Production of recombinant human proliferating cellular nuclear antigen (PCNA) for structural and biophysical characterization.

Human proliferating cell nuclear antigen (hPCNA) is a DNA replication processivity factor, which acts as a docking platform, allowing proteins to have access to the replication fork and increasing the affinity of DNA interacting proteins, making it critical for cell survival. The trimer forms a ring-shaped oligomer allowing DNA to pass through the middle and interacting proteins to dock on the outside of the ring. Without this structural formation, there is a loss of DNA replication and repair in the cell. Due to the location of subunit-subunit termini, the addition of a purification tag can hamper crystallography and biophysical experiments, as the trimer complex folding can be impeded. To avoid these complications, a tag-less, step-wise purification was implemented, which resulted in 17.6 mg from 2 L culture of pure hPCNA with a 260 nm/280 nm value of 0.43. The produced crystal structure reveals a correctly formed oligomer. The clear depletion of the tracer binding and probe protein interaction in a fluorescence polarisation competition-based assay demonstrates the purification method produces a protein structure with a functional binding site. This purification method presents a reliable and simple method for producing hPCNA for biophysical characterisation.

Liver Transplantation After Ex Vivo Normothermic Machine Preservation: A Phase 1 (First-in-Man) Clinical Trial.

The number of donor organs suitable for liver transplantation is restricted by cold preservation and ischemia-reperfusion injury. We present the first patients transplanted using a normothermic machine perfusion (NMP) device that transports and stores an organ in a fully functioning state at 37°C. In this Phase 1 trial, organs were retrieved using standard techniques, attached to the perfusion device at the donor hospital, and transported to the implanting center in a functioning state. NMP livers were matched 1:2 to cold-stored livers. Twenty patients underwent liver transplantation after NMP. Median NMP time was 9.3 (3.5-18.5) h versus median cold ischaemia time of 8.9 (4.2-11.4) h. Thirty-day graft survival was similar (100% NMP vs. 97.5% control, p = 1.00). Median peak aspartate aminotransferase in the first 7 days was significantly lower in the NMP group (417 IU [84-4681]) versus (902 IU [218-8786], p = 0.03). This first report of liver transplantation using NMP-preserved livers demonstrates the safety and feasibility of using this technology from retrieval to transplantation, including transportation. NMP may be valuable in increasing the number of donor livers and improving the function of transplantable organs.

Colovaginal and colovesical fistulae: the diagnostic paradigm.

BACKGROUND: Colovaginal and colovesical fistulae (CVF) are relatively uncommon conditions, most frequently resulting from diverticular disease or colorectal cancer. A high suspicion of a CVF can usually be obtained from an accurate clinical history. Demonstrating CVF radiologically is often challenging, and patients frequently undergo a multitude of investigations prior to definitive management. The aim of this study was to develop an algorithm for the investigation of suspected CVF in order to improve diagnosis and subsequent management. METHODS: Thirty-seven patients from a single NHS Trust with a diagnosis of colovaginal or colovesical fistula were included in the study. Clinical records and imaging were reviewed retrospectively, and data on demographics, symptoms, investigations, management and outcome were collated. RESULTS: A total of 87.5% patients with a colovesical fistula presented with pathognomic symptoms of faecaluria or pneumaturia. The commonest aetiologies were diverticular disease (72.9%), colonic and gynaecological neoplasia (10.8% each). Computerised tomography (CT) was the most frequently performed investigation (91.9%) and was most sensitive in detecting the fistula (76.5%) and underlying aetiology (94.1%). Colonoscopy was most sensitive in detecting an underlying colonic malignancy (100%). Resectional surgery was performed in 62.1% of cases, although morbidity and 1-year mortality was significant, with rates of 21.7 and 17.4%, respectively. CONCLUSIONS: The diagnosis of CVF is predominately a clinical one, and patients with a suspected CVF are over-investigated. Investigations should be focused on determining aetiology rather than demonstrating the fistulous tract itself. We propose that, in the majority of cases, CT and lower gastrointestinal endoscopy should suffice.

Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of Escherichia coli.

The influences of the antibacterial magainin 2 and PGLa from the African clawed frog (Xenopus laevis) and the hemolytic bee venom melittin on Escherichia coli as the target cell were studied by atomic force microscopy (AFM). Nanometer-scale images of the effects of the peptides on this gram-negative bacterium's cell envelope were obtained in situ without the use of fixing agents. These high-resolution AFM images of the surviving and intact target cells before and after peptide treatment showed distinct changes in cell envelope morphology as a consequence of peptide action. Although all three peptides are lytic to E. coli, it is clear from this AFM study that each peptide causes distinct morphological changes in the outer membrane and in some cases the inner membrane, probably as a consequence of different mechanisms of action.

The development of a clinical skills centre.

Guidelines produced by the General Medical Council of Great Britain have emphasised the importance of the development of the skills and attitudes appropriate for a junior doctor. Medical schools are in the process of reforming their curricula accordingly. The development of these skills is made increasingly difficult by changes such as short admissions to hospital, increased care in the community, and reduced resources. This article outlines the development of a clinical skills centre as a multidisciplinary unit to improve clinical skills teaching with the aid of up-to-date technology and educational practices. By sharing our experience we aim to provide a practical guide for the development of such units in other medical and nursing colleges.

Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600.

Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39.5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component.