Fluorescence polarization reveals a possible displacement model of competition in PRC2:RNA:DNA interactions

PRC2 is a histone methyltransferase that acts on histone subunit H3 at lysine 27 to repress chromatin state and inhibit gene expression. The interactions between PRC2 and RNA have been heavily studied in vivo and in vitro with conflicting results reported on the relationship, but there is less data on interactions between PRC2 and DNA. Fluorescence polarization-based methodology was used with various RNA and DNA species to study the binding kinetics of PRC2 with RNA and DNA. Previous data indicate that PRC2 has a relatively high affinity for DNA species that are rich in consecutive G and C nucleotides. Double-stranded DNA species with lengths of 50-60 bp were designed for this project. Fluorescence polarization binding experiments were used to identify the general binding affinity of the DNA and RNA species to PRC2 by calculating the Kd apparent for the binding curve. After the apparent binding affinities were determined, various FP-competition experiments were performed to determine if each DNA species could be competed off by an RNA species or itself, and vice versa. These results showed that the DNA species were more effective competitors although the RNA species were stronger binders. These results suggest a mechanism for RNA-mediated PRC2 regulation that could reconcile the conflicting experimental results and interpretations of past experiments. Further FP-based Kd experiments under varying salt concentrations revealed PRC2 has ionic interactions with DNA that are not seen with RNA. This suggests PRC2 has extra unique interactions with DNA, which could explain the differing effectiveness of DNA versus RNA as competitors. Altogether, the results imply that DNA has additional contacts with PRC2 that limit its displacement by RNA, allowing for RNA to guide PRC2 to its target genetic loci and then for PRC2 to deposit its methyl marks without being sequestered by RNA.&nbsp;</p

The medical licensing examination debate

National licensing examinations are typically large-scale examinations taken early in a career or near the point of graduation, and, importantly, success is required to subsequently be able to practice. They are becoming increasingly popular as a method of quality assurance in the medical workforce, but debate about their contribution to patient safety and the improvement of healthcare outcomes continues. A systematic review of the national licensing examination literature demonstrates that there is disagreement between assessment experts about the strengths and challenges of licensing examinations. This is characterized by a trans-Atlantic divide between the dominance of psychometric reliability assurance in North America and the wider interpretations of validity, to include consequences, in Europe. We conclude that the debate might benefit from refocusing to what a national licensing examination should assess: to achieve a balance between assessing a breadth of skills and the capacity for such skills in practice, and focusing less on reproducibility

The International Landscape of Medical Licensing Examinations: A Typology Derived From a Systematic Review

Background National licensing examinations (NLEs) are large-scale examinations usually taken by medical doctors close to the point of graduation from medical school. Where NLEs are used, success is usually required to obtain a license for full practice. Approaches to national licensing, and the evidence that supports their use, varies significantly across the globe. This paper aims to develop a typology of NLEs, based on candidacy, to explore the implications of different examination types for workforce planning. Methods A systematic review of the published literature and medical licensing body websites, an electronic survey of all medical licensing bodies in highly developed nations, and a survey of medical regulators. Results The evidence gleaned through this systematic review highlights four approaches to NLEs: where graduating medical students wishing to practice in their national jurisdiction must pass a national licensing exam before they are granted a license to practice; where all prospective doctors, whether from the national jurisdiction or international medical graduates, are required to pass a national licensing exam in order to practice within that jurisdiction; where international medical graduates are required to pass a licensing exam if their qualifications are not acknowledged to be comparable with those students from the national jurisdiction; and where there are no NLEs in operation. This typology facilitates comparison across systems and highlights the implications of different licensing systems for workforce planning. Conclusion The issue of national licensing cannot be viewed in isolation from workforce planning; future research on the efficacy of national licensing systems to drive up standards should be integrated with research on the implications of such systems for the mobility of doctors to cross borders

Disaster Exposure and Mental Health Among Puerto Rican Youths After Hurricane Maria

Importance: Quantifying the magnitude of disaster exposure and trauma-related symptoms among youths is critical for deployment of psychological services in underresourced settings. Hurricane Maria made landfall in Puerto Rico on September 20, 2017, resulting in massive destruction and unprecedented mortality. Objective: To determine the magnitude of disaster exposure and mental health outcomes among Puerto Rican youths after Hurricane Maria. Design, Setting, and Participants: Survey study in which a school-based survey was administered to each public school student at all schools in Puerto Rico between February 1 and June 29, 2018 (5-9 months after Hurricane Maria). Of the 226 808 students eligible to participate, 96 108 students completed the survey. Main Outcomes and Measures: Participants were assessed for exposure to hurricane-related stressors, posttraumatic stress disorder (PTSD), and depressive symptoms, using standardized self-report measures administered in Spanish. Descriptive statistics were compiled for all outcome variables, as was the frequency of individuals reporting clinically elevated symptoms of PTSD or depression. Differences in these statistics across sexes were also examined via t tests. Correlations between demographic, geographic, and main outcome variables were also calculated, and regressions were conducted to examine their association with symptoms of PTSD. Results: A total of 96 108 students participated in the study (42.4% response rate; 50.3% female), representative of grades 3 to 12 across all 7 educational regions of Puerto Rico. As a result of the hurricane, 83.9% of youths saw houses damaged, 57.8% had a friend or family member leave the island, 45.7% reported damage to their own homes, 32.3% experienced shortages of food or water, 29.9% perceived their lives to be at risk, and 16.7% still had no electricity 5 to 9 months after the hurricane. Overall, 7.2% of youths (n = 6900) reported clinically significant symptoms of PTSD; comparison of the frequency of reporting clinically elevated symptoms of PTSD across sex yielded a significant difference (t = 12.77; 95% CI of the difference, 0.018-0.025; P \u3c .001), with girls (8.2%) exceeding the clinical cutoff score more often than boys (6.1%). Finally, similar analysis of differences in depression between sexes was also significant (t = 17.56; 95% CI of the difference, 0.31-0.39; P \u3c .001), with girls displaying higher mean (SD) scores (2.72 [3.14]) than boys (2.37 [2.93]). Demographic and risk variables accounted for approximately 20% of variance in symptoms of PTSD (r2 = 0.195; 95% CI, 0.190-0.200). Conclusions and Relevance: Survey results indicate that Hurricane Maria exposed Puerto Rican youths to high levels of disaster-related stressors, and youths reported high levels of PTSD and depressive symptoms. Results are currently being used by the Puerto Rico Department of Education to inform targeted and sustainable evidence-based practices aimed at improving mental health outcomes for Puerto Rico\u27s youths

Senior Recital: Noah Berkshier, Percussion; Seth Berkshier, Alto Sax; Lynn Kavelman, Piano; Regan Baxter, Bass; Aaron Kavelman, Drums; February 4, 2023

Kemp Recital HallFebruary 4, 2023Saturday Afternoon4:00 p.m

Giant anharmonicity and non-linear electron-phonon coupling in MgB2_{2}; A combined first-principles calculations and neutron scattering study

We report first-principles calculations of the electronic band structure and lattice dynamics for the new superconductor MgB$_{2}$. The excellent agreement between theory and our inelastic neutron scattering measurements of the phonon density of states gives confidence that the calculations provide a sound description of the physical properties of the system. The numerical results reveal that the in-plane boron phonons (with E$_{2g}$ symmetry) near the zone-center are very anharmonic, and are strongly coupled to the partially occupied planar B $\sigma$ bands near the Fermi level. This giant anharmonicity and non-linear electron-phonon coupling is key to explaining the observed high T$_{c}$ and boron isotope effect in MgB$_{2}$Comment: In this revised version (to appear in PRL) we also discuss the boron isotope effect. Please visit http://www.ncnr.nist.gov/staff/taner/mgb2 for detail

Genotyping concordance in DNA extracted from formalin‐fixed paraffin embedded (FFPE) breast tumor and whole blood for pharmacogenetic analyses

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135708/1/mol22015991868.pd

The International Landscape of Medical Licensing Examinations: A Typology Derived From a Systematic Review

Abstract Background: National licensing examinations (NLEs) are large-scale examinations usually taken by medical doctors close to the point of graduation from medical school. Where NLEs are used, success is usually required to obtain a license for full practice. Approaches to national licensing, and the evidence that supports their use, varies significantly across the globe. This paper aims to develop a typology of NLEs, based on candidacy, to explore the implications of different examination types for workforce planning. Methods: A systematic review of the published literature and medical licensing body websites, an electronic survey of all medical licensing bodies in highly developed nations, and a survey of medical regulators. Results: The evidence gleaned through this systematic review highlights four approaches to NLEs: where graduating medical students wishing to practice in their national jurisdiction must pass a national licensing exam before they are granted a license to practice; where all prospective doctors, whether from the national jurisdiction or international medical graduates, are required to pass a national licensing exam in order to practice within that jurisdiction; where international medical graduates are required to pass a licensing exam if their qualifications are not acknowledged to be comparable with those students from the national jurisdiction; and where there are no NLEs in operation. This typology facilitates comparison across systems and highlights the implications of different licensing systems for workforce planning. Conclusion: The issue of national licensing cannot be viewed in isolation from workforce planning; future research on the efficacy of national licensing systems to drive up standards should be integrated with research on the implications of such systems for the mobility of doctors to cross borders

Evolving strategies for enzyme engineering.

Directed evolution is a common technique to engineer enzymes for a diverse set of applications. Structural information and an understanding of how proteins respond to mutation and recombination are being used to develop improved directed evolution strategies by increasing the probability that mutant sequences have the desired properties. Strategies that target mutagenesis to particular regions of a protein or use recombination to introduce large sequence changes can complement full-gene random mutagenesis and pave the way to achieving ever more ambitious enzyme engineering goals. Introduction Enzymes are Nature&apos;s catalysts, tremendously accelerating the rates of a wide range of biochemical reactions, often with exquisite specificity. Harnessing enzymes for other purposes usually requires engineering them to improve their activity or stability. One approach to engineering enzymes is to make specific modifications, but this demands a detailed and frequently unattainable understanding of the relationship between sequence and function. Directed evolution bypasses this problem in much the same way as natural evolution, by combining mutation with selection or screening to identify improved variants. Because it is never possible to test more than an infinitesimal fraction of the vast number of possible protein sequences, it is essential to have a strategy for creating directed evolution sequence libraries that are rich in proteins with the desired enzymatic function. Such libraries can be designed by drawing on our knowledge of how proteins respond to mutation Directed evolution strategies Directed evolution works when the researcher can find at least one enzyme with improved properties in the sequence library. The most naïve strategy of creating a library of random protein sequences is not useful for most enzyme engineering goals. Although sequences with simple functions such as ATP binding Most directed evolution strategies involve making relatively small changes to existing enzymes. This takes advantage of the fact that enzymes often have a range of weak promiscuous activities that are quickly improved with just a few mutations Random mutagenesis The most straightforward strategy for library construction is to randomly mutate the full gene of an enzyme with a function close to the desired function. This approach requires no structural or mechanistic information, and can uncover unexpected beneficial mutations. Using sequential rounds of error-prone PCR to make an average of a few mutations per gene, followed by screening or selection for improved variants, is effective for a wide range of engineering goals. The creation of enantioselective catalysts from an enzyme whose structure is unknown is one such application. A single round of error-prone PCR produced several dozen cyclohexane monooxygenases with R or S selectivity Beneficial mutations found by random mutagenesis can be combined by DNA shuffling. A study with b-glucuronidase showed that beneficial mutations drive each other to extinction during recursive random mutagenesis, but that this problem can be eliminated by DNA shuffling Random mutagenesis can also uncover additional beneficial mutations in rationally designed enzymes. The Withers laboratory Targeted mutagenesis Some engineering goals, such as dramatically altering an enzyme&apos;s specificity or regioselectivity, may require mul- Random mutagenesis, targeted mutagenesis and recombination are three strategies for producing sequence libraries for directed evolution. (a) Random mutagenesis introduces amino acid substitutions throughout the protein and can uncover beneficial mutations distant from the active site. The red residues in the structure at top show four mutations uncovered by random mutagenesis that enhanced the activity of mammalian cytochrome P450 2B1 on several substrates Using a high-resolution crystal structure to target mutagenesis to three active site residues, Hill et al. [23] created a triple mutant of phosphotriesterase with a rate enhancement of three orders of magnitude for the degradation of organic triesters such as those used in chemical warfare agents. Crucially, two of the corresponding single mutants did not increase activity and so would not have been identified if they had been explored one at a time. The problem of inverting the enantioselectivity of a lipase offers an interesting comparison between full-gene random mutagenesis and targeted mutagenesis. Reetz and co-workers [24] used several rounds of full-gene random mutagenesis and DNA shuffling to invert the enantioselectivity of a lipase of unknown structure from S to R. Another lipase was engineered for the same goal by simultaneous mutation of four active site residues A variety of other enzymes have recently been engineered by targeted mutagenesis. Mutating three active site residues of penicillin acylase created six variants with improved activity, five of which were triple mutants [27]. Juillerat et al. [28] targeted four active site residues to engineer an O6-alkylguanine-DNA alkyltransferase for the efficient in vivo labeling of fusion proteins. They developed a selection system that allowed them to examine over 20,000 mutants and found that the best variants were triple mutants, suggesting the importance of simultaneously exploring multiple mutations. Novel DNA and RNA polymerases have also been engineered by targeted mutagenesis. Chelliserrykattil and Ellington [29] mutated four amino acids in RNA polymerase to engineer the enzyme to transcribe 2 0 -O-methyl RNA. Using a screen that selected variants that generated more RNA, they identified several mutants that incorporated nucleotides modified at the 2 0 position. Fa et al. [30] used targeted mutagenesis to engineer a DNA polymerase to specifically incorporate 2 0 -O-methyl ribonucleoside triphosphates by mutating six amino acids and selecting improved variants using phage display. Targeted mutagenesis of two active site residues was used to engineer a thioredoxin protein to replace the disulfide bond formation system in Escherichia coli Schultz and co-workers have created tRNA synthetases that charge orthogonal tRNAs with non-natural amino acids by targeting mutagenesis to five or six amino acids involved in substrate recognition. They then performed a positive selection for recognition of the non-natural amino acid and a negative selection against recognition of other amino acids The best mutants discovered by targeted mutagenesis almost always contain multiple mutations. These mutations are often beneficial as single mutants, but evidence is accumulating that at least some of them are beneficial only in combination Recombination Recombining structurally similar proteins can access larger degrees of sequence change than random mutagenesis The family shuffling protocol relies on regions of sequence identity to create crossovers that recombine the sequences of related proteins. This protocol is therefore limited to proteins with more than 70-75% identity, because libraries created from more diverged sequences tend to yield mostly parent sequences. A variety of methods have been developed to avoid this problem in the recombination of divergent sequences by using mismatched PCR primer pairs Although the studies described above demonstrate that recombining highly diverged but homologous sequences can produce libraries of diverse folded sequences, so far there has been little work to test whether it is also a useful method for discovering new functions. A tantalizing hint is that four out of fourteen chimeras of two cytochrome P450 proteins with 64% sequence identity show new product profiles Non-homologous recombination that combines fragments of unrelated proteins is another way to introduce large sequence changes. A new methodology was used to recombine the non-homologous chorismate mutase and fumarase proteins A striking application of non-homologous recombination is Ostermeier and co-workers&apos; creation of a protein that combines the activity of a b-lactamase with the maltoseinduced conformational change of maltose-binding protein. In one experiment, they randomly inserted the lactamase sequence into the maltose-binding protein and screened for mutants with enhanced lactamase activity in the presence of maltose Conclusions Directed evolution is now an established method to engineer enzymes for a wide range of uses. Full-gene random mutagenesis continues to be a straightforward and powerful tool, and studies using this approach repeatedly illustrate that beneficial mutations can occur at unexpected sites. Targeted mutagenesis and recombination can extend directed evolution to the engineering of enzyme properties that require more than a few uncoupled changes in a protein&apos;s sequence (which are easily obtained by sequential rounds of random mutagenesis and screening). The increasing incorporation of structural and chemical knowledge will undoubtedly enhance the utility of these methods. The growing use of rational design in conjunction with directed evolution offers the exciting promise of generating libraries containing a high frequency of sequences with the desired functional properties. Update Recent work has emphasized the tendency of directed evolution to improve weak promiscuous functions by broadening specificity, as discussed i