SOX transcription factors direct TCF-independent WNT/ß-catenin responsive transcription to govern cell fate in human pluripotent stem cells.

WNT/ß-catenin signaling controls gene expression across biological contexts from development and stem cell homeostasis to diseases including cancer. How ß-catenin is recruited to distinct enhancers to activate context-specific transcription is unclear, given that most WNT/ß-catenin-responsive transcription is thought to be mediated by TCF/LEF transcription factors (TFs). With time-resolved multi-omic analyses, we show that SOX TFs can direct lineage-specific WNT-responsive transcription during the differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm and neuromesodermal progenitors. We demonstrate that SOX17 and SOX2 are required to recruit ß-catenin to lineage-specific WNT-responsive enhancers, many of which are not occupied by TCFs. At TCF-independent enhancers, SOX TFs establish a permissive chromatin landscape and recruit a WNT-enhanceosome complex to activate SOX/ß-catenin-dependent transcription. Given that SOX TFs and the WNT pathway are critical for specification of most cell types, these results have broad mechanistic implications for the specificity of WNT responses across developmental and disease contexts.

Development of a Standard Reference Material for metabolomics research.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Serum concentrations of an aflatoxin-albumin adduct in the National Health and Nutrition Examination Survey (NHANES) 1999-2000.

BACKGROUND: During 1998, weather conditions in the United States favored the growth of Aspergillus species leading to widespread contamination of Midwestern and Southern corn with hepatotoxic and hepatocarcinogenic aflatoxins. We designed a study to provide the first national prevalence estimate of aflatoxin exposure using the National Health and Nutrition Examination Survey (NHANES), a representative cross-sectional survey of the noninstitutionalized civilian population of the US. METHODS: Isotope dilution liquid chromatography-tandem mass spectrometry was used to quantitate serum concentrations of aflatoxin B1-lysine in a one-third random subset of participants from NHANES 1999-2000. RESULTS: About 1% of the U.S. population had detectable levels (≥0.02µg/l) of aflatoxin B1-lysine. Of those with detectable levels, the geometric mean (95% confidence interval) was 0.038 (0.024-0.060) µg/l (equivalent to 0.842 (0.530-1.34) pg/mg albumin). The highest value was 0.2µg/l (4.43pg/mg albumin). Based on liver function biomarkers, there was no evidence of increased liver dysfunction in these persons. CONCLUSIONS: During a time when exposure to aflatoxins in food products might have been expected to be increased, we identified few exposed persons. Although none of the subgroup analyses provided reliable estimates due to high relative standard errors, they suggested that additional targeted surveillance may be warranted.

Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum.

BACKGROUND: An ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D(2) (25OHD(2)), 25-hydroxyvitamin D(3) (25OHD(3)) and the C-3 epimer of 25OHD(3) (epi-25OHD(3)) in human serum. METHODS: Tri- and hexa-deuterated internal standards were added to serum (100 µl) to monitor recovery. Liquid-liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8-100 nmol/L 25OHD(2,) 12-150 nmol/L 25OHD(3), and 4-50 nmol/L epi-25OHD(3)] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1×100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min per sample; 25OHD(3) and epi-25OHD(3) were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD(2), m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD(3), m/z 383.3>365.3 (105.1 qualifier); d(3)-25OHD(2), m/z 398.3>380.3; and d(6)-25OHD(3), m/z 389.3>371.3. RESULTS: Recovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20 nmol/l. Bias averaged <5%. Detection limits were <5 nmol/l. Median (nmol/l) 25OHD(2), 25OHD(3) and epi-25OHD(3) were quantitated in 98 blood donors (

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry.

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

Measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method.

BACKGROUND: Measurement of vitamin D molecules are important in the management of patients with bone disease. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 25OHD(3) and 25OHD(2) in human serum and compared it to the traditionally used DiaSorin radioimmunoassay (RIA). METHODS: Serum samples (200 microl) were treated with acetonitrile and centrifuged to remove protein. An online solid-phase extraction procedure was used. Calibration solutions (5-100 ng/ml) of 25OHD(2) and 25OHD(3) were prepared using 4% albumin in phosphate-buffered saline. Chromatography: C18 column, isocratic ethanol/water (83/17, v/v). Mass spectrometry system: atmospheric pressure chemical ionization in the positive ion mode. Transitions: 25OHD(3), m/z 401.4-->383.4; 25OHD(2), m/z 413.4-->395.4; and the internal standard hexadeuterated-25OHD(3), m/z 407.7-->389.7. RESULTS: Detection limits were 0.49 ng/ml (25OHD(3)) and 1.86 ng/ml (25OHD(2)). Intra- and inter-assay coefficients of variation (CV) were <7% and <11%, respectively, for 25OHD(3) and <9% and <16%, respectively, for 25OHD(2). Recovery averaged (SD) 99% (2%) for 25OHD(3) and 95% (0.8%) for 25OHD(2). In a method comparison of 551 specimens from the National Health and Nutrition Examination Survey, the LC-MS/MS method gave values that were on average 13% higher (95%CI: 11-15%) than RIA results. CONCLUSIONS: This high throughput candidate reference method requires minimal sample preparation and is suitable for routine use for analysis of vitamin D status.

Workgroup report: public health strategies for reducing aflatoxin exposure in developing countries.

Consecutive outbreaks of acute aflatoxicosis in Kenya in 2004 and 2005 caused > 150 deaths. In response, the Centers for Disease Control and Prevention and the World Health Organization convened a workgroup of international experts and health officials in Geneva, Switzerland, in July 2005. After discussions concerning what is known about aflatoxins, the workgroup identified gaps in current knowledge about acute and chronic human health effects of aflatoxins, surveillance and food monitoring, analytic methods, and the efficacy of intervention strategies. The workgroup also identified public health strategies that could be integrated with current agricultural approaches to resolve gaps in current knowledge and ultimately reduce morbidity and mortality associated with the consumption of aflatoxin-contaminated food in the developing world. Four issues that warrant immediate attention were identified: a) quantify the human health impacts and the burden of disease due to aflatoxin exposure; b) compile an inventory, evaluate the efficacy, and disseminate results of ongoing intervention strategies; c) develop and augment the disease surveillance, food monitoring, laboratory, and public health response capacity of affected regions; and d) develop a response protocol that can be used in the event of an outbreak of acute aflatoxicosis. This report expands on the workgroup's discussions concerning aflatoxin in developing countries and summarizes the findings.

Case-control study of an acute aflatoxicosis outbreak, Kenya, 2004.

OBJECTIVES: During January-June 2004, an aflatoxicosis outbreak in eastern Kenya resulted in 317 cases and 125 deaths. We conducted a case-control study to identify risk factors for contamination of implicated maize and, for the first time, quantitated biomarkers associated with acute aflatoxicosis. DESIGN: We administered questionnaires regarding maize storage and consumption and obtained maize and blood samples from participants. PARTICIPANTS: We recruited 40 case-patients with aflatoxicosis and 80 randomly selected controls to participate in this study. EVALUATIONS/MEASUREMENTS: We analyzed maize for total aflatoxins and serum for aflatoxin B1-lysine albumin adducts and hepatitis B surface antigen. We used regression and survival analyses to explore the relationship between aflatoxins, maize consumption, hepatitis B surface antigen, and case status. RESULTS: Homegrown (not commercial) maize kernels from case households had higher concentrations of aflatoxins than did kernels from control households [geometric mean (GM) = 354.53 ppb vs. 44.14 ppb; p = 0.04]. Serum adduct concentrations were associated with time from jaundice to death [adjusted hazard ratio = 1.3; 95% confidence interval (CI), 1.04-1.6]. Case patients had positive hepatitis B titers [odds ratio (OR) = 9.8; 95% CI, 1.5-63.1] more often than controls. Case patients stored wet maize (OR = 3.5; 95% CI, 1.2-10.3) inside their homes (OR = 12.0; 95% CI, 1.5-95.7) rather than in granaries more often than did controls. CONCLUSION: Aflatoxin concentrations in maize, serum aflatoxin B1-lysine adduct concentrations, and positive hepatitis B surface antigen titers were all associated with case status. RELEVANCE: The novel methods and risk factors described may help health officials prevent future outbreaks of aflatoxicosis.

Analysis of aflatoxin B1-lysine adduct in serum using isotope-dilution liquid chromatography/tandem mass spectrometry.

A method for quantitative analysis of aflatoxin B1-lysine adduct (B1-Lys) in serum by liquid chromatography using tandem mass spectrometry (LC/MS/MS) is presented. The protein in a 250-microL sample was digested in the presence of a stable-isotope internal standard during a 4-h incubation at 37 degrees C with Pronasetrade mark. B1-Lys and the internal standard were extracted using mixed-mode solid-phase extraction cartridges and eluted with 2% formic acid in methanol. Following evaporation and reconstitution, extracts were injected onto a Luna C-18(2) column and eluted with a step gradient of acetonitrile and 0.06% formic acid. The B1-Lys and the internal standard were detected in a positive ionization selective reaction monitoring mode with a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer. Calibration curves were linear for concentrations from 0.05-8.0 ng/mL. The method was validated with aflatoxin B1 dosed rat serum diluted to anticipated high and low concentrations. Total imprecision determined from 30 measurements over 15 days was 5.6% and 9.1%, respectively. Recoveries of 78.8 +/- 6.4% for B1-Lys and 85.4 +/- 12.4% for the internal standard were based on the full extraction and reconstitution processes. The method can be used to quantitate B1-Lys at the 0.5 pg/mg albumin level and is suitable for routine analysis.