Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1.

We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects.

The hereditary stomatocytoses are a group of dominantly inherited conditions in which the osmotic stability of the red cell is compromised by abnormally high cation permeability. This report demonstrates the very marked similarities between the cryohydrocytosis form of hereditary stomatocytosis and the common tropical condition south-east Asian ovalocytosis (SAO). We report two patients, one showing a novel cryohydrocytosis variant (Ser762Arg in SLC4A1) and a case of SAO. Both cases showed a mild haemolytic state with some stomatocytes on the blood film, abnormal intracellular sodium and potassium levels which were made markedly abnormal by storage of blood at 0°C, increased cation 'leak' fluxes at 37°C and increased Na(+) K(+) pump activity. In both cases, the anion exchange function of the mutant band 3 was destroyed. Extensive electrophysiological studies comparing the cation leak and conductance in Xenopus laevis oocytes expressing the two mutant genes showed identical patterns of abnormality. These data are consistent with the cryohydrocytosis form of hereditary stomatocytosis and we conclude that the cation leak in SAO is indistinguishable from that in cryohydrocytosis, and that SAO should be considered to be an example of hereditary stomatocytosis.

Recipient-derived HPA-1a antibodies: a cause of prolonged thrombocytopenia after unrelated donor stem cell transplantation.

BACKGROUND: Patients with human platelet antigen (HPA) specific antibodies in cases of neonatal alloimmune thrombocytopenia and platelet (PLT) refractoriness derive clinical benefit from the use of HPA-selected PLTs. STUDY DESIGN AND METHODS: This study describes three patients with underlying diagnoses of acute myeloid leukemia, chronic lymphocytic leukemia, and myelodysplasia, respectively, who underwent allogeneic bone marrow transplantation (BMT) with unrelated donors matched at the HLA-A, B, C, Dr, and DQ loci but who failed to achieve an adequate PLT count. Investigation using PLT immunofluorescence test, monoclonal antibody immobilization of PLT antigens assay, and genotyping revealed the presence of recipient-derived HPA-1a antibodies. RESULTS: In two patients, anti-HPA-1a was detected post-BMT and in the third patient, anti-HPA-1a was detected during pre-BMT chemotherapy. Despite apparent 100% engraftment of donor cells, the patients' PLT counts failed to recover 9-10 months posttransplant. The patients remained PLT-transfusion dependent and failed to achieve satisfactory increments following random donor or HLA-matched PLT transfusions. After the identification of HPA-1a antibodies, the patients were supported by HPA-1a(-) PLTs and satisfactory posttransfusion PLT increments were obtained. These cases illustrate that HPA-1a antibodies may remain detectable for 10 months following apparently successful donor engraftment and the disappearance of recipient-derived HLA antibodies. The prolonged persistence of recipient-derived PLT-specific antibodies following BMT has to our knowledge not been described previously. CONCLUSION: HPA-1a antibodies were associated with protracted PLT-transfusion dependence and significant hemorrhagic complications. Appropriate and timely laboratory investigation for HPA-specific antibodies followed by transfusion support with HPA-selected PLTs provided the cornerstone of the hemostatic management in these cases.

Engaging Children as Simulated Patients in Healthcare Education.

STATEMENT: The value of "simulation" as a learning strategy is well established among healthcare professionals (Educ Prim Care 2015; 26(4):242-7). The use of "simulated patients (SPs)" to present real-world scenarios provides opportunities for students to develop "soft skills," including interpersonal communication, critical thinking, and problem solving. These skills are particularly relevant in pediatric care, where healthcare providers must consider the patient's cognitive development, emotional state, and familial context. This article focuses on middle childhood (6-12 years) a distinctive developmental stage between 2 major developmental transition stages: infancy and adolescence. Middle childhood is associated with compulsory school attendance, developing skills in self-discipline, conflict resolution, and decision-making. Child SPs can play an important role in healthcare education providing direct insight into this unique period of development. They can contribute to the design and delivery of simulations to increase fidelity and provide meaningful real-time feedback to learners on children's experience of the healthcare system. Despite children's expertise and particular care delivery needs, documented simulations using child SPs are limited. This article considers the role of child SPs to support a case for further research into the value of engaging middle years children in the development and delivery of "simulation-based learning experiences". It addresses the gap in child-focused education, the challenges inherent in working with children and outlines strategies and guidelines for effective practice.

Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis.

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.

Students take the lead for learning in practice: A process for building self-efficacy into undergraduate nursing education.

To prepare graduate nurses for practice, the curriculum and pedagogy need to facilitate student engagement, active learning and the development of self-efficacy. This pilot project describes and explores an initiative, the Check-in and Check-out process, that aims to engage students as active partners in their learning and teaching in their clinical preparation for practice. Three interdependent elements make up the process: a check-in (briefing) part; a clinical practice part, which supports students as they engage in their learning and practise clinical skills; and a check-out (debriefing) part. A student evaluation of this initiative confirmed the value of the process, which has subsequently been embedded in the preparation for practice and work-integrated learning courses in the undergraduate nursing programs at the participating university. The introduction of a singular learning process provides consistency in the learning approach used across clinical learning spaces, irrespective of their location or focus. A consistent learning process-including a common language that easily transfers across all clinical courses and clinical settings-arguably enhances the students' learning experience, helps them to actively manage their preparation for clinical practice and to develop self-efficacy.

Evaluating an interview preparation process for student nurses.

The aim of this article is to share innovative practice in relation to the recruitment process for nurses. Quality of preparation offered to student nurses prior to interview was variable, therefore, a study day to equip student nurses with the necessary knowledge and skills for interviews was devised. The strategies employed include a range of teaching approaches. Student evaluation indicates that this interview preparation day was successful and popular, with a majority of students receiving immediate offers of posts. Recommendations for educators include addressing the recruitment process earlier within the curriculum and adopting some of the practises of a business school.

Gastrointestinal care for older people.

This article discusses gastrointestinal (GI) healthcare in older people. It outlines the physiological changes that occur in the GI tract as a result of ageing, and discusses common GI disorders in older people. These GI disorders include dysphagia, gastrointestinal reflux disease, colorectal cancer, diverticular disease, constipation and anaemia. Healthcare professionals should be aware of the factors that may influence gastrointestinal health in older people, including nutrition, hydration and alcohol use, which are important considerations when delivering person-centred care.

Solution structure of prosurvival Mcl-1 and characterization of its binding by proapoptotic BH3-only ligands.

The B cell lymphoma-2 (Bcl-2) homologs myeloid cell leukemia-1 (Mcl-1) and A1 are prosurvival factors that selectively bind a subset of proapoptotic Bcl homology (BH) 3-only proteins. To investigate the molecular basis of the selectivity, we determined the solution structure of the C-terminal Bcl-2-like domain of Mcl-1. This domain shares features expected of a prosurvival Bcl-2 protein, having a helical fold centered on a core hydrophobic helix and a surface-exposed hydrophobic groove for binding its cognate partners. A number of residues in the binding groove differentiate Mcl-1 from its homologs, and in contrast to other Bcl-2 homologs, Mcl-1 has a binding groove in a conformation intermediate between the open structures characterized by peptide complexes and the closed state observed in unliganded structures. Mutagenesis of potential binding site residues was used to probe the contributions of groove residues to the binding properties of Mcl-1. Although mutations in Mcl-1 had little impact on binding, a single mutation in the BH3-only ligand Bad enabled it to bind both Mcl-1 and A1 while retaining its binding to Bcl-2, Bcl-xL, and Bcl-w. Elucidating the selective action of certain BH3-only ligands is required for delineating their mode of action and will aid the search for effective BH3-mimetic drugs.

The structure of Bcl-w reveals a role for the C-terminal residues in modulating biological activity.

Pro-survival Bcl-2-related proteins, critical regulators of apoptosis, contain a hydrophobic groove targeted for binding by the BH3 domain of the pro-apoptotic BH3-only proteins. The solution structure of the pro-survival protein Bcl-w, presented here, reveals that the binding groove is not freely accessible as predicted by previous structures of pro-survival Bcl-2-like molecules. Unexpectedly, the groove appears to be occluded by the C-terminal residues. Binding and kinetic data suggest that the C-terminal residues of Bcl-w and Bcl-x(L) modulate pro-survival activity by regulating ligand access to the groove. Binding of the BH3-only proteins, critical for cell death initiation, is likely to displace the hydrophobic C-terminal region of Bcl-w and Bcl-x(L). Moreover, Bcl-w does not act only by sequestering the BH3-only proteins. There fore, pro-survival Bcl-2-like molecules probably control the activation of downstream effectors by a mechanism that remains to be elucidated.