Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-ß-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

A Crosswalk Analysis Between Magnet® Standards and Clinical Nurse Specialist Practice Competencies.

OBJECTIVE: The purpose of this study was to determine the alignment between the American Nurses Credentialing Center's Magnet Recognition Program® standards and clinical nurse specialist (CNS) practice competencies. BACKGROUND: Despite documentation of CNS contributions to achieving and sustaining Magnet Recognition®, there is a lack of evidence clearly aligning Magnet® standards and CNS practice competencies. METHODS: Using a crosswalk method, an expert panel of CNSs and chief nursing executives analyzed alignment of the 50 Magnet standards with the 44 National Association of Clinical Nurse Specialists core practice competencies. RESULTS: CNS practice competencies are aligned closely with Magnet standards: 86% of the 50 Magnet standards aligned with at least 1 CNS competency and 81.8% of CNS competencies aligned with at least 1 Magnet® standard. CONCLUSIONS: The alignment between Magnet standards and CNS competencies supports evidence of CNS contributions to organizational achievement of Magnet Recognition and will assist nurse executives in identifying a full scope of opportunities for CNSs to contribute to nursing excellence.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

Multi-institutional validation of a modified scheme for subcategorizing salivary gland neoplasm of uncertain malignant potential (SUMP).

BACKGROUND: The salivary gland neoplasm of uncertain malignant potential (SUMP) category in the Milan System is diagnostically challenging. This study aims to validate a modified scheme for subcategorizing SUMP in a large multi-institutional cohort. METHODS: Retrospective review of salivary gland fine-needle aspirations (FNAs) from 10 institutions were classified based on the Milan System. Cases diagnosed as SUMP with available cytology slides and surgical follow-up were retrieved for review and subcategorized based on a modified scheme as follows: basaloid SUMP (B1: absent/scant nonfibrillary matrix; B2: presence of nonfibrillary/mixed-type matrix), oncocytic/oncocytoid SUMP (O1: with mucinous background; O2: without mucinous background), and SUMP not otherwise specified (NOS). RESULTS: A total of 742 (7.5%) cases from 9938 consecutive salivary gland FNAs were classified as SUMP. Among them, 525 (70.8%) had surgical follow-up and 329 (62.7%) were available for review. The overall risk of malignancy (ROM) of SUMP was 40.4%. There were 156 cases (47.4%) subcategorized as basaloid SUMP with a ROM of 36.5%, 101 (30.7%) as oncocytic/oncocytoid SUMP with a ROM of 52.5%, and 72 (21.9%) as SUMP NOS with a ROM of 31.9%. The ROM of oncocytic/oncocytoid SUMP was significantly higher than basaloid SUMP (P = .0142) and SUMP NOS (P = .0084). No significant differences in ROM were noted between B1 and B2 (36.7% vs 36.4%, P = 1.0000) and O1 and O2 (65.2% vs 48.7%, P = .2349). CONCLUSIONS: The ROM of oncocytic/oncocytoid SUMP was 52.5% and significantly higher than that of basaloid SUMP (36.5%, P = .0142) and SUMP NOS (31.9%, P = .0084), whereas no significant differences in ROM were noted for cases with different types of extracellular matrix or background material.

A study of Indiana University Health's spirit of inquiry and innovation during COVID.

BACKGROUND: COVID-19 has required nursing innovations to meet patient care needs not previously encountered. PURPOSE: The purpose of this study was to describe nursing innovations conceived, implemented, and desired during the first COVID-19 surge. METHODS: The investigators invited registered nurses employed across 16 Midwest hospitals (6,207) to complete the survey. Respondents provided demographics and written descriptions of innovations they conceived, witnessed, and desired. Investigators analyzed text responses using standard content analytic procedures and summarized quantitative demographics using percentages. FINDINGS: Nurses reported seven types of innovations that would (a) improve personal protective equipment (PPE), (b) limit the need to repeatedly don and doff PPE, (c) ensure safer practice, (d) conserve and access supplies, (e) provide patient and family education and support, (f) make team member communication more efficient, and (g) improve peer support. DISCUSSION: Nurses are in a unique position to generate innovative solutions to meet patient care needs under adverse and rapidly changing situations.

Sepsis-induced cardiomyopathy is associated with higher mortality rates in patients with sepsis.

BACKGROUND: Patients with sepsis are at risk for developing sepsis-induced cardiomyopathy (SIC). Previous studies offer inconsistent results regarding the association of SIC and mortality. This study sought to assess whether SIC is linked to mortality in patients with sepsis and to evaluate predictors of the development of SIC. METHODS: In this retrospective study, patients admitted to the medical intensive care unit with a diagnosis of sepsis in the absence of acute coronary syndrome were included. SIC was identified using transthoracic echo and was defined by a new onset decline in left ventricular ejection fraction (LVEF) ≤50%, or ≥10% decline in LVEF compared to baseline in patients with a history of heart failure with reduced ejection fraction. Multivariable logistic regression analysis was performed using the R software program. RESULTS: Of the 359 patients in the final analysis, 19 (5.3%) had SIC. Eight (42.1%) of the 19 patients in the SIC group and 60 (17.6%) of the 340 patients in the non-SIC group died during hospitalization. SIC was associated with an increased risk for all-cause in-hospital mortality (odds ratio [OR], 4.46; 95% confidence interval [CI], 1.15-18.69; P=0.03). Independent predictors for the development of SIC were albumin level (OR, 0.47; 95% CI, 0.23-0.93; P=0.03) and culture positivity (OR, 8.47; 95% CI, 2.24-55.61; P=0.006). Concomitant right ventricular hypokinesis was noted in 13 (68.4%) of the 19 SIC patients. CONCLUSIONS: SIC was associated with an increased risk for all-cause in-hospital mortality. Low albumin level and culture positivity were independent predictors of SIC.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Neurofilament Light Chain as a Biomarker of Hereditary Transthyretin-Mediated Amyloidosis.

OBJECTIVE: To identify changes in the proteome associated with onset and progression of hereditary transthyretin-mediated (hATTR) amyloidosis, also known as ATTRv amyloidosis, we performed an observational, case-controlled study that compared proteomes of patients with ATTRv amyloidosis and healthy controls. METHODS: Plasma levels of >1,000 proteins were measured in patients with ATTRv amyloidosis with polyneuropathy who received either placebo or patisiran in a Phase 3 study of patisiran (APOLLO), and in healthy controls. The effect of patisiran on the time profile of each protein was determined by linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) was further assessed with an orthogonal quantitative approach. RESULTS: Levels of 66 proteins were significantly changed with patisiran vs placebo, with NfL change most significant (p < 10-20). Analysis of changes in protein levels demonstrated that the proteome of patients treated with patisiran trended toward that of healthy controls at 18 months. Healthy controls' NfL levels were 4-fold lower than in patients with ATTRv amyloidosis with polyneuropathy (16.3 pg/mL vs 69.4 pg/mL, effect -53.1 pg/mL [95% confidence interval -60.5 to -45.9]). NfL levels at 18 months increased with placebo (99.5 pg/mL vs 63.2 pg/mL, effect 36.3 pg/mL [16.5-56.1]) and decreased with patisiran treatment (48.8 pg/mL vs 72.1 pg/mL, effect -23.3 pg/mL [-33.4 to -13.1]) from baseline. At 18 months, improvement in modified Neuropathy Impairment Score +7 score after patisiran treatment significantly correlated with reduced NfL (R = 0.43 [0.29-0.55]). CONCLUSIONS: Findings suggest that NfL may serve as a biomarker of nerve damage and polyneuropathy in ATTRv amyloidosis, enable earlier diagnosis of patients with ATTRv amyloidosis, and facilitate monitoring of disease progression. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that NfL levels may enable earlier diagnosis of polyneuropathy in patients with ATTRv amyloidosis and facilitate monitoring of disease progression.

Nonclinical Safety Profile of Revusiran, a 1st-Generation GalNAc-siRNA Conjugate for Treatment of Hereditary Transthyretin-Mediated Amyloidosis.

Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the "ENDEAVOUR" phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.

A Multisite Health System Survey to Assess Organizational Context to Support Evidence-Based Practice.

BACKGROUND: Implementation and sustainability of a culture of evidence-based practice (EBP) require a systematic approach. A baseline assessment of the organizational context can inform implementation efforts. AIMS: To examine organizational hospital context and provider characteristics associated with EBP readiness and to describe EBP context across hospitals. METHODS: A nonexperimental descriptive correlational design was used to conduct a web-based survey of direct-care registered nurses (N = 701) and nurse managers (N = 94) across a large Midwestern multisite healthcare system using the Alberta Context Tool (ACT). RESULTS: Many significant relationships existed among nurse characteristics and ACT domains, including age (lower age had higher Leadership, Evaluation, and Formal Interactions), education (graduate education had lower Social Capital than a bachelor's or associate degree), role (direct-care nurses had lower Culture than managers and lower Social Capital), and work status (full-time employees had lower Evaluation and Social Capital). EBP context across type of hospitals is similar, with marginal differences in Social Capital and Organizational Slack (higher in critical access hospitals). LINKING EVIDENCE TO ACTION: Assessing organizational context to support EBP is the first step in developing and enhancing a sustainable culture of inquiry. The ACT has been tested across countries, settings, and healthcare disciplines to measure perception of readiness of the practice environment toward EBP. Optimal organizational context is essential to support EBP and sustain the use of evidence in professional nursing practice. Nursing leaders can use baseline assessment information to identify strengths and opportunities to enhance EBP implementation. Enhancing organizational context across nurse characteristics (e.g., age, role, and work status) to acknowledge nurses' contributions, balance nurses' personal and work life, enhance connectedness, and support work culture is beneficial. Fostering development of Social Capital in nurses is needed to influence EBP readiness. A systematic and standardized approach to foster EBP across health systems is key to successful implementation.

Globally optimal volume-trap arrangements for the narrow-capture problem inside a unit sphere.

The determination of statistical characteristics for particles undergoing Brownian motion in constrained domains has multiple applications in various areas of research. This work presents an attempt to systematically compute globally optimal configurations of traps inside a three-dimensional domain that minimize the average of the mean first passage time (MFPT) for the narrow capture problem, the average time it takes a particle to be captured by any trap. For a given domain, the mean first passage time satisfies a linear Poisson problem with Dirichlet-Neumann boundary conditions. While no closed-form general solution of such problems is known, approximate asymptotic MFPT expressions for small traps in a unit sphere have been found. These solutions explicitly depend on trap parameters, including locations, through a pairwise potential function. After probing the applicability limits of asymptotic formulas through comparisons with numerical and available exact solutions of the narrow capture problem, full three-dimensional global optimization was performed to find optimal trap positions in the unit sphere for 2≤N≤100 identical traps. The interaction energy values and geometrical features of the putative optimal trap arrangements are presented.

Organizational intellectual capital and the role of the nurse manager: A proposed conceptual model.

BACKGROUND: Nurse managers must leverage both the human capital and social capital of the teams they lead in order to produce quality outcomes. Little is known about the relationship between human capital and social capital and how these concepts may work together to produce organizational outcomes through leadership of nurses. PURPOSE: The purpose of this article was to explore the concepts of human capital and social capital as they relate to nursing leadership in health care organizations. Specific aims included (a) to synthesize the literature related to human capital and social capital in leadership, (b) to refine the conceptual definitions of human capital and social capital with associated conceptual antecedents and consequences, and (c) to propose a synthesized conceptual model guiding further empirical research of social capital and human capital in nursing leadership. METHODS: A systematic integrative review of leadership literature using criteria informed by Whittemore and Knafl (2005) was completed. CINAHL Plus with Full Text, Academic Search Premier, Business Source Premier, Health Business FullTEXT, MEDLINE, and PsychINFO databases were searched for the years 1995 to 2016 using terms "human capital," "social capital," and "management." DISCUSSION: Analysis of conceptual definitions, theoretical and conceptual models, antecedents and consequences, propositions or hypotheses, and empirical support for 37 articles fitting review criteria resulted in the synthesis of the proposed Gilbert Conceptual Model of Organizational Intellectual Capital. CONCLUSION: The Gilbert Conceptual Model of Organizational Intellectual Capital advances the propositions of human capital theory and social capital theory and is the first model to conceptualize the direct and moderating effects that nurse leaders have on the human capital and social capital of the teams they lead. This model provides a framework for further empirical study and may have implications for practice, organizational policy, and education related to nursing leadership.

Constraining Solar Wind Heating Processes by Kinetic Properties of Heavy Ions.

We analyze the heavy ion components (A>4 amu) in collisionally young solar wind plasma and show that there is a clear, stable dependence of temperature on mass, probably reflecting the conditions in the solar corona. We consider both linear and power law forms for the dependence and find that a simple linear fit of the form T_{i}/T_{p}=(1.35±.02)m_{i}/m_{p} describes the observations twice as well as the equivalent best fit power law of the form T_{i}/T_{p}=(m_{i}/m_{p})^{1.07±.01}. Most importantly we find that current model predictions based on turbulent transport and kinetic dissipation are in agreement with observed nonthermal heating in intermediate collisional age plasma for m/q<3.5, but are not in quantitative or qualitative agreement with the lowest collisional age results. These dependencies provide new constraints on the physics of ion heating in multispecies plasmas, along with predictions to be tested by the upcoming Solar Probe Plus and Solar Orbiter missions to the near-Sun environment.

GDF11 Increases with Age and Inhibits Skeletal Muscle Regeneration.

Age-related frailty may be due to decreased skeletal muscle regeneration. The role of TGF-ß molecules myostatin and GDF11 in regeneration is unclear. Recent studies showed an age-related decrease in GDF11 and that GDF11 treatment improves muscle regeneration, which were contrary to prior studies. We now show that these recent claims are not reproducible and the reagents previously used to detect GDF11 are not GDF11 specific. We develop a GDF11-specific immunoassay and show a trend toward increased GDF11 levels in sera of aged rats and humans. GDF11 mRNA increases in rat muscle with age. Mechanistically, GDF11 and myostatin both induce SMAD2/3 phosphorylation, inhibit myoblast differentiation, and regulate identical downstream signaling. GDF11 significantly inhibited muscle regeneration and decreased satellite cell expansion in mice. Given early data in humans showing a trend for an age-related increase, GDF11 could be a target for pharmacologic blockade to treat age-related sarcopenia.

An HMGA2-IGF2BP2 axis regulates myoblast proliferation and myogenesis.

A group of genes that are highly and specifically expressed in proliferating skeletal myoblasts during myogenesis was identified. Expression of one of these genes, Hmga2, increases coincident with satellite cell activation, and later its expression significantly declines correlating with fusion of myoblasts into myotubes. Hmga2 knockout mice exhibit impaired muscle development and reduced myoblast proliferation, while overexpression of HMGA2 promotes myoblast growth. This perturbation in proliferation can be explained by the finding that HMGA2 directly regulates the RNA-binding protein IGF2BP2. Add-back of IGF2BP2 rescues the phenotype. IGF2BP2 in turn binds to and controls the translation of a set of mRNAs, including c-myc, Sp1, and Igf1r. These data demonstrate that the HMGA2-IGF2BP2 axis functions as a key regulator of satellite cell activation and therefore skeletal muscle development.