RESUMEN

Dynamic nuclear polarization (DNP) is a method for achieving high levels of nuclear spin polarization by transferring spin polarization from electrons to nuclei by microwave irradiation, resulting in higher sensitivity in NMR/MRI. In particular, DNP using photoexcited triplet electron spins (triplet-DNP) can provide a hyperpolarized nuclear spin state at room temperature and in low magnetic field. In this review article, we highlight recent developments in materials and instrumentation for the application of triplet-DNP. First, a brief history and principles of triplet-DNP will be presented. Next, important advances in recent years will be outlined: new materials to hyperpolarize water and biomolecules; high-sensitivity solution NMR by dissolution triplet-DNP; and strategies for further improvement of the polarization. In view of these developments, future directions to widen the range of applications of triplet-DNP will be discussed.

RESUMEN

Hyperphosphorylation of the protein tau is one of the biomarkers of neurodegenerative diseases in the category of tauopathies. However, the molecular level, mechanistic, role of this common post-translational modification (PTM) in enhancing or reducing the aggregation propensity of tau is unclear, especially considering that combinatorial phosphorylation of multiple sites can have complex, non-additive, effects on tau protein aggregation. Since tau proteins stack in register and parallel to elongate into pathological fibrils, phosphoryl groups from adjacent tau strands with 4.8 Å separation must find an energetically favorable spatial arrangement. At first glance, this appears to be an unfavorable configuration due to the proximity of negative charges between phosphate groups from adjacent neighboring tau fibrils. However, this study tests a counterhypothesis that phosphoryl groups within the fibril core-forming segments favorably assemble into highly ordered, hydrogen-bonded, one-dimensionally extended wires under biologically relevant conditions. We selected two phosphorylation sites associated with neurodegeneration, serine 305 (S305p) and tyrosine 310 (Y310p), on a model tau peptide jR2R3-P301L (tau295-313) spanning the R2/R3 splice junction of tau, that readily aggregate into a fibril with characteristics of a seed-competent mini prion. Using multiple quantum spin counting (MQ-SC) by 31P solid-state NMR of phosphorylated jR2R3-P301L tau peptide fibrils, enhanced by dynamic nuclear polarization, we find that at least six phosphorous spins must neatly arrange in 1D within fibrils or in 2D within a protofibril to yield the experimentally observed MQ-coherence orders of four. We found that S305p stabilizes the tau fibrils and leads to more seeding-competent fibrils compared to jR2R3 P301L or Y310p. This study introduces a new concept that phosphorylation of residues within a core forming tau segment can mechanically facilitate fibril registry and stability due a hitherto unrecognized role of phosphoryl groups to form highly ordered, extended, 1D wires that stabilize pathological tau fibrils.

RESUMEN

Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. DNPjuice comprising of glycerol-d8/D2O/H2O has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the invitro preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the DNP juice, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at ∼72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for 13C-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with DNP juice, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.

Asunto(s)

RESUMEN

Background: The Doctor of Nursing Practice (DNP) degree prepares nurses for engagement in practice scholarship to improve health outcomes at multiple system levels. Organizational leaders' and employers' perceptions and expectations of DNP-prepared nurses require further study. Objective: To explore the perception of organizational leaders regarding expectations and engagement of DNP-prepared nurses in practice scholarship activities. Methods: Guided by the Actualized DNP Model, a cross-sectional design was used to survey organizational leaders (N = 87) regarding DNP-prepared nurses' engagement in practice scholarship. Results: Findings indicate that 92% (n = 80) of participants believed practice scholarship should be an expectation of DNP-prepared nurses serving in a variety of roles. Of the 77 respondents to the scholarship engagement questions, 97.4% (n = 75) reported that DNP-prepared nurses engaged in one or more practice scholarship activities over the past year. Conclusions: Aligning DNP practice scholarship competencies, as outlined in the American Association of Colleges of Nursing 2021 Essentials, with organizational needs, expectations, and provision of sufficient time to support these scholarship activities is needed. Implications for Nursing: Opportunities exist for DNP-prepared nurses to articulate and demonstrate their value by conducting practice scholarship through innovative advanced nursing roles to realize organizational goals. Organizational support is necessary for practice scholarship activities to transform DNP education.

Asunto(s)

RESUMEN

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, α-glucan, and ß-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

RESUMEN

Lipid nanoparticles (LNPs) are increasingly finding applications in targeted drug delivery, including for subcutaneous, intravenous, inhalation, and vaccine administration. While a variety of microscopy techniques are widely used for LNP characterization, their resolution does not allow for characterization of the spatial organization of different components, such as the excipients, targeting agents, or even the active ingredient. Herein, an approach is presented to probe the spatial organization of individual constituent groups of LNPs used for siRNA-based drug delivery, currently in clinical trials, by multinuclear solid-state magic-angle-spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Dynamic nuclear polarization is exploited (DNP) for sensitivity enhancement, together with judicious 2H labeing, to detect functionally important LNP constituents, the siRNA and the targeting agent (<1-2 w/v%), respectively, and achieve a structural model of the LNP locating the siRNA in the core, the targeting agent below the surface, and the sugars above the lipid bilayer at the surface. The integrated approach presented here is applicable for structural analysis of LNPs and can be extended more generally to other multi-component biological formulations.

RESUMEN

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.

RESUMEN

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.

Asunto(s)

RESUMEN

Hyperpolarized water in dissolution dynamic nuclear polarization (dDNP) experiments has emerged as a promising method for enhancing nuclear magnetic resonance (NMR) signals, particularly in studies of proteins and peptides. Herein, we focus on the application of "proton exchange-doubly relayed" nuclear Overhauser effects (NOE) from hyperpolarized water to achieve positive signal enhancement of methyl groups in the side chain of an alanine-glycine peptide. In particular, we show a cascade hyperpolarization transfer. Initial proton exchange between solvent and amide introduces hyperpolarization into the peptide. Subsequently, intermolecular NOE relays the hyperpolarization first to Ala-Hα and then in a second step to the Ala-CH3 moiety. Both NOEs have negative signs. Hence, the twice-relayed NOE pathway leads to a positive signal enhancement of the methyl group with respect to the thermal equilibrium magnetization. This effect might indicate a way towards hyperpolarized water-based signal enhancement for methyl groups, which are often used for NMR studies of large proteins in solution.

RESUMEN

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.

RESUMEN

BACKGROUND: The incidence of elderly people experiencing falls is currently increasing, which results in serious medical issues, such as fear of falling, limited physical activity, disability, and bone fractures, especially hip fractures. This study aimed to investigate balancing ability using a multi-directional reach test (MDRT) in older adults with and without diabetes mellitus. METHODS: 72 older adults with and without diabetes mellitus were recruited, and divided into two groups, including older adults without diabetes mellitus (n = 36) and older adults with diabetes mellitus (n = 36). All subjects completed all directions of the MDRT. RESULTS: There were no significant differences in MDRT scores in all directions between the two groups (p>0.05). Both groups achieved the highest MDRT scores in the forward direction. In contrast, the two groups had the lowest scores of MDRT in a backward direction. Furthermore, older adults with diabetes mellitus had lower MDRT scores in all directions than older adults without diabetes mellitus. CONCLUSION: The current study indicated that MDRT could be used to investigate the ability of balance in individuals with diabetes mellitus.

Asunto(s)

RESUMEN

Anecdotally, from personal experience as a growing editor and in talking with experienced editors, rejecting evidence-based project manuscripts that do not include patient outcomes is routine. Phrased differently, it is typic.

Asunto(s)

RESUMEN

Nuclear spin hyperpolarization techniques, such as dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP), have revolutionized nuclear magnetic resonance and magnetic resonance imaging. In these methods, a readily available source of high spin order, either electron spins in DNP or singlet states in hydrogen for PHIP, is brought into close proximity with nuclear spin targets, enabling efficient transfer of spin order under external quantum control. Despite vast disparities in energy scales and interaction mechanisms between electron spins in DNP and nuclear singlet states in PHIP, a pseudo-spin formalism allows us to establish an intriguing equivalence. As a result, the important low-field polarization transfer regime of PHIP can be mapped onto an analogous system equivalent to pulsed-DNP. This establishes a correspondence between key polarization transfer sequences in PHIP and DNP, facilitating the transfer of sequence development concepts. This promises fresh insights and significant cross-pollination between DNP and PHIP polarization sequence developers.

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

School nurses who are considering a terminal degree in nursing have two options, a Doctor of Nursing Practice (DNP) or a Doctor of Philosophy in Nursing (PhD). There are several factors for school nurses to consider when determining which terminal degree is best suited for them. This article focuses on the relevance of a DNP degree to school nursing, by first reviewing the evolution of DNP programs and then pivoting to discussions by three school nurses on why they chose this terminal degree and the projects completed during their courses of study. A main focus of a DNP project is to gather, analyze, synthesize, and translate research into practice, often operationalized as quality improvement (QI) in clinical settings. School nurses, practicing independently from other healthcare professionals while often managing large workloads, stand to benefit from obtaining or working with a nurse prepared at this level.

Asunto(s)

RESUMEN

We describe the design of a low-field portable magnet, based on two ceramic magnets, separated by a distance, with their magnetic poles aligned to create a large homogeneous region with a field strength of 425 gauss. Ceramic magnets are an uncommon choice compared to Neodymium Iron Boron magnets for low-field magnetic resonance but are preferable for our purposes to create a homogeneous region at lower field strength. The low cost of large ceramic magnets results in an inexpensive design with a large measurement volume. The magnets rest in a 3D-printed structure, which allows for the magnets to be moved by hand so the experimentalist has control over the field topology. To test the utility of the design, we explored an Overhauser dynamic nuclear polarization experiment with an aqueous solution of 4-Hydroxy-TEMPO. We also explored a simple flow measurement employing the ceramic magnets at a 6-degree pitch, creating a 14.6 gauss/cm constant gradient.

RESUMEN

Objective:To explore the feature of FOS expression in oxytocin-and vasopressin-positive neurons in the hypothalamic paraventricular nucleus(PVN)under different status of diabetes mellitus(DM).Methods:Intraperito-neal injection of vehicle or STZ in mice was conducted to establish control or diabetes model.Mechanical sensitivity was evaluated by von Frey filament tests to distinguish diabetic neuropathic pain(DNP)from without-pain group(DWP).The expression of FOS,oxytocin(OXT)-and vasopressin(VP)-positive neurons,as well as their double labeling was detected by immunohistochemical and immunofluorescent staining.Cell counting and comparison were made in groups.Results:FOS expression was easily detected in the PVN in the three groups(Control group,DNP group and DWP group)at 7 days,while that in DWP and DNP groups at 28 days was hardly detectable,with the number being signifi-cantly different from the 7 days group(P＜0.05 or 0.001).Likewise,compared with the control group,immunofluo-rescent signals for VP and OXT staining in the DNP and DWP groups also showed a trend of weakening as the modeling time increased(P＜0.05).The cell counting after double staining for VP or OXT with FOS showed that,in the DWP group at 7 days,the number of VP and FOS double-labeled neurons was 74.33±22.10,accounting for(56.64± 7.52)％of VP-positive cells,whereas the double labeling rate for OXT and FOS was only(10.44±3.14)％.In the DNP group at 7 days,the number of OXT and FOS double-labeled neurons was 51.00±31.80,accounting for(18.50 ±9.51)％of OXT-positive neurons,whereas the double labeling rate for VP and FOS was only(9.34±3.27)％.In contrast to these changes in 7 days group,the expression of FOS decreased sharply in the group of 28 days,thereby al-most no double-labeled neurons.Conclusion:The plasticity changes of oxytocin-and vasopressin-positive neurons in the PVN are different depending on the status of pain and non-pain,and the stage of disease progression.Understanding the changes is of great significance for unravelling the neural mechanism of diabetes and its complications.

RESUMEN

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.

Asunto(s)