Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C'-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

Peculiarities of DNP-INT and DBMIB as inhibitors of the photosynthetic electron transport.

Inhibitory analysis is a useful tool for studying cytochrome b6f complex in the photosynthetic electron transport chain. Here, we examine the inhibitory efficiency of two widely used inhibitors of the plastoquinol oxidation in the cytochrome b6f complex, namely 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT) and 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). Using isolated thylakoids from pea and arabidopsis, we demonstrate that inhibitory activity of DNP-INT and DBMIB is enhanced by increasing irradiance, and this effect is due to the increase in the rate of electron transport. However, the accumulation of protons in the thylakoid lumen at low light intensity has opposite effects on the inhibitory activity of DNP-INT and DBMIB, namely increasing the activity of DNP-INT and restricting the activity of DBMIB. These results allow for the refinement of the conditions under which the use of these inhibitors leads to the complete inhibition of plastoquinol oxidation in the cytochrome b6f complex, thereby broadening our understanding of the operation of the cytochrome b6f complex under conditions of steady-state electron transport.

In Situ Generation and High Bioresistance of Trityl-based Semiquinone Methide Radicals Under Anaerobic Conditions in Cellular Systems.

Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQM⋅ and CTQM⋅) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQM⋅ which was most likely mediated by flavoenzymes. Importantly, the resulting OXQM⋅ exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide, respectively. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQM⋅ moiety. Owing to extremely high resistance of OXQM⋅ towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments.

Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

The quality of clinician and student quality improvement reports: An analysis of 8 years of submissions.

INTRODUCTION: Many papers reporting on QI projects are not publishable for a variety of reasons. We compared manuscripts submitted as QI reports between June 2014 and June 2016 (prior to publication of the revised Standards for Quality Improvement Reporting Excellence (SQUIRE 2.0) with papers submitted to the American Journal of Nursing between July 2016 and December 2022). The aim was to evaluate any changes in the quality of manuscripts and identify problems that led to rejection; we also compared the quality of students with non-student submissions. METHODS: We conducted a non-randomized descriptive study to evaluate 349 papers submitted as QI project reports between June 2014 and December 2022 using screening templates based on the SQUIRE 2.0 checklist and findings of the INANE Working Group on Student Papers. RESULTS: Manuscripts designated as QI reports accepted for publication increased from 4% during 2014-2016 (T1) to 14% during 2016-2022 (T2); one student submission was accepted. There was a slight decrease in submissions designated as QI that were not QI: 36% of student submissions during T1 and 31% of student submissions during T2. Among clinician submissions, 44% in T1 designated as QI reports were not QI versus 31% submitted during T2. There was a decrease in student submissions that followed the SQUIRE guidelines (36% during T1 to 24% during T2). CONCLUSIONS: Findings demonstrate that by following the SQUIRE 2.0 guidelines, authors submit more complete manuscripts with fewer missing components. However, there are still misconceptions about what constitutes QI versus research and how to report QI initiatives. After comparing the findings from both periods, it is noteworthy that there is essentially the same level of inaccuracy and lack of acceptable manuscripts. CLINICAL RELEVANCE: Sharing findings from QI activities through presentations and publications is a vital way of helping spread the learnings from these projects and improve health care for a wider audience. Clinicians, academicians, and students must understand the elements of the SQUIRE guidelines and ensure that this framework is used for both designing and submitting QI projects for publication.

Student-led Doctor of Nursing Practice projects in the clinical environment: Viewpoint from hospital-based nurse scientists and leaders.

BACKGROUND: Exponential increases in Doctor of Nursing Practice (DNP) program enrollment have come with a rapid rise in the number of capstone projects conducted in clinical environments. However, misaligned priorities between students, faculty, and clinician leaders have created significant challenges. PURPOSE: Identify opportunities to strengthen collaboration between academic and clinical stakeholders to better support DNP projects and education. METHODS: Experienced hospital-based nurse leaders engaged in scholarly discourse supplemented by policy and research in DNP education. FINDINGS: Facilitating a DNP project requires significant investment of time, resources, and funds from the healthcare institution. Discord has arisen due to unclear responsibilities or decision-making ability for clinical stakeholders, ethical dilemmas for students who are also employees of the clinical site, and mismatched priorities between clinical need and student/academic project desires. Clinical leaders have raised significant concerns about DNP project proposals that are research-focused, diverge from healthcare institution goals, and lack a sustainability plan. DISCUSSION: Fortification of academic-practice partnerships and clarification of roles in the DNP student project are necessary to ensure that the project is of educational value to the student, a demonstration of learning for faculty, and of sustained clinical value to the healthcare system.

Assessment of nursing faculty retirement projections.

BACKGROUND: Nursing faculty retirement is a critical factor contributing to the nursing faculty shortage. PURPOSE: To assess the accuracy of projections on 2016 to 2025 nursing faculty retirements made in a previous study by Fang and Kesten (2017). METHODS: The 2016 to 2022 full-time nursing faculty data collected by American Association of Colleges of Nursing were used to examine the accuracy of the retirement projections for the same years. DISCUSSION: The study found that the mean age of full-time nursing faculty decreased for the first time; the number of faculty retirees and their age distributions projected by Fang and Kesten (2017) were accurate; there was a larger loss of nursing faculty at senior ranks to retirements than was anticipated; nursing faculty aged 50 to 59 in 2015 have made significant progress in doctoral attainment, senior rank, and graduate-level teaching by 2022, but they were still underrepresented in senior ranks compared to the 2016 to 2022 retirees; and for nursing faculty with a PhD degree, their growth was slower than their loss to retirements. CONCLUSION: The findings demonstrate the usefulness of the specific methods for faculty retirement projections. The decline in the mean age of nursing faculty is a positive sign that there is an increased recruitment of younger nurses into academia. The increase in the number of younger nurses entering academia with Doctor of Nursing Practice (DNP)-degree preparation can be leveraged through PhD-DNP collaboration to prepare practice-ready nursing graduates who contribute to health care improvements. Nursing schools need to implement innovative strategies to mentor younger faculty for their successful succession.

Frontiers in the Application of RF Vacuum Electronics.

The application of radio frequency (RF) vacuum electronics for the betterment of the human condition began soon after the invention of the first vacuum tubes in the 1920s and has not stopped since. Today, microwave vacuum devices are powering important applications in health treatment, material and biological science, wireless communication-terrestrial and space, Earth environment remote sensing, and the promise of safe, reliable, and inexhaustible energy. This article highlights some of the exciting application frontiers of vacuum electronics.

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by 13C-13C correlation DNP-enhanced NMR.

We show that multidimensional solid-state NMR 13C-13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Phytoremediation of dinitrophenol from wastewater by atriplex lentiformis: effect of salicylic acid.

Quail bush [Atriplex lentiformis (Torr.) S. Wats] plants were used in removing 2, 4-dinitrophenol (DNP) from wastewater in a hydroponic experiment. The hydroponic system contained three doses of DNP, i.e., 0, 10, and 20 mg L-1. Quail bush plants were sprayed with 0.1 mM salicylic acid (SA) to study its role in resisting DNP toxicity. DNP significantly (p < 0.05) reduced plant growth. Exposure of A. lentiformis plants to 20 mg L-1 of DNP reduced the total chlorophyl and relative water content by 39 and 24%, respectively. SA improved the antioxidant defense in terms of ascorbate peroxidase (APX) and polyphenol oxidase (PPO) activities. SA alleviated DNP toxicity by enhancing the production of osmoprotectants, e.g.,proline, phenols, and carbohydrates. SA enhanced the removal efficiency of DNP and the highest removal efficiency (96%) was recorded in the plants sprayed with SA and grown on 10 mg L-1 of DNP. A. lentiformis is a halophytic plant that has good physiological characteristics to resist 2, 4-dinitrophenol toxicity in wastewaters and is qualified to purify water from these harmful compounds. Exogenous application of 0.1 mM SA increased the defense system in A. lentiformis against 2, 4-dinitrophenol toxicity and enhanced the removal efficiency.

2, 4-dinitrophenol inhibited the synthesis of photosynthetic pigments.Salicylic acid protects the vital bio-compounds in plant cells.Atriplex plants are able to remove (96%) of 2, 4-dinitrophenol from the wastewater.Atriplex plants have a strong antioxidant defense enable them to survive in wastewater.

Design, synthesis, and biological evaluation of thienopyrimidine derivatives as multifunctional agents against Alzheimer's disease.

A series of 12 S-substituted tetrahydrobenzothienopyrimidines were designed and synthesized based on the donepezil scaffold. All the newly synthesized compounds were evaluated for their acetylcholinesterase (AChE) inhibitory activity and the most active compounds were tested for their butyrylcholinesterase (BuChE) inhibitory activity. Moreover, all the synthesized compounds were evaluated for their inhibitory effects against Aß aggregation and antioxidant activity using the oxygen radical absorbance capacity method. Compounds 4b, 6b, and 8b displayed the most prominent AChE inhibitory action comparable to donepezil. Compound 6b showed the greatest AChE inhibitory action (IC50 = 0.07 ± 0.003 µM) and the most potent BuChE inhibitory action (IC50 = 0.059 ± 0.004 µM). Furthermore, the three compounds exhibited significant antioxidant activity. Compounds 6b and 8b exerted more inhibitory action on Aß aggregation than donepezil. The cytotoxic activity of compounds 4b, 6b, and 8b against the WI-38 cell line in comparison with donepezil was examined using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. The results revealed that compounds 6b and 8b were less cytotixic than donepezil, while compound 4b showed nonsignificant cytotoxicity compared to donepezil. For more insights about the binding patterns of the most promising compounds (4b, 6b, and 8b) with the AChE at molecular levels; molecular docking and molecular dynamics simulations were performed. The density functional theory calculations and absorption, distribution, metabolism, excretion and toxicity properties were described as well. The results highlighted compound 6b, which incorporates a phenylpiperazine moiety coupled to a thienopyrimidone scaffold via two-atom spacer, to be a promising multifunctional therapeutic agent for the treatment of Alzheimer's disease. It is a potent dual AChE and BuChE inhibitor. Furthermore, it had stronger Aß aggregation inhibitory action than donepezil. Additionally, compound 6b exerted significant antioxidant activity.

Conversion of Similar Xenochemicals to Dissimilar Products: Exploiting Competing Reactions in Whole-Cell Catalysis.

Many enzymes have latent activities that can be used in the conversion of non-natural reactants for novel organic conversions. A classic example is the conversion of benzaldehyde to a phenylacetyl carbinol, a precursor for ephedrine manufacture. It is often tacitly assumed that purified enzymes are more promising catalysts than whole cells, despite the lower cost and easier maintenance of the latter. Competing substrates inside the cell have been known to elicit currently hard-to-predict selectivities that are not easily measured inside the living cell. We employ NMR spectroscopic assays to rationally combine isomers for selective reactions in commercial S. cerevisiae. This approach uses internal competition between alternative pathways of aldehyde clearance in yeast, leading to altered selectivities compared to catalysis with the purified enzyme. In this manner, 4-fluorobenzyl alcohol and 2-fluorophenylacetyl carbinol can be formed with selectivities in the order of 90%. Modification of the cellular redox state can be used to tune product composition further. Hyperpolarized NMR shows that the cellular reaction and pathway usage are affected by the xenochemical. Overall, we find that the rational construction of ternary or more complex substrate mixtures can be used for in-cell NMR spectroscopy to optimize the upgrading of similar xenochemicals to dissimilar products with cheap whole-cell catalysts.

Nursing practice leaders' perspectives on continuing challenges related to the Doctor of Nursing Practice Degree.

In 2020, deans from top-ranked nursing schools authored a Nursing Outlook article titled, "Doctor of Nursing Practice (DNP) Degree in the United States: Reflecting, Readjusting, and Getting Back on Track." In 2022, the American Association of Colleges of Nursing published the report, "State of the Doctor of Nursing Practice Education."- Both have been critical to advancing national discussions on the implementation of a universal DNP practiceentry standard in nursing. This paper, written by Chief Nursing Officers from top-ranked academic medical centers, reports on perspectives from practice settings/employers regarding issues raised by educators and deans in those documents. Barriers to acceptance of the DNP degree in practice include a lack of degree standardization, a need for DNP outcomes data, and a desire for a clearer return on investment for the DNP degree among graduates and employers.

The Doctor of Nursing Practice help desk: An innovative approach to developing evidence-based practice competency and providing DNP project support.

BACKGROUND: The 2021 American Association for Colleges of Nursing (AACN) Essentials for Professional Nursing Education identifies evidence-based practice (EBP) as a core concept meant to be integrated across curricula. Integrating EBP in Doctor of Nursing Practice (DNP) programs is challenging because of barriers including persistent confusion between research, EBP, and quality improvement; low faculty confidence and competence in their EBP skills and mentoring; increased faculty workload with growing program enrollments; and limited opportunities for students and faculty to practice EBP skills. AIMS: The aim of this initiative was to implement and evaluate an innovative education strategy to build DNP students' and faculty's EBP competency and confidence while mitigating barriers faced by DNP programs. METHODS: A DNP Help Desk was created and implemented at a large, Midwestern college of nursing with participation and support from EBP experts. Student and faculty participation were tracked, and perceptions of the help desk were evaluated. Opportunities to evolve the help desk were identified and implemented. RESULTS: Outcomes included a self-reported increase in student and faculty confidence and EBP competency, increased integration, and utilization of EBP in project work, elevated DNP project quality, and decreased demands on DNP project advisors. LINKING EVIDENCE TO ACTION: Engaging in innovative strategies aimed at increasing EBP competency and confidence may lead to increased EBP engagement and positive outcomes for students, faculty, and DNP programs.

Development of Dissolution Dynamic Nuclear Polarization of [15 N3 ]Metronidazole: A Clinically Approved Antibiotic.

We report dissolution Dynamic Nuclear Polarization (d-DNP) of [15 N3 ]metronidazole ([15 N3 ]MNZ) for the first time. Metronidazole is a clinically approved antibiotic, which can be potentially employed as a hypoxia-sensing molecular probe using 15 N hyperpolarized (HP) nucleus. The DNP process is very efficient for [15 N3 ]MNZ with an exponential build-up constant of 13.8 min using trityl radical. After dissolution and sample transfer to a nearby 4.7 T Magnetic Resonance Imaging scanner, HP [15 N3 ]MNZ lasted remarkably long with T1 values up to 343 s and 15 N polarizations up to 6.4 %. A time series of HP [15 N3 ]MNZ images was acquired in vitro using a steady state free precession sequence on the 15 NO2 peak. The signal lasted over 13 min with notably long T2 of 20.5 s. HP [15 N3 ]MNZ was injected in the tail vein of a healthy rat, and dynamic spectroscopy was performed over the rat brain. The in vivo HP 15 N signals persisted over 70 s, demonstrating an unprecedented opportunity for in vivo studies.

Surface Sites and Ligation in Amine-capped CdSe Nanocrystals.

Converting colloidal nanocrystals (NCs) into devices for various applications is facilitated by designing and controlling their surface properties. One key strategy for tailoring surface properties is thus to choose tailored surface ligands. In that context, amines have been universally used, with the goal to improve NCs synthesis, processing and performances. However, understanding the nature of surface sites in amine-capped NCs remains challenging, due to the complex surface compositions as well as surface ligands dynamic. Here, we investigate both surface sites and amine ligation in CdSe NCs by combining advanced NMR spectroscopy and computational modelling. Notably, dynamic nuclear polarization (DNP) enhanced 113 Cd and 77 Se 1D NMR helps to identify both bulk and surface sites of NCs, while 113 Cd 2D NMR spectroscopy enables to resolve amines terminated sites on both Se-rich and nonpolar surfaces. In addition to directly bonding to surface sites, amines are shown to also interact through hydrogen-bonding with absorbed water as revealed by 15 N NMR, augmented with computations. The characterization methodology developed for this work provides unique molecular-level insight into the surface sites of a range of amine-capped CdSe NCs, and paves the way to identify structure-function relationships and rational approaches towards colloidal NCs with tailored properties.

Chemical manipulation of mitochondrial function affects metabolism of red carotenoids in a marine copepod (Tigriopus californicus).

The shared-pathway hypothesis offers a cellular explanation for the connection between ketocarotenoid pigmentation and individual quality. Under this hypothesis, ketocarotenoid metabolism shares cellular pathways with mitochondrial oxidative phosphorylation such that red carotenoid-based coloration is inextricably linked mitochondrial function. To test this hypothesis, we exposed Tigriopus californicus copepods to a mitochondrially targeted protonophore, 2,4-dinitrophenol (DNP), to induce proton leak in the inner mitochondrial membranes. We then measured whole-animal metabolic rate and ketocarotenoid accumulation. As observed in prior studies of vertebrates, we observed that DNP treatment of copepods significantly increased respiration and that DNP-treated copepods accumulated more ketocarotenoid than control animals. Moreover, we observed a relationship between ketocarotenoid concentration and metabolic rate, and this association was strongest in DNP-treated copepods. These data support the hypothesis that ketocarotenoid and mitochondrial metabolism are biochemically intertwined. Moreover, these results corroborate observations in vertebrates, perhaps suggesting a fundamental connection between ketocarotenoid pigmentation and mitochondrial function that should be explored further.

Dynamic Nuclear Polarization in battery materials.

The increasing need for portable and large-scale energy storage systems requires development of new, long lasting and highly efficient battery systems. Solid state NMR spectroscopy has emerged as an excellent method for characterizing battery materials. Yet, it is limited when it comes to probing thin interfacial layers which play a central role in the performance and lifetime of battery cells. Here we review how Dynamic Nuclear Polarization (DNP) can lift the sensitivity limitation and enable detection of the electrode-electrolyte interface, as well as the bulk of some electrode and electrolyte systems. We describe the current challenges from the point of view of materials development; considering how the unique electronic, magnetic and chemical properties differentiate battery materials from other applications of DNP in materials science. We review the current applications of exogenous and endogenous DNP from radicals, conduction electrons and paramagnetic metal ions. Finally, we provide our perspective on the opportunities and directions where battery materials can benefit from current DNP methodologies as well as project on future developments that will enable NMR investigation of battery materials with sensitivity and selectivity under ambient conditions.

MAS-DNP enables NMR studies of insect wings.

NMR is a valuable tool for studying insects. Solid-state NMR has been used to obtain the chemical composition and gain insight into the sclerotization process of exoskeletons. There is typically little difficulty in obtaining sufficient sample quantity for exoskeletons. However, obtaining enough sample of other insect components for solid-state NMR experiments can be problematic while isotopically enriching them is near impossible. This is especially the case for insect wing membranes which is of interest to us. Issues with obtaining sufficient sample are the thickness of wing membranes is on the order of microns, each membrane region is surrounded by veins and occupies a small area, and the membranes are separated from the wing by physical dissection. Accordingly, NMR signal enhancement methods are needed. MAS-DNP has a track record of providing significant signal enhancements for a wide variety of materials. Here we demonstrate that MAS-DNP is useful for providing high quality one-dimensional and two-dimensional solid-state NMR spectra on cicada wing membrane at natural isotopic abundance.