NMR Dynamics Investigation of Ligand-Induced Changes of Main and Side-Chain Arginine N-H’s in Human Phosphomevalonate Kinase

Phosphomevalonate kinase (PMK) catalyzes phosphoryl transfer from adenosine triphosphate (ATP) to mevalonate 5-phosphate (M5P) on the pathway for synthesizing cholesterol and other isoprenoids. To permit this reaction, its substrates must be brought proximal, which would result in a significant and repulsive buildup of negative charge. To facilitate this difficult task, PMK contains 17 arginines and eight lysines. However, the way in which this charge neutralization and binding is achieved, from a structural and dynamics perspective, is not known. More broadly, the role of arginine side-chain dynamics in binding of charged substrates has not been experimentally defined for any protein to date. Herein we report a characterization of changes to the dynamical state of the arginine side chains in PMK due to binding of its highly charged substrates, ATP and M5P. These studies were facilitated by the use of arginine-selective labeling to eliminate spectral overlap. Model-free analysis indicated that while substrate binding has little effect on the arginine backbone dynamics, binding of either substrate leads to significant rigidification of the arginine side chains throughout the protein, even those that are \u3e8 Å from the binding site. Such a global rigidification of arginine side chains is unprecedented and suggests that there are long-range electrostatic interactions of sufficient strength to restrict the motion of arginine side chains on the picosecond-to-nanosecond time scale. It will be interesting to see whether such effects are general for arginine residues in proteins that bind highly charged substrates, once additional studies of arginine side-chain dynamics are reported

Experiential Learning in Patient Safety: A Multi-Center Study Examining Emergency Medicine Residents\u27 Situational Awareness of Hazards via Simulation

Introduction The 2016 Clinical Learning Environment Review established that experiential patient safety curricula for residents are uncommon. Moreover, these curricula do not incorporate non-technical skills linked to safety, such as situational awareness (SA). We developed an in-situ patient safety simulation exercise incorporating core SA concepts and subsequently assessed exercise feasibility and acceptability, and measured residents\u27 safety SA. Methods A simulation scenario and mock chart were designed, incorporating 16 patient safety hazards. Residents at two institutions reviewed the chart and had 10 minutes in an emergency department room with the simulated scenario to document identified hazards, followed by a facilitated debriefing. Pre- and post-exercise surveys were completed. We used regression analyses to compare exercise performance and survey responses by training year, and measures of proportional difference and association for survey responses. Results This study included 76 of 104 eligible residents (73.1%). Around 56.5% initially reported being comfortable identifying hazards. During the exercise, hazards requiring higher SA were identified less frequently. Senior residents identified more hazards (OR 2.26; 95%CI 1.56-3.28) (mean 8.28, SD1.45); 93.4% expressed satisfaction with the session, and residents reporting comfort increased significantly (89.5%,

Dynamics and functional diversity of the smallest phytoplankton on the Northeast US shelf

Author Posting. © National Academy of Sciences, 2020. This article is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of the United States of America 117(22), (2020): 12215-12221, doi: 10.1073/pnas.1918439117.Picophytoplankton are the most abundant primary producers in the ocean. Knowledge of their community dynamics is key to understanding their role in marine food webs and global biogeochemical cycles. To this end, we analyzed a 16-y time series of observations of a phytoplankton community at a nearshore site on the Northeast US Shelf. We used a size-structured population model to estimate in situ division rates for the picoeukaryote assemblage and compared the dynamics with those of the picocyanobacteria Synechococcus at the same location. We found that the picoeukaryotes divide at roughly twice the rate of the more abundant Synechococcus and are subject to greater loss rates (likely from viral lysis and zooplankton grazing). We describe the dynamics of these groups across short and long timescales and conclude that, despite their taxonomic differences, their populations respond similarly to changes in the biotic and abiotic environment. Both groups appear to be temperature limited in the spring and light limited in the fall and to experience greater mortality during the day than at night. Compared with Synechococcus, the picoeukaryotes are subject to greater top-down control and contribute more to the region’s primary productivity than their standing stocks suggest.We thank E. T. Crockford, E. E. Peacock, J. Fredericks, Z. Sandwith, the MVCO Operations Team, and divers of the Woods Hole Oceanographic Institution diving program. This work was supported by NSF Grants OCE-0119915 (to R.J.O. and H.M.S.) and OCE-1655686 (to M.G.N., R.J.O., A.R.S., and H.M.O.); NASA Grants NNX11AF07G (to H.M.S.) and NNX13AC98G (to H.M.S.); Gordon and Betty Moore Foundation Grant GGA#934 (to H.M.S.); and Simons Foundation Grant 561126 (to H.M.S.).2020-11-1

Comparative Genome-Wide Screening Identifies a Conserved Doxorubicin Repair Network That Is Diploid Specific in Saccharomyces cerevisiae

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases

An analysis of fast photochemistry over high northern latitudes during spring and summer using in-situ observations from ARCTAS and TOPSE

Observations of chemical constituents and meteorological quantities obtained during the two Arctic phases of the airborne campaign ARCTAS (Arctic Research of the Composition of the Troposphere from Aircraft and Satellites) are analyzed using an observationally constrained steady state box model. Measurements of OH and HO2 from the Penn State ATHOS instrument are compared to model predictions. Forty percent of OH measurements below 2 km are at the limit of detection during the spring phase (ARCTAS-A). While the median observed-to-calculated ratio is near one, both the scatter of observations and the model uncertainty for OH are at the magnitude of ambient values. During the summer phase (ARCTAS-B), model predictions of OH are biased low relative to observations and demonstrate a high sensitivity to the level of uncertainty in NO observations. Predictions of HO2 using observed CH2O and H2O2 as model constraints are up to a factor of two larger than observed. A temperature-dependent terminal loss rate of HO2 to aerosol recently proposed in the literature is shown to be insufficient to reconcile these differences. A comparison of ARCTAS-A to the high latitude springtime portion of the 2000 TOPSE campaign (Tropospheric Ozone Production about the Spring Equinox) shows similar meteorological and chemical environments with the exception of peroxides; observations of H2O2 during ARCTAS-A were 2.5 to 3 times larger than those during TOPSE. The cause of this difference in peroxides remains unresolved and has important implications for the Arctic HOx budget. Unconstrained model predictions for both phases indicate photochemistry alone is unable to simultaneously sustain observed levels of CH2O and H2O2; however when the model is constrained with observed CH2O, H2O2 predictions from a range of rainout parameterizations bracket its observations. A mechanism suitable to explain observed concentrations of CH2O is uncertain. Free tropospheric observations of acetaldehyde (CH3CHO) are 2–3 times larger than its predictions, though constraint of the model to those observations is sufficient to account for less than half of the deficit in predicted CH2O. The box model calculates gross O3 formation during spring to maximize from 1–4 km at 0.8 ppbv d−1, in agreement with estimates from TOPSE, and a gross production of 2–4 ppbv d−1 in the boundary layer and upper troposphere during summer. Use of the lower observed levels of HO2 in place of model predictions decreases the gross production by 25–50%. Net O3 production is near zero throughout the ARCTAS-A troposphere, and is 1–2 ppbv in the boundary layer and upper altitudes during ARCTAS-B

GM 16. Sire effects on postweaning growth and carcass characteristics in pubered Brahman cattle

A progeny test for postweaning growth and carcass characteristics was conducted on five purebred Brahman sires. Data presented here are from the progeny of five sires that were tested the first year of a multiyear study that is planned to evaluate approximately 25 sires. There was no effect of sire on initial body weight, but sire effects on average daily gain and final body weight were significant (P<.01). Hot carcass weight (P<.001) and ribeye area (P<.01). Sire affected dressing percentage (P<.001), but there was no effect of sire on USDA yield grade. There were no differences among sires in tenderness scored by sensory panel on steaks aged for 14 days or measured by Warner-Bratzler shear on steaks aged for 7, 14 or 21 days. This progeny test of five Brahman sires revealed genetic differences in postweaning rate of gain and carcass quality grade. Data from additional sires will be required to demonstrate genetic differences in tenderness

Current Knowledge and Considerations Regarding Survey Refusals: Executive Summary of the AAPOR Task Force Report on Survey Refusals

The landscape of survey research has arguably changed more significantly in the past decade than at any other time in its relatively brief history. In that short time, landline telephone ownership has dropped from some 98 percent of all households to less than 60 percent; cell-phone interviewing went from a novelty to a mainstay; address-based designs quickly became an accepted method of sampling the general population; and surveys via Internet panels became ubiquitous in many sectors of social and market research, even as they continue to raise concerns given their lack of random selection. Among these widespread changes, it is perhaps not surprising that the substantial increase in refusal rates has received comparatively little attention. As we will detail, it was not uncommon for a study conducted 20 years ago to have encountered one refusal for every one or two completed interviews, while today experiencing three or more refusals for every one completed interview is commonplace. This trend has led to several concerns that motivate this Task Force. As refusal rates have increased, refusal bias (as a component of nonresponse bias) is an increased threat to the validity of survey results. Of practical concern are the efficacy and cost implications of enhanced efforts to avert initial refusals and convert refusals that do occur. Finally, though no less significant, are the ethical concerns raised by the possibility that efforts to minimize refusals can be perceived as coercive or harassing potential respondents. Indeed, perhaps the most important goal of this document is to foster greater consideration by the reader of the rights of respondents in survey research

Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples

It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses’ Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (greater than 97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (greater than 95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform

Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples

It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses’ Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (>97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (>95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform

Introduction and evaluation of Romosinuano in the U.S.A.

In the warm regions of the U.S.A., adapted breeds of cattle are primarily limited to Bos indicus (Zebu), mainly American Brahman, and to breeds developed by crossing with Brahman. Some characteristics of purebred Brahman cattle that have been criticized include reproductive and feedlot performance, carcass quality and meat tenderness, temperament, and calf survivability. Therefore, the major niche for Brahman cattle in the U.S.A. has been in crossbreeding systems that combine the heat tolerance of the Brahman with the desirable reproductive and carcass traits of temperate Bos taurus breeds. The Romosinuano is a tropically adapted, criollo beef breed native to Colombia. The breed derived its name from its origin in the Sinu river region (sinuano) of northern Colombia and its polled (romo) character. Romosinuanos are purported to be highly fertile, and are noted for their longevity, docile temperament, and combining ability with Bos indicus. The long term objectives in our evaluation of the Romosinuano are to investigate its growth, reproductive performance, and carcass quality. Short term objectives have been to determine the effect of breed of recipient dam and estrous synchronization treatment on pregnancy and preweaning performance of Romosinuano embryo transfer calves, to evaluate postweaning growth and reproductive development in bulls through 20 months of age, to evaluate postweaning growth, to measure heat tolerance, and to determine age and body weight at puberty in heifers raised in central Florida