Determination of Absolute Protein Content of Hepatic CYP4F Enzymes in Human Liver Microsomes Using LC/MS/MS Methodology and Comparison with Immunoquantification and Enzyme Activity

The purpose of the work presented herein was to determine the absolute protein content of multiple cytochrome P450 (CYP) enzymes in individual donor human liver microsome (HLM) samples. The CYP4F subfamily of enzymes have recently been identified to be involved in the metabolism of endogenous compounds (arachidonic acid, leukotriene B4), nutrients (vitamin K1 and vitamin E), and xenobiotics (parfuramidine (DB289), DB1230, fingolimod). The CYP3A subfamily of enzymes are known to metabolize a wide variety of physiologically and pharmaceutically important substances. The determination of the absolute enzyme protein content and the inter-individual variability in the expression of these enzymes is important in understanding the effects they may have on the disposition of both endogenous and exogenous substances. Therefore, the absolute enzyme protein content in 20 individual donor HLM was determined by LC/MS/MS for CYP4F2, CYP4F3B, CYP3A, CYP3A4, and CYP3A5. The contribution of CYP4F enzymes to the overall hepatic CYP "pie" was also determined. The observed enzyme protein values were then correlated with immunoquantified protein content and enzyme activity. The enzyme protein contents determined by LC/MS/MS were well correlated with immunoquantification results (r2 ≥ 0.60) for both CYP4F and CYP3A. The CYP4F enzymes displayed significantly less (~2- to 4-fold) variability than did the CYP3A enzymes (~7- to 20-fold). The CYP4F protein contents did not correlate with primary metabolite formation rates for DB289 or DB1230. Chemical inhibition experiments were performed which provided additional evidence for the metabolism for DB289 and DB1230 by enzymes other than CYP4F enzymes. However, a lack of improved correlation for the chemical inhibition experiments suggest that the poor correlation observed for CYP4F protein content and metabolic activity is not solely due to the contributions of additional enzymes. The LC/MS/MS methodology used for the current study has thus been shown to provide a rapid and reproducible (CV ≤ 24%) method for quantitation of enzyme protein expression level that obviates the need for specific antibodies for immunoquantification, a major problem for the less commonly studied enzymes that share significant sequence homology such as the CYP4F enzymes

Phasing the Mirror Segments of the Keck Telescopes: The Broadband Phasing Algorithm

To achieve its full diffraction limit in the infrared, the primary mirror of the Keck telescope (now telescopes) must be properly phased: The steps or piston errors between the individual mirror segments must be reduced to less than 100 nm. We accomplish this with a wave optics variation of the Shack–Hartmann test, in which the signal is not the centroid but rather the degree of coherence of the individual subimages. Using filters with a variety of coherence lengths, we can capture segments with initial piston errors as large as ± 30 µm and reduce these to 30 nm—a dynamic range of 3 orders of magnitude. Segment aberrations contribute substantially to the residual errors of ~75 nm

FLOWERING LOCUS C -dependent and -independent regulation of the circadian clock by the autonomous and vernalization pathways

Background The circadian system drives pervasive biological rhythms in plants. Circadian clocks integrate endogenous timing information with environmental signals, in order to match rhythmic outputs to the local day/night cycle. Multiple signaling pathways affect the circadian system, in ways that are likely to be adaptively significant. Our previous studies of natural genetic variation in Arabidopsis thaliana accessions implicated FLOWERING LOCUS C (FLC) as a circadian-clock regulator. The MADS-box transcription factor FLC is best known as a regulator of flowering time. Its activity is regulated by many regulatory genes in the "autonomous" and vernalization-dependent flowering pathways. We tested whether these same pathways affect the circadian system. Results Genes in the autonomous flowering pathway, including FLC, were found to regulate circadian period in Arabidopsis. The mechanisms involved are similar, but not identical, to the control of flowering time. By mutant analyses, we demonstrate a graded effect of FLC expression upon circadian period. Related MADS-box genes had less effect on clock function. We also reveal an unexpected vernalization-dependent alteration of periodicity. Conclusion This study has aided in the understanding of FLC's role in the clock, as it reveals that the network affecting circadian timing is partially overlapping with the floral-regulatory network. We also show a link between vernalization and circadian period. This finding may be of ecological relevance for developmental programing in other plant species

ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing.

Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications, including DNA methylation, histone H3 dimethylation at Lys9 (H3K9me2) and monomethylation at Lys27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified; alterations in these proteins lead to reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6 have H3K27 monomethyltransferase activity, and atxr5 atxr6 double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Notably, H3K9me2 and DNA methylation are unaffected in double mutants. These results indicate that ATXR5 and ATXR6 form a new class of H3K27 methyltransferases and that H3K27me1 represents a previously uncharacterized pathway required for transcriptional repression in Arabidopsis

Pleiotropy of FRIGIDA enhances the potential for multivariate adaptation.

An evolutionary response to selection requires genetic variation; however, even if it exists, then the genetic details of the variation can constrain adaptation. In the simplest case, unlinked loci and uncorrelated phenotypes respond directly to multivariate selection and permit unrestricted paths to adaptive peaks. By contrast, 'antagonistic' pleiotropic loci may constrain adaptation by affecting variation of many traits and limiting the direction of trait correlations to vectors that are not favoured by selection. However, certain pleiotropic configurations may improve the conditions for adaptive evolution. Here, we present evidence that the Arabidopsis thaliana gene FRI (FRIGIDA) exhibits 'adaptive' pleiotropy, producing trait correlations along an axis that results in two adaptive strategies. Derived, low expression FRI alleles confer a 'drought escape' strategy owing to fast growth, low water use efficiency and early flowering. By contrast, a dehydration avoidance strategy is conferred by the ancestral phenotype of late flowering, slow growth and efficient water use during photosynthesis. The dehydration avoidant phenotype was recovered when genotypes with null FRI alleles were transformed with functional alleles. Our findings indicate that the well-documented effects of FRI on phenology result from differences in physiology, not only a simple developmental switch

The use of a cap-mounted tri-axial accelerometer for measurement of distance, lap times and stroke rates in swim training

This paper will report some of the findings from a trial which recorded accelerometer data from six elite level swimmers (three female and three male, varying primary event stroke and distance) over the course of a regular 15 week training block. Measurements from a headmounted accelerometer are used to determine when the athlete is swimming, marking of turning points (and therefore distance and lap-time measurements), and is processed by frequency analysis to determine stroke-rate. Comparison with video where available, and with training plans and literature where not, have proven this method to be accurate and reliable for determining these performance metrics. The primary objective of this project was to develop a low-cost, simple and highly usable system for use in swim coaching, feedback from elite coaches has indicated that development of this could be an extremely useful addition to their training regime

State Assessment of Demographic Data and Youth Development to Advance 4-H Programs

4-H youth development programs throughout the United States can be planned and delivered more effectively in their states by assessing demographic data and following research-based theories and models of positive youth development. A review of the research literature determined current youth development theories and models to effectively guide statewide 4-H program implementation. A state assessment was conducted for demographic areas of youth population age, race, socioeconomic status, health factors, child poverty (includes parent-guardian job status at the onset of COVID), and household structure. The Ohio 4-H Youth Development program utilized the demographic data to establish goals of becoming more diverse and inclusive. In addition, demographic data points help for targeted recruitment of youth to include families from diverse socioeconomic backgrounds, household structures, and those with health risks. Finally, implications and conclusions are presented to serve as an illustration for other states to advance their state 4-H programs and practices

An assessment of the use of sediment traps for estimating upper ocean particle fluxes

Author Posting. © Sears Foundation for Marine Research, 2007. This article is posted here by permission of Sears Foundation for Marine Research for personal use, not for redistribution. The definitive version was published in Journal of Marine Research 65 (2007): 345–416, doi: 10.1357/002224007781567621This review provides an assessment of sediment trap accuracy issues by gathering data to address trap hydrodynamics, the problem of zooplankton "swimmers," and the solubilization of material after collection. For each topic, the problem is identified, its magnitude and causes reviewed using selected examples, and an update on methods to correct for the potential bias or minimize the problem using new technologies is presented. To minimize hydrodynamic biases due to flow over the trap mouth, the use of neutrally buoyant sediment traps is encouraged. The influence of swimmers is best minimized using traps that limit zooplankton access to the sample collection chamber. New data on the impact of different swimmer removal protocols at the US time-series sites HOT and BATS are compared and shown to be important. Recent data on solubilization are compiled and assessed suggesting selective losses from sinking particles to the trap supernatant after collection, which may alter both fluxes and ratios of elements in long term and typically deeper trap deployments. Different methods are needed to assess shallow and short- term trap solubilization effects, but thus far new incubation experiments suggest these impacts to be small for most elements. A discussion of trap calibration methods reviews independent assessments of flux, including elemental budgets, particle abundance and flux modeling, and emphasizes the utility of U-Th radionuclide calibration methods.WG meetings and production of this report was partially supported by the U.S. National Science Foundation via grants to the SCOR. Individuals and science efforts discussed herein were supported by many national science programs, including the U.S. National Science Foundation, Swedish Research Council, the International Atomic Energy Agency through its support of the Marine Environmental Laboratory that also receives support from the Government of the Principality of Monaco, and the Australian Antarctic Science Program. K.B. was supported in part by a WHOI Ocean Life Institute Fellowship