Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

OBJECTIVE: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma. METHODS: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent OC and FTE FFPE tissues. MDMs were tested using Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) assays in pre-treatment plasma from women newly diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. RESULTS: Thirty-three MDMs showed marked methylation fold changes (10 to >1000) across all OC subtypes vs FTE. Eleven MDMs (GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, RIPPLY3, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (73 (80%) high-grade serous (HGS)) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95% CI, 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86-0.96)). Among the 5 stage I/II HGS OCs included, all were correctly identified. CONCLUSIONS: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded candidate MDMs for OC that performed with high sensitivity and specificity in plasma. Larger plasma-based OC MDM studies, including testing of pre-diagnostic specimens, are warranted.

Detection of organophosphate pesticides using a prototype liquid crystal monitor.

The precision and accuracy of a prototype wearable liquid crystal monitor (LCM) for the measurement of airborne organophosphate pesticide concentrations was explored in a series of laboratory experiments. LCM response to vapor-phase and aerosol diazinon was compared to concentrations obtained using a standard reference method (NIOSH 5600) at concentrations ranging from approximately 8 to 108 ppb (parts per billion) over durations of 2 to 80 hours. Temperature ( approximately 25, 30, and 35 degrees C) and relative humidity (15, 50, and 85%) were varied to estimate the effect of these factors on LCM performance. The LCM response to vapor phase pesticide exposure was linear for concentrations in the range of 8-20 ppb. At exposure concentrations above approximately 20 ppb, however, there was a decline in monitor response and measurement precision. Elevated temperatures improved diazinon vapor-only measurement precision, while increased relative humidity reduced LCM response at the extremes of tested temperatures. Compared to vapor-only exposures, the LCM was less sensitive to diazinon aerosol concentrations, but displayed reasonable precision over a relatively large range of exposures (29 to 1190 ppb-hr). Further efforts to characterize temperature and humidity effects and improve low-end sensitivity would likely provide a portable personal exposure monitor or environmental sensor for this widely used class of pesticides.

Seed phosphorus and inositol phosphate phenotype of barley low phytic acid genotypes.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (Ins P(6) or "phytic acid") typically represents approximately 75% of the total phosphorus and >80% of soluble myo-inositol (Ins) phosphates in seeds. The seed phosphorus and Ins phosphate phenotypes of four non-lethal barley (Hordeum vulgare L.) low phytic acid mutations are described. In seeds homozygous for M 635 and M 955 reductions in Ins P(6), approximately 75 and >90% respectively, are accompanied by reductions in other Ins phosphates and molar-equivalent increases in Pi. This phenotype suggests a block in supply of substrate Ins. In seeds homozygous for barley low phytic acid 1-1 (lpa1-1), a 45% decrease in Ins P(6) is mostly matched by an increase in Pi but also accompanied by small increases in Ins(1,2,3,4,6)P(5). In seeds homozygous for barley lpa2-1, reductions in seed Ins P(6) are accompanied by increases in both Pi and in several Ins phosphates, a phenotype that suggests a lesion in Ins phosphate metabolism, rather than Ins supply. The increased Ins phosphates in barley lpa2-1 seed are: Ins(1,2,3,4,6)P(5); Ins(1,2,4,6)P(4) and/or its enantiomer Ins(2,3,4,6)P(4); Ins(1,2,3,4)P(4) and/or its enantiomer Ins(1,2,3,6)P(4); Ins(1,2,6)P(3) and/or its enantiomer Ins(2,3,4)P(3); Ins(1,5,6)P(3) and/or its enantiomer Ins(3,4,5)P(3) (the methods used here cannot distinguish between enantiomers). This primarily "5-OH" series of Ins phosphates differs from the "1-/3-OH" series observed at elevated levels in seed of the maize lpa2 genotype, but previous chromosomal mapping data indicated that the maize and barley lpa2 loci might be orthologs of a single ancestral gene. Therefore one hypothesis that might explain the differing lpa2 phenotypes is that their common ancestral gene encodes a multi-functional, Ins phosphate kinase with both "1-/-3-" and "5-kinase" activities. A putative pyrophosphate-containing Ins phosphate, possibly an Ins P(7), was also observed in the mature seed of all barley genotypes except lpa2-1. Barley M 955 indicates that at least for this species, the ability to accumulate Ins P(6) can be nearly abolished while retaining at least short-term ( approximately 1.0 years) viability.