Biochemical, kinetic, and spectroscopic characterization of Ruegeria pomeroyi DddW - A mononuclear iron-dependent DMSP lyase

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW

Comparison of genetic variances in F2 and backcross populations of maize

Second cycle breeding is characterized by the improvement of an elite line through the addition of favorable alleles from a complimentary line. Second cycle breeding, utilizing backcross and F[subscript]2 generations, has become the predominant type of line development program in the U.S. maize industry. Epistasis and linkage have been identified as possible limits to progress from selection in F[subscript]2 and backcross populations;Objectives of this study were: (1) to provide empirical data comparing the testcross progenies of the F[subscript]2, F[subscript]2-Syn 8, and first backcross generations for means, genotypic variances, heritabilities, frequency distributions, selection responses, and trait correlations, (2) to examine the importance of epistasis in the generation testcrosses derived from inbred lines B73, B84, and Mo17, (3) to examine changes in the testcross population derived from the F[subscript]2 generation following eight generations of random mating, and (4) to recommend the superior type of segregating population to develop from the cross of elite inbred lines B73 and B84;Significant positive digenic epistatic effects were detected for grain yield. The estimate of epistasis obtained by weighted least squares regression of generation means was 0.20 Mg ha[superscript]-1. Ear height also contained significant digenic epistatic effects and evidence for higher order epistasis was detected. Epistatic effects were not significant contributors to genetic variation for any other traits. Due to a lack of significant genetic variance in the superior backcross generation, epistasis was concluded to be of minor importance in the choice of segregating population to develop for selection of superior yielding lines;The F[subscript]2 population had significantly greater mean grain yield (0.27 Mg ha[superscript]-1) while significantly lower genetic variance from 5.80 x 10[superscript]-2 to 9.02 x 10[superscript]-2 in the F[subscript]2-Syn 8 population. Random mating was considered of little value, however, considering the increased time to produce progeny for evaluation when selection is for individuals with increased grain yield;Applied breeding programs generally utilize a high selection intensity for grain yield in elite by elite segregating populations. The superior type of segregating population to develop from the cross of inbreds B73 by B84 under high selection intensity for grain yield was the F[subscript]2 generation. The backcross to B84 was the superior type of segregating population under low selection intensities

Interpopulation Genetic Variance after Reciprocal Recurrent Selection in BSSS and BSCB1 Maize Populations

Reciprocal recurrent selection is a breeding procedure designed to improve two populations simultaneously by selecting for performance in the interpopulation cross. The objective of this study was to evaluate the effects of II cycles of selection on the mean, genetic variance, and correlations amongtr aits in the interpopulationc ross of the Iowa Stiff Stalk Synthetic (BSSS) and Iowa Corn Borer Synthetic no. 1 (BSCBI) maize (Zea mays L.) populations. Oneh undredr eciprocal full-sib families from Cycle 0, Cycle 5, and Cycle II of the interpopulation cross between BSSS and BSCBI were evaluated at two locations in 1989 and three locations in 1990. Grain yield increased 0.25 Mg ha-1 per cycle (6.46%) after II cycles of selection, grain moisture increased 0.85 g kg-1 per cycle, root lodging decreased 0.23%, and stalk lodging decreased 1.64% (all changes significant at P = 0.01). Estimates of genetic variance for all traits were significantly different from zero, except for root lodging in Cycle II. Genetic variance for grain yield decreased after II cycles of selection, although the differences among cycles were not significant. Root lodging, stalk lodging, anthesis date, and silking date had significant decreases in genetic variance after II cycles of selection. Changeisn heritability generally paralleled changes in genetic variance. Genotypic and phenotypic correlations were seemingly unchangeda cross cycles of selection. The results indicate that reciprocal recurrent selection has been effective in increasing the mean performance of the population cross while maintaining genetic variance

Epistasis in an Elite Maize Hybrid and Choice of Generation for Inbred Line Development

Second cycle breeding, utilizing backcross and F2 generations, has become the predominant type of line development program in the U.S. maize (Zea mays L.) industry. Epistasis and linkage have been identified as possible limits to progress from selection in F2 and backcross populations. Objectives of this study were to determine the importance of epistasis in an elite maize hybrid and to determine the appropriate generation for initiation of inbred line development. Six generations of progeny were developed from B73 and B84: the two parental generations, Pl (B73) and P2 (B84); the F2 generation [(B73 × B84) F2]; the BCP1 generation [(B73 × B84) × B73]; the BCP2 generation [(B73 × B84) × B84]; and the F2-Syn 8 generation (the F2 generation random mated for eight generations). Testcross progeny were produced by crossing random S9 plants from each of the six generations onto the inbred tester Mo17. One-hundred progeny of F2 × Mo17 and F2-Syn 8 × Mo17 and 50 progeny of BCP1 × Mo17 and BCP2 × Mo17, as well as the parental testcrosses, were harvested. The experiment was evaluated at four locations in 1990 and three locations in 1991. Epistatic effects were significant for grain yield and grain moisture, and accounted for 21 and 18% of the variation among generation means, respectively. The genetic variance and heritability for grain yield ranked F2-Syn 8 \u3e F2 \u3e BCP1 \u3e BCP2, but differences among generations were not significant. The predicted gains for each generation ranked in agreement with the heritability. Under low selection intensities (a = 20%), the predicted mean (usefulness) ranked the generations BCP22 \u3e F2 \u3e\u3e F2-Syn 8 \u3e BCPI. Under high selection intensity (a = 1%), usefulness ranked the generations F2 \u3e F2-Syn 8 \u3e BCP2 \u3e BCP1. The choice between F2and backcrosses as source populations is primarily a function of selection intensity. Our results suggest little to no advantage of random mating the F2 before initiating selection and inbreeding

A Community Needs Assessment for the Perry Community School District

76 leaves. Advisor: Richard D. BrooksThe problem. The purpose of this study was to identify the major educational needs of the Perry Community School District as perceived by parents, teachers, students, and other community patrons, thereby assisting the Board and administration in making changes in the schools' programs necessary to meet those needs and aspirations of the people of the community. Procedure. The community meeting technique was employed to determine the reactions by parents, teachers, students, and other community patrons. At the community meeting participants were asked to rate the importance of eighteen educational goals. Participants were then placed in groups of four and each group was required to obtain group consensus on the rating of each goal. Finally, individual participants were asked to rate the schools' performance in meeting each of the eighteen goals. The results were analyzed by computing the mean and variance of the group consensus ratings of each goal. Also computed for each goal were the median of the individual performance ratings, the relative percentages of responses for each of five rating categories used, and the median performance rating of each goal for each of the different subpopulations. Findings and Conclusions. The immediate purpose of this study was to identify the major educational goals and needs of the school district and to obtain community perceptions of how well present school programs are doing in meeting those goals. Sixty-seven people participated in the community meeting. The goals ranked most important by the participants centered around developing the basic skills of written and oral communication, gaining a general education, and developing a desire for learning. These goals received average ratings of 4.471, 4.235, and 3.941 respectively on a fivepoint scale with five the highest possible score. Community members are in agreement regarding the importance of the educational goals and are generally satisfied that present school programs are meeting their expectations in achieving those goals. There are virtually no discrepancies between present school programs and those desired by the community. Recommendations. The results of this study can be helpful to the Board in making those decisions for allocating resources and initiating programs and may serve as indicators as to what kinds of Board decisions will or will not receive public support

Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate-catabolizing bacteria

Dimethylsulfoniopropionate (DMSP) cleavage, yielding dimethyl sulfide (DMS) and acrylate, provides vital carbon sources to marine bacteria, is a key component of the global sulfur cycle and effects atmospheric chemistry and potentially climate. Acrylate and its metabolite acryloyl-CoA are toxic if allowed to accumulate within cells. Thus, organisms cleaving DMSP require effective systems for both the utilization and detoxification of acrylate. Here, we examine the mechanism of acrylate utilization and detoxification in Roseobacters. We propose propionate-CoA ligase (PrpE) and acryloyl-CoA reductase (AcuI) as the key enzymes involved and through structural and mutagenesis analyses, provide explanations of their catalytic mechanisms. In most cases, DMSP lyases and DMSP demethylases (DmdAs) have low substrate affinities, but AcuIs have very high substrate affinities, suggesting that an effective detoxification system for acylate catabolism exists in DMSP-catabolizing Roseobacters. This study provides insight on acrylate metabolism and detoxification and a possible explanation for the high Km values that have been noted for some DMSP lyases. Since acrylate/acryloyl-CoA is probably produced by other metabolism, and AcuI and PrpE are conserved in many organisms across all domains of life, the detoxification system is likely relevant to many metabolic processes and environments beyond DMSP catabolism

Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate-catabolizing bacteria

Dimethylsulfoniopropionate (DMSP) cleavage, yielding dimethyl sulfide (DMS) and acrylate, provides vital carbon sources to marine bacteria, is a key component of the global sulfur cycle and effects atmospheric chemistry and potentially climate. Acrylate and its metabolite acryloyl-CoA are toxic if allowed to accumulate within cells. Thus, organisms cleaving DMSP require effective systems for both the utilization and detoxification of acrylate. Here, we examine the mechanism of acrylate utilization and detoxification in Roseobacters. We propose propionate-CoA ligase (PrpE) and acryloyl-CoA reductase (AcuI) as the key enzymes involved and through structural and mutagenesis analyses, provide explanations of their catalytic mechanisms. In most cases, DMSP lyases and DMSP demethylases (DmdAs) have low substrate affinities, but AcuIs have very high substrate affinities, suggesting that an effective detoxification system for acylate catabolism exists in DMSP-catabolizing Roseobacters. This study provides insight on acrylate metabolism and detoxification and a possible explanation for the high Km values that have been noted for some DMSP lyases. Since acrylate/acryloyl-CoA is probably produced by other metabolism, and AcuI and PrpE are conserved in many organisms across all domains of life, the detoxification system is likely relevant to many metabolic processes and environments beyond DMSP catabolism

Evaluation of a Motivation and Psycho-Educational Guided Self-Help Intervention for People with Eating Disorders (MOPED)

This is the peer reviewed version of the following article: BREWIN, N. ... et al, 2016. Evaluation of a motivation and psycho-educational guided self-help intervention for people with eating disorders (MOPED). European Eating Disorders Review, 24(3), pp. 241–246, which has been published in final form at http://dx.doi.org/10.1002/erv.2431. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.High dropout rates and poor levels of engagement are well documented for patients with eating disorders. Utilising motivational techniques and providing psycho-education have been suggested as ways to reduce treatment disengagement. This study aimed to evaluate the effect of a newly developed motivational and psycho-educational guided self-help intervention (MOPED) for people with eating disorders on engagement and retention in therapy. Patients who received MOPED pre-treatment (n = 79) were compared to a diagnosis matched group of patients receiving treatment as usual (TAU; n = 79). The study found that patients receiving MOPED had a higher engagement rate than those within the TAU group. Specifically, patients in the anorexic spectrum were found to present with both higher rates of engagement and completion of therapy when issued with MOPED in comparison to TAU. Self-help packages using motivational style could be a valuable and cost effective intervention for patients with eating disorders

Eicosanoid signalling blockade protects middle-aged mice from severe COVID-19

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D–PGD2/PTGDR pathway is a useful target for therapeutic interventions.This work is supported in part by grants from the National Institutes of Health USA (NIH; P01 AI060699 (S.P. and P.B.M.) and R01 AI129269 (S.P.)) and BIOAGE Labs (S.P.). The Pathology Core is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH grant P30 DK-54759) and the Cystic Fibrosis Foundation. P.B.M. is supported by the Roy J. Carver Charitable Trust. L.-Y.R.W. is supported by Mechanism of Parasitism Training Grant (T32 AI007511). We thank M. Gelb (University of Washington) for Pla2g2d−/− mice.Peer reviewe