Pre-operative resuscitation discussion with patients undergoing fractured neck of femur repair: a service evaluation and discussion of current standards.

INTRODUCTION: The majority of neck of femur (NOF) fracture patients are frail and at a higher risk of cardiac arrest. This makes discussion of treatment escalation vital to informed care. The optimal time for these discussions is prior to admission or trauma. However, when this has not occurred, it is vital that these discussions happen early in the patient's admission when family is often present and before further deterioration in their condition. We undertook a service evaluation to evaluate and discuss the effect of clinician education on improving rates of timely discussion amongst orthopaedic doctors. MATERIALS AND METHODS: The first cycle included 94 patients. Their notes were reviewed for presence of a ReSPECT (Recommend Summary Plan for Emergency Care and Treatment) form prior to operation and whether this it countersigned by a consultant. Following this, clinician education was undertaken and a re-audit was carried out involving 57 patients. RESULTS: ReSPECT form completion rates rose from 23% in cycle 1-32% in cycle 2 following intervention. The proportion which consultants signed rose from 41% to 56% following intervention. CONCLUSION: This project demonstrates how a basic education program can prove limited improvements in the rates of timely resuscitation discussions. We discuss a current lack in quality research into educational programs for discussion of treatment escalation for orthopaedic trainees. We suggest there is room to improve national best practice guidelines and training to ensure these discussions are carried out more frequently and to a better standard.

Kindlin binds migfilin tandem LIM domains and regulates migfilin focal adhesion localization and recruitment dynamics.

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.

Using FRET to analyse signals controlling cell adhesion and migration.

Cell adhesion and migration are dynamic, complex processes that require tight temporal control of signalling cascades at defined subcellular sites to occur. Decades of research have used biochemical methods to identify numerous signalling pathways that are involved in the coordination of adhesion, membrane protrusion and contractility that all contribute to migration. However, understanding the way in which these signals are controlled within discrete sites in individual cells undergoing migration is essential to enable a clearer understanding of how these pathways are integrated during motility. Recent advances in the generation of fluorescent protein variants have enabled the development of probes to analyse localized changes in protein conformation, activation or interactions in single cells using microscopy. Many of these probes are based on the use of fluorescence resonance energy transfer that permits precise determination of interacting species based on the emission or lifetime properties of the fluorophores involved. Here, we provide an overview of some of the recent studies that have developed such probes and examples of how these biosensors have been used to further our understanding of cell migration signalling.

The interchain disulfide linkage of T-cell antigen receptor-alpha and -beta chains is a prerequisite for T-cell activation.

Complementary DNAs encoding the T-cell antigen receptor (TCR)-alpha and mutant TCR-beta chains, lacking the interchain disulfide bond-related cysteine, were introduced into a TCR-alpha and -beta protein-deficient T-cell line. TCR-alpha and the mutant TCR-beta chains assembled with the CD3-epsilon, -gamma, -delta, and -zeta subunits and were efficiently transported to the cell surface; however, the hybrid TCR molecules exhibited a diminished response to T-cell activation by major histocompatibility complex-bound antigen, superantigen, and TCR cross-linking. These results suggest that the interchain disulfide bond between the TCR clonotypic chains is not required for TCR assembly and cell surface expression, but it plays an important role in maintaining the functional integrity of the TCR complex.

Conformation of the T-cell antigen receptor-beta chain C-domain contributes to V beta 3 epitope recognition by monoclonal antibody KJ25.

The clonotypic T-cell antigen receptor (TCR)-beta chain contains two extracellular intrachain disulfide bonds. It belongs to the immunoglobulin gene superfamily and is subdivided into variable (V), joining (J), diversity (D) and constant (C) region. Monoclonal antibody (MoAb) KJ25 is believed to recognize an epitope in the V-domain of TCR-beta (V beta 3) chain, but its epitope requirements are unknown. In this study of TCR-alpha beta chain interactions using chimeric recombinant TCR-beta chains, the authors found that partial substitution of the C beta-domain with that of interleukin-2 receptor alpha chain (Tac) sequences led to the loss of TCR-V beta 3 epitope recognition by KJ25. These results suggest that epitope recognition of the TCR-V beta 3 by KJ25 MoAb is dependent not only on the V-domain, but also on the close contact with the extracellular C-domain which influences the conformation and epitope recognition of the V beta 3-region. This may not be unique to V beta 3 and may be a general feature of TCR-beta protein folding.

The T cell antigen receptor alpha and beta chains interact via distinct regions with CD3 chains.

Selective pairwise interactions between CD3 chains and the clonotypic T cell antigen receptor (TCR)-alpha, -beta chains has recently been established. In this study, the region of interaction between clonotypic and CD3 chains involved with assembly was examined. To determine the site of protein interaction a variety of genetically altered TCR chains were constructed. These included: truncated proteins, lacking transmembrane and or cytosolic domains; chimeric proteins, in which extracellular, transmembrane or cytosolic domains were replaced with similar domains derived from either the Tac antigen or CD4; and point mutagenized TCR chains. COS-1 cells were transfected with cDNA, metabolically labeled, and immunoprecipitates analyzed using non-equilibrium pH gel electrophoresis (NEPHGE)-SDS/PAGE. The results demonstrated that assembly between TCR-alpha and TCR-beta chains occurred at the extracellular level. Assembly of the TCR-alpha chain with CD3-delta, and CD3-epsilon was localized to an eight-amino acid motif within the transmembrane domain of TCR-alpha. Site-specific mutations of the TCR-alpha charged residues within this motif (arginine, lysine) to leucine and similar point mutations of the transmembrane CD3-epsilon and CD3-delta charge groups resulted in the abrogation of assembly. In contrast, TCR-beta and CD3-epsilon binary complexes interacted via their extracellular domain. Analogous to TCR-alpha, the site of TCR-beta and CD3-delta assembly was at the transmembrane region. Despite multiple genetic manipulations on CD3-gamma and zeta these proteins failed to assemble with TCR-alpha. Similarly, there was no interaction between TCR-beta and zeta. These findings when coupled with the information on pairwise interactions and formation of higher order subcomplexes extend our model for the structure of the TCR complex.