIV-associated dementia (HAD) is not firmly established in patients with circulating recombinant form (CRF) 01_AE HIV-1. In this study, we compared neuropsychological performance among 15 Thai individuals with HAD, 15 Thai individuals without HAD, and 30 HIV-negative control subjects. HIV-1 participants were highly active anti-retroviral therapy naive and matched by age, education, and CD4 count. Neuropsychological testing abnormalities were identified in most cognitive domains among HAD vs HIV-negative participants, confirming the presence of HAD in CRF01_AE.