Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH.

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.

A Luciferase Immunoprecipitation System (LIPS) assay for profiling human norovirus antibodies.

A luciferase immunoprecipitation systems (LIPS) assay was developed to define the antigenic specificity and titer of antibodies directed against human norovirus (HuNoV). Recombinant proteins, expressed by plasmid constructs encoding Renilla luciferase (Ruc) fused to the full-length HuNoV major capsid protein (VP1) (Ruc-antigen), were generated for ten HuNoV strains. In addition, subdomain constructs Ruc-Shell (S) and Ruc-Protruding (P) were engineered for a representative GII.4 norovirus (strain GII.4/2006b). The LIPS assay measured antibody levels in a well-defined panel of HuNoV-specific sera, and the results were compared to an ELISA standard. In hyperimmune sera, the LIPS produced titers similar to or higher than those measured by the ELISA of HuNoV-specific antibodies. The specificity of antibodies in various sera was profiled by LIPS with a panel of diverse Ruc-antigens containing full-length HuNoV VP1 proteins or VP1 subdomains, and the assay detected both specific and cross-reactive antibodies. Competition assays, in which antibodies were pre-incubated with one or more intact VLPs representing different genotypes, proved useful in further assessment of the antibody specificity detected by LIPS in complex polyclonal sera. The profiling of HuNoV-specific antibodies in the high-throughput LIPS format may prove useful in defining the strength or specificity of the adaptive immune response following natural infection or vaccination.

Improving the safety of chemotherapy administration: an oncology nurse-led failure mode and effects analysis.

PURPOSE/OBJECTIVES: To assess and improve the safety of hospital-based adult chemotherapy administration. DESIGN: Prospective, systems-focused clinical risk assessment. SETTING: An adult inpatient and outpatient oncology unit in a large urban hospital in the United Kingdom. SAMPLE: 8-person nurse-led multidisciplinary team, which included managerial staff and patient safety researchers. METHODS: Failure mode and effects analysis (FMEA), a prospective, systems-focused risk assessment methodology, was undertaken in biweekly team meetings and included mapping the chemotherapy administration process, identifying and numerically prioritizing potential errors (failure modes) for each process step, and generating remedial strategies to counteract them. MAIN RESEARCH VARIABLES: The analysis aimed to identify chemotherapy administration failure modes and to generate remedial strategies to address them. User feedback on the FMEA process also was collected. FINDINGS: Several specific chemotherapy failure modes were identified, the majority of which had not previously been recognized, and several novel strategies to counteract them were generated. Many of the strategies were specific, environment-focused actions, which are simple, quick, and inexpensive to implement; however, more substantive, longer-term initiatives also were generated. User feedback generally was very positive, and the process of undertaking the analysis improved multidisciplinary teamwork and communication. CONCLUSIONS: Although time and resource intensive, FMEA is a useful safety improvement tool. IMPLICATIONS FOR NURSING: Nurses should be aware of and informed about FMEA as a tool they can use in partnership with management and other disciplines to proactively and collectively improve the safety of high-risk oncology procedures such as chemotherapy administration.

DNA sequence recognition in the minor groove by hairpin pyrrole polyamide-Hoechst 33258 analogue conjugate.

A hairpin pyrrole polyamide conjugated to a Hoechst 33258 (Ht) analogue, PyPyPy-gamma-PyPyPy-gamma-Ht, was synthesized on solid-phase by adaptation of an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. Sequence selectivity and complex stabilities were characterized by spectrofluorometric titrations and thermal melting studies. The polyamide of the conjugate was observed to bind in a hairpin motif forming 1:1 conjugate:dsDNA complexes. The conjugate is able to recognize nine contiguous A/T bps, discriminating from the sequences containing fewer than nine contiguous A/T bps.