Explore the vast range of titles available.

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.

Arranging biological processes into “compartments” is a key feature of all eukaryotic cells. Through this mechanism, cells can drastically increase metabolic efficiency and manage complex cellular processes more efficiently, saving space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cytochrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabolons around P450s on the surface of the ER. We␣outline direct and indirect experimental data that describes P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors.

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4'-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3beta-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.

Capsidiol is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. We developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by two well-characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well-conserved domains found within most P450 enzymes, cDNA fragments representing four distinct P450 families were amplified by PCR from a cDNA library prepared against mRNA from elicitor-treated cells. The PCR fragments were used to isolate full-length cDNAs, whose corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system. Only yeast expressing the CYP71 D20 cDNA was capable of converting both 5-epi-anstolochene and 1-deoxycapsidiol to capsidiol in vitro. Full-length 5-epi-aristolochene-1,3-dihydroxylase (CYP71D20) and CYP92A5 cDNAs were transformed into tobacco plants in both the sense and antisense orientation under the constitutive CaMV 35S promoter. Overexpression and antisense expression of the 5-epi-aristolochene-1,3-dihydroxylase, should help to discern whether this terminal step in the production of capsidiol might also be the limiting factor in capsidiol biosynthesis. Three distinct P450 genes were isolated after screening a tobacco genomic DNA library with full-length CYP71D20 and CYP92A5 cDNA probes. Two of the genes corresponded to CYP71D20 and 21, respectively. The other clone corresponded exactly to CYP92A2, a cDNA previously isolated from tobacco challenged with phytopathogenic bacteria. Molecular analysis was performed on the putative promoters of these three P450 genes as well as on the promoter of EAS4 to find common motifs which might serve as cis-elements in a conserved signal transduction pathway. To better study the regulation of individual genes and identify putative regulatory domains, a series of 5′ deletion constructs was generated from the 5′ flanking regions of each of the P450 genes isolated in this study.

Retrieving information from memory increases the likelihood the information will be remembered later. The strategic use of retrieval to enhance memory is known as retrieval practice. Teachers can exert considerable control over students’ retrieval practice, dictating when and how much students practice. Laboratory research has shown that retention benefits from increasing the amount of practice (i.e., the number of times information is retrieved) and from spacing practice out over time. Although retrieval practice is a prominent part of the learning experience in certain educational domains, such as mathematics, relatively little research has examined how retention of actual classroom content is affected by increasing the amount and spacing of retrieval practice. Here, we implemented a complete within-subjects crossing of practice amount (baseline versus increased) and practice spacing (baseline versus increased) in a precalculus course for engineering students. Practice consisted of answering quiz questions. We assessed retention of precalculus knowledge at two educationally relevant time points: the end of the precalculus course (within-semester) and the beginning of a calculus course 4 weeks later (across-semester). Within-semester retention benefited significantly from practicing more and from spacing out practice, although some evidence suggested that the effect of amount of practice was less robust than the effect of spacing. Across-semester retention benefited exclusively from increasing spacing. Given that retaining precalculus knowledge across semesters is crucial for success in higher-level mathematics, these findings support increasing spacing in real-world mathematics education. We discuss how our findings fit within the larger literature on the memory-enhancing effects of retrieval practice.

A major challenge college students face is retaining the knowledge they acquire in their classes, especially in cumulative disciplines such as engineering, where ultimate success depends on long-term retention of foundational content. Cognitive psychologists have recently recommended various techniques educators might use to increase retention. One technique (spaced retrieval practice) involves extending opportunities to retrieve course content beyond a customarily short temporal window following initial learning. Confirming the technique's utility requires demonstrating that it increases retention in real classroom settings, with commonly encountered educational content, and that gains endure into subsequent semesters. We manipulated spaced versus massed retrieval practice in a precalculus course for engineering students and followed a subset of students who proceeded into a calculus class the following semester. Spacing versus massing was manipulated within- and between-subjects. Within-subjects, students retained spaced content better than massed content in the precalculus course. Between-subjects, students for whom some retrieval practice was spaced, compared to those for whom all practice was massed, performed better on the final exam in the precalculus class and on exams in the calculus class. These findings suggest that spaced retrieval practice can have a meaningful, long-lasting impact on educational outcomes.

After being taught how to perform a new mathematical operation, students are often given several practice problems in a single set, such as a homework assignment or quiz (i.e., massed practice). An alternative approach is to distribute problems across multiple homeworks or quizzes, increasing the temporal interval between practice (i.e., spaced practice). Spaced practice has been shown to increase the long-term retention of various types of mathematics knowledge. Less clear is whether spacing decreases performance during practice, with some studies indicating that it does and others indicating it does not. To increase clarity, we tested whether spacing produces long-term retention gains, but short-term practice costs, in a calculus course. On practice quizzes, students worked problems on various learning objectives in either massed fashion (3 problems on a single quiz) or spaced fashion (3 problems across 3 quizzes). Spacing increased retention of learning objectives on an end-of-semester test but reduced performance on the practice quizzes. The reduction in practice performance was nuanced: Spacing reduced performance only on the first two quiz questions, leaving performance on the third question unaffected. We interpret these findings as evidence that spacing led to more protracted, but ultimately more robust, learning. We, therefore, conclude that spacing imposes a desirable form of difficulty in calculus learning.

Background Undergraduate STEM instructors want to help students learn and retain knowledge for their future courses and careers. One promising evidence-based technique that is thought to increase long-term memory is spaced retrieval practice, or repeated testing over time. The beneficial effect of spacing has repeatedly been demonstrated in the laboratory as well as in undergraduate mathematics courses, but its generalizability across diverse STEM courses is unknown. We investigated the effect of spaced retrieval practice in nine introductory STEM courses. Retrieval practice opportunities were embedded in bi-weekly quizzes, either massed on a single quiz or spaced over multiple quizzes. Student performance on practice opportunities and a criterial test at the end of each course were examined as a function of massed or spaced practice. We also conducted a single-paper meta-analysis on criterial test scores to assess the generalizability of the effectiveness of spaced retrieval practice across introductory STEM courses. Results Significant positive effects of spacing on the criterial test were found in only two courses (Calculus I for Engineers and Chemistry for Health Professionals), although small positive effect sizes were observed in two other courses (General Chemistry and Diversity of Life). Meta-analyses revealed a significant spacing effect when all courses were included, but not when calculus was excluded. The generalizability of the spacing effect across STEM courses therefore remains unclear. Conclusions Although we could not clearly determine the generalizability of the benefits of spacing in STEM courses, our findings indicate that spaced retrieval practice could be a low-cost method of improving student performance in at least some STEM courses. More work is needed to determine when, how, and for whom spaced retrieval practice is most beneficial. The effect of spacing in classroom settings may depend on some design features such as the nature of retrieval practice activities (multiple-choice versus short answer) and/or feedback settings, as well as student actions (e.g., whether they look at feedback or study outside of practice opportunities). The evidence is promising, and further pragmatic research is encouraged.

Background: The questionnaires used in clinical research have often been developed and validated using paper, and in such cases it is necessary to show that the electronic versions are equivalent to the originals. Objective: To determine if electronic versions of questionnaires assessing severity and impact of rheumatoid arthritis (RA) are equivalent to the original paper versions. Methods: Patients (n = 43; 31 female) aged 32–83 years (25 aged <60 years) took part in a single session during which they completed paper and electronic assessments in randomized order, with an interval of 45 minutes between the two modes. Electronic assessments were set up on a Palm® TX handheld device. Assessments included measures of pain, fatigue, disability, and health status. Results: Scores were similar between the two modes. All effect sizes for electronic-paper differences were <0.2, and there was no overall tendency for one mode to show higher scores than the other. Intraclass correlation coefficients (ICCs) ranged from 0.72 to 0.96, and were generally similar to reported retest reliabilities of the scales in their paper versions. The Disability Index of the Health Assessment Questionnaire (HAQ-DI) showed an ICC of 0.96. ICCs for the EQ-5D health status scale were utility: 0.79; profile: 0.91; visual analog scale: 0.75. Most patients reported that both modes were easy to use. In general, patients preferred the electronic version over the paper, and this was true for the older as well as the younger patients. Conclusions: Electronic versions of questionnaires assessing severity and impact of RA provide data that correspond closely to those of paper originals, are easy to use, and are acceptable to patients.

Mathematical competency is vital to success in engineering. Competency requires not only short-term mastery of mathematical concepts, but also long-term retention. Research in cognitive psychology shows that the more times information is retrieved from memory, the more likely it is to be remembered over the long-term. Research also shows that increasing the amount of time between retrievals increases long-term retention. Which of these interventions—increasing the amount or the temporal spacing of retrieval practice—has the greater impact on long-term retention? Supported by NSF’s Improving Undergraduate STEM Education (IUSE) program, we answered this question by independently manipulating the amount and the spacing of retrieval practice in a precalculus course for engineering students. This was done by varying the number of quiz questions concerning key precalculus objectives (amount of practice) and varying whether those questions were massed on a single quiz or distributed across quizzes in multiple weeks (spacing of practice). Long-term retention was assessed in a test of precalculus knowledge administered one month after the end of the precalculus course. We found that students were significantly more likely to retain precalculus objectives when quiz questions had been spaced versus massed. Increasing the number of quiz questions did not significantly affect retention. These findings suggest that educators wishing to increase students’ long-term retention of mathematics knowledge should increase the spacing, rather than the amount, of retrieval practice in their courses.