Affimers targeting proteins in the cardiomyocyte Z-disc: Novel tools that improve imaging of heart tissue.

Dilated Cardiomyopathy is a common form of heart failure. Determining how this disease affects the structure and organization of cardiomyocytes in the human heart is important in understanding how the heart becomes less effective at contraction. Here we isolated and characterised Affimers (small non-antibody binding proteins) to Z-disc proteins ACTN2 (α-actinin-2), ZASP (also known as LIM domain binding protein 3 or LDB3) and the N-terminal region of the giant protein titin (TTN Z1-Z2). These proteins are known to localise in both the sarcomere Z-discs and the transitional junctions, found close to the intercalated discs that connect adjacent cardiomyocytes. We use cryosections of left ventricles from two patients diagnosed with end-stage Dilated Cardiomyopathy who underwent Orthotopic Heart Transplantation and were whole genome sequenced. We describe how Affimers substantially improve the resolution achieved by confocal and STED microscopy compared to conventional antibodies. We quantified the expression of ACTN2, ZASP and TTN proteins in two patients with dilated cardiomyopathy and compared them with a sex- and age-matched healthy donor. The small size of the Affimer reagents, combined with a small linkage error (the distance from the epitope to the dye label covalently bound to the Affimer) revealed new structural details in Z-discs and intercalated discs in the failing samples. Affimers are thus useful for analysis of changes to cardiomyocyte structure and organisation in diseased hearts.

Affimers and nanobodies as molecular probes and their applications in imaging.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

Introduction to Interdisciplinary Roles in a Nursing Leadership Course: If I Understand You, Then I Know How to Work With You.

ABSTRACT: Active learning experiences for undergraduate nursing students build upon their understanding of concepts that transfer to new learning skills throughout the curriculum. Faculty implemented a leadership simulation in the classroom focusing on communication skills as a nurse leader to better understand the role of the nurse in the interdisciplinary team. The classroom setting is an ideal platform to teach interdisciplinary collaboration when opportunities do not present in the clinical setting. Scaffolding simulation experiences throughout the curriculum helps bridge the knowledge and skills gap between classroom and clinical experiences.

Disease mutations in striated muscle myosins.

Over 1000 disease-causing missense mutations have been found in human ß-cardiac, α-cardiac, embryonic and adult fast myosin 2a myosin heavy chains. Most of these are found in human ß-cardiac myosin heavy chain. Mutations in ß-cardiac myosin cause hypertrophic cardiomyopathy predominantly, whereas those in α-cardiac are associated with many types of heart disease, of which the most common is dilated cardiomyopathy. Mutations in embryonic and fast myosin 2a affect skeletal muscle function. This review provides a short overview of the mutations in the different myosin isoforms and their disease-causing effects.

Actin Mutations and Their Role in Disease.

Actin is a widely expressed protein found in almost all eukaryotic cells. In humans, there are six different genes, which encode specific actin isoforms. Disease-causing mutations have been described for each of these, most of which are missense. Analysis of the position of the resulting mutated residues in the protein reveals mutational hotspots. Many of these occur in regions important for actin polymerization. We briefly discuss the challenges in characterizing the effects of these actin mutations, with a focus on cardiac actin mutations.

A1603P and K1617del, Mutations in ß-Cardiac Myosin Heavy Chain that Cause Laing Early-Onset Distal Myopathy, Affect Secondary Structure and Filament Formation In Vitro and In Vivo.

Over 20 mutations in ß-cardiac myosin heavy chain (ß-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase-light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged ß-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged ß-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease.

Factors Associated With First-Time NCLEX-RN Success: A Descriptive Research Study.

BACKGROUND: The purpose of this study was to identify variables associated with scores achieved on the Health Education Systems, Inc. (HESI) exit examination and successful first-time NCLEX-RN® pass rates. METHOD: A retrospective descriptive study examined the administrative data of 211 baccalaureate nursing students. RESULTS: Students who completed the program in sequence and scored higher in certain HESI course examinations were more likely to have a better performance on the HESI exit examination. The higher the scores students achieved on the HESI exit examination, the more likely they were to pass the NCLEX-RN on their first attempt. CONCLUSION: These findings add to the growing body of literature seeking to identify variables associated with success in first-time NCLEX-RN success. Further research is needed to identify strategies that can be implemented to ensure timely progression, program completion, and licensure examination success. [J Nurs Educ. 2017;56(9):542-545.].

Promoting differentiation of cultured myoblasts using biomimetic surfaces that present alpha-laminin-2 peptides.

Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15-155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts.

Mission Possible CD ROM: instructional tool for preceptors.

Senior student preceptorship is a vital clinical component of many baccalaureate nursing curricula providing a one-on-one partnership with a registered nurse with clinical expertise. Experiential data and literature review support the preceptor's need for guidance with student assessment and evaluation strategies. This article reports on research conducted to assess the perceived increase in a preceptor's knowledge after viewing an author developed instructional CD, Mission Possible CD ROM: Instructional Tool for Preceptors. The results suggest that perceived knowledge increased; however, the lack of time to view the CD was an issue for the majority of the nurse preceptor participants. More research is warranted to build on current literature regarding instructional strategies of benefit to a nurse in the preceptor role.

Reforming perspectives: MSN graduates' knowledge, attitudes and awareness of self-transformation.

Socialization includes internalization of attitudes and values critical to the development of a professional identity. Resocialization relates to the returning registered nurse student's development of professional values and roles. Nurse educators' challenge is to consider socialization/resocialization as a formation/re-formation process. The purpose of this article is to report findings from a replication qualitative descriptive study that examined the process of change in knowledge and attitudes of recent MSN graduates using Mezirow's (1994)Transformative Learning Theory. Findings substantiated the changes in attitudes, knowledge and skills of MSN graduates identified by Cragg and Andrusyszyn (2005). Results further inform nurse educators of the process of transformation in MSN graduates according to Mezirow's Phases of Perspective Transformation.

Shared decision-making in nursing education.

Shared decision-making is an effective management strategy that may have positive implications for nurse educators facing curricular and course delivery issues. Use of shared or participative decision-making recognizes that decisions made for the overall good of the organization should include those integrally involved, i.e., faculty, students and administration. Ultimately, effective student learning should be the outcome of decisions related to curricular and content delivery. In this anecdotal paper, the authors present shared decision-making (SDM) as a management strategy that may be effectively utilized in a range of situations in educational settings. An exemplar is presented regarding changes in course delivery methods at two sister schools of nursing. Strategies to promote successful implementation, as well as challenges in initiating SDM, are discussed. The information presented in this paper can benefit nurse educators by offering a collaborative approach to the issues of evolving nursing curricula and content delivery.

Lights, camera, action: using feature films to stimulate emancipatory learning in the RN to BSN student.

Nurse educators are continually challenged to develop and implement effective activities to stimulate reflective learning in the RN to BSN student. The authors discuss the successful use of the feature film My Life as a reflective learning activity for a family health systems course.While feature films have been used constructively to teach family systems and social development, there is scant literature on the use of feature film as a teaching strategy within the discipline of nursing. The authors present evidence of how a film promoted stimulating and powerful transformative learning.