The St Thomas' Hospital Emergency Department Homeless Health Initiative: improving the quality, safety and equity of healthcare provided for homeless patients attending the ED.

We carried out a quality improvement (QI) project (QIP), aiming to improve the quality, safety and equity of healthcare provided for homeless patients attending the emergency department (ED). We used QI methodology to identify areas for improvement, and introduced and modified interventions over four Plan, Do, Study, Act cycles. We launched a departmental 'Homeless Health Initiative' (HHI), the chief intervention being the provision of 'Homeless Health Boxes' in the ED, which contained a 'Safe Discharge Checklist for Homeless Patients', maps to specialist homeless general practitioner surgeries and homeless day centres, information on other inclusion health services, copies of a local rough sleepers' magazine and oral hygiene supplies. Voluntary Homeless Link Nurses and a number of informal 'Homeless Health Champions' were appointed. The HHI was embedded in departmental awareness through regular presentations to staff and incorporation into the induction programme for new doctors. Staff satisfaction, in terms of how satisfied staff members were with the care they were able to provide for homeless patients in the ED on a 0-10 scale, improved modestly over the course of the QIP from median 6/10 to median 7/10. The number of staff who were severely dissatisfied with the care they were able to provide for homeless patients improved more markedly: first quartile staff satisfaction improved from 3.875/10 to 6.125/10. Staff compliance with the checklist was poor, with full compliance observed in only 15% of cases by the end of the QIP. An HHI is a cheap and worthwhile QI project, with the potential to significantly improve the quality, safety and equity of healthcare provided for homeless patients, while improving staff satisfaction concurrently. Similar initiatives should be considered in any ED which sees a significant number of homeless patients.

Meso scale discovery-based assays for the detection of aggregated huntingtin.

Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.

Site-specific recombination strategies for engineering actinomycete genomes.

The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific loxLE and loxRE sites was similar to its activity on wild-type loxP sites. Moreover, an apramycin resistance marker flanked by the loxLERE sites was eliminated from the Streptomyces coelicolor M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from Streptomyces sp. strain Tü6071, Streptomyces cinnamonensis A519, and Streptomyces aureofaciens Tü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.

Functions of genes and enzymes involved in phenalinolactone biosynthesis.

Phenalinolactones are novel terpene glycoside antibiotics produced by Streptomyces sp. Tü6071. Inactivation of three oxygenase genes (plaO2, plaO3 and plaO5), two dehydrogenase genes (plaU, plaZ) and one putative acetyltransferase gene (plaV) led to the production of novel phenalinolactone derivatives (PL HS6, PL HS7, PL HS2 and PL X1). Furthermore, the exact biosynthetic functions of two enzymes were determined, and their in vitro activities were demonstrated. PlaO1, an Fe(II)/alpha-ketoglutarate-dependent dioxygenase, is responsible for the key step in gamma-butyrolactone formation, whereas PlaO5, a cytochrome P450-dependent monooxygenase, catalyses the 1-C-hydroxylation of phenalinolactone D. In addition, stable isotope feeding experiments with biosynthetic precursors shed light on the origin of the carbons in the gamma-butyrolactone moiety.

Genes and enzymes involved in bacterial isoprenoid biosynthesis.

Isoprenoids produced by bacteria are of particular interest as they encompass huge structural and functional diversity. A rapidly increasing number of bacterial derived isoprenoids have been reported in recent years, as many of the genes and biosynthetic gene clusters are responsible for their biosynthesis. Polyprenyl chains, synthesized by the condensation of isopentenyl diphosphate units, serve as the substrates for cyclases and subsequent tailoring enzymes. It is these enzymes, particularly the cyclases, which are responsible for the structural diversity of this chemical class. Recent studies have revealed novel insights into isoprenoid biosynthesis, and in several cases enzymatic mechanisms have been redefined.