A link between the cytoplasmic engulfment protein Elmo1 and the Mediator complex subunit Med31.

The cytoplasmic Elmo1:Dock180 complex acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac and functions downstream of the phagocytic receptor BAI1 during apoptotic cell clearance, and in the entry of Salmonella and Shigella into cells. We discovered an unexpected binding between Elmo1 and the Mediator complex subunit Med31. The Mediator complex is a regulatory hub for nearly all gene transcription via RNA polymerase II, bridging the general transcription machinery with gene-specific regulatory proteins. Med31 is the smallest and the most evolutionarily conserved Mediator subunit, and knockout of Med31 results in embryonic lethality in mice; however, Med31 function in specific biological contexts is poorly understood. We observed that in primary macrophages, during Salmonella infection, Elmo1 and Med31 specifically affected expression of the cytokine genes Il10 and Il33 among the >25 genes monitored. Although endogenous Med31 is predominantly nuclear localized, Elmo1 increased the cytoplasmic localization of Med31. We identify ubiquitination as a novel posttranslational modification of Med31, with the cytoplasmic monoubiquitinated form of Med31 being enhanced by Elmo1. These data identify Elmo1 as a novel regulator of Med31, revealing a previously unrecognized link between cytoplasmic signaling proteins and the Mediator complex.

Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions.

The spectral processed Förster resonance energy transfer (psFRET) imaging method provides an effective and fast method for measuring protein-protein interactions in living specimens. The commercially available linear unmixing algorithms efficiently remove the contribution of donor spectral bleedthrough to the FRET signal. However, the acceptor contribution to spectral bleedthrough in the FRET image cannot be similarly removed, since the acceptor spectrum is identical to the FRET spectrum. Here, we describe the development of a computer algorithm that measures and removes the contaminating ASBT signal in the sFRET image. The new method is characterized in living cells that expressed FRET standards in which the donor and acceptor fluorescent proteins are tethered by amino acid linkers of specific lengths. The method is then used to detect the homo-dimerization of a transcription factor in the nucleus of living cells, and then to measure the interactions of that protein with a second transcription factor.

Perchlorate exposure of the US Population, 2001-2002.

Perchlorate is commonly found in the environment and can impair thyroid function at pharmacological doses. As a result of the potential for widespread human exposure to this biologically active chemical, we assessed perchlorate exposure in a nationally representative population of 2,820 US residents, ages 6 years and older, during 2001 and 2002 as part of the National Health and Nutrition Examination Survey (NHANES). We found detectable levels of perchlorate (>0.05 microg/l) in all 2,820 urine samples tested, indicating widespread human exposure to perchlorate. Urinary perchlorate levels were distributed in a log normal fashion with a median of 3.6 microg/l (3.38 microg/g creatinine) and a 95th percentile of 14 microg/l (12.7 microg/g creatinine). When geometric means of urinary perchlorate levels were adjusted for age, fasting, sex and race-ethnicity, we found significantly higher levels of urinary perchlorate in children compared with adolescents and adults. We estimated total daily perchlorate dose for each adult (ages 20 years and older), based on urinary perchlorate, urinary creatinine concentration and physiological parameters predictive of creatinine excretion rate. The 95th percentile of the distribution of estimated daily perchlorate doses in the adult population was 0.234 microg/kg-day [CI 0.202-0.268 microg/kg-day] and is below the EPA reference dose (0.7 microg/kg-day), a dose estimated to be without appreciable risk of adverse effects during a lifetime of exposure. These data provide the first population-based assessment of the magnitude and prevalence of perchlorate exposure in the US.

Analysis of perchlorate in human urine using ion chromatography and electrospray tandem mass spectrometry.

Because of health concerns surrounding widespread exposure to perchlorate, we developed a sensitive and selective method for quantifying perchlorate in human urine using ion chromatography coupled with electrospray ionization tandem mass spectrometry. Perchlorate was quantified using a stable isotope-labeled internal standard ((18)O(4)-perchlorate) with excellent assay precision (coefficient of variation <5% for repetitively analyzed quality control material). Analytical accuracy was established by blind analysis of certified proficiency testing materials prepared in synthetic urine matrix; calculated amounts deviated minimally from true amounts, with percent differences ranging from 2% to 5%. Selective chromatography and tandem mass spectrometry reduced the need for sample cleanup, resulting in a rugged and rapid method capable of routinely analyzing 75 samples/day. The lowest reportable level (0.025 ng/mL) was sufficiently sensitive to detect perchlorate in all human urine samples evaluated to date, with a linear response range from 0.025 to 100 ng/mL. This selective, sensitive, and rapid method will help elucidate any potential associations between human exposure to low levels of perchlorate and adverse health effects.

Identification and elimination of polysiloxane curing agent interference encountered in the quantification of low-picogram per milliliter methyl tert-butyl ether in blood by solid-phase microextraction headspace analysis.

Widespread use of the gasoline additive methyl tert-butyl ether (MTBE) and the subsequent human exposure that follows have led to the need to quantify MTBE in a variety of complex biological matrixes. In this work, we demonstrate our latest MTBE quantification assay for whole blood and uncover previously unidentified contamination sources that prevented routine quantification in the low picogram per milliliter (parts per trillion, ppt) range despite a sensitive and selective analytical approach. The most significant and unexpected sources of contamination were found in reagents and laboratory materials most relevant to sample preparation and quantification. In particular, significant levels of MTBE were identified in sample vial septa that use poly(dimethylsiloxane) (PDMS)-based polymers synthesized with peroxide curing agents having tert-butyl side groups. We propose that MTBE is one of the byproducts of these curing agents, which cross-link PDMS via the methyl side groups. Residual MTBE levels of approximately 20 microg/septa are seen in septa whose formulations use these curing agents. Fortunately, these levels can be significantly reduced (i.e., <0.2 ng/septa) by additional processing. Performance achieved with this sample preparation approach is demonstrated using a mass spectrometry-based method to quantify blood MTBE levels in the low-ppt range.