Pseudomonas aeruginosa lectin LecB inhibits tissue repair processes by triggering β-catenin degradation

AbstractPseudomonas aeruginosa is an opportunistic pathogen that induces severe lung infections such as ventilator-associated pneumonia and acute lung injury. Under these conditions, the bacterium diminishes epithelial integrity and inhibits tissue repair mechanisms, leading to persistent infections. Understanding the involved bacterial virulence factors and their mode of action is essential for the development of new therapeutic approaches.In our study we discovered a so far unknown effect of the P. aeruginosa lectin LecB on host cell physiology. LecB alone was sufficient to attenuate migration and proliferation of human lung epithelial cells and to induce transcriptional activity of NF-κB. These effects are characteristic of impaired tissue repair. Moreover, we found a strong degradation of β-catenin, which was partially recovered by the proteasome inhibitor lactacystin. In addition, LecB induced loss of cell–cell contacts and reduced expression of the β-catenin targets c-myc and cyclin D1. Blocking of LecB binding to host cell plasma membrane receptors by soluble l-fucose prevented these changes in host cell behavior and signaling, and thereby provides a powerful strategy to suppress LecB function.Our findings suggest that P. aeruginosa employs LecB as a virulence factor to induce β-catenin degradation, which then represses processes that are directly linked to tissue recovery

Effectiveness of balance training exercise in people with mild to moderate severity Alzheimer's disease: protocol for a randomised trial

BACKGROUND: Balance dysfunction and falls are common problems in later stages of dementia. Exercise is a well-established intervention to reduce falls in cognitively intact older people, although there is limited randomised trial evidence of outcomes in people with dementia. The primary objective of this study is to evaluate whether a home-based balance exercise programme improves balance performance in people with mild to moderate severity Alzheimer's disease. METHODS/DESIGN: Two hundred and fourteen community dwelling participants with mild to moderate severity Alzheimer's disease will be recruited for the randomised controlled trial. A series of laboratory and clinical measures will be used to evaluate balance and mobility performance at baseline. Participants will then be randomized to receive either a balance training home exercise programme (intervention group) from a physiotherapist, or an education, information and support programme from an occupational therapist (control group). Both groups will have six home visits in the six months following baseline assessment, as well as phone support. All participants will be re-assessed at the completion of the programme (after six months), and again in a further six months to evaluate sustainability of outcomes. The primary outcome measures will be the Limits of Stability (a force platform measure of balance) and the Step Test (a clinical measure of balance). Secondary outcomes include other balance and mobility measures, number of falls and falls risk measures, cognitive and behavioural measures, and carer burden and quality of life measures. Assessors will be blind to group allocation. Longitudinal change in balance performance will be evaluated in a sub-study, in which the first 64 participants of the control group with mild to moderate severity Alzheimer's disease, and 64 age and gender matched healthy participants will be re-assessed on all measures at initial assessment, and then at 6, 12, 18 and 24 months. DISCUSSION: By introducing a balance programme at an early stage of the dementia pathway, when participants are more likely capable of safe and active participation in balance training, there is potential that balance performance will be improved as dementia progresses, which may reduce the high falls risk at this later stage. If successful, this approach has the potential for widespread application through community based services for people with mild to moderate severity Alzheimer's disease. TRIAL REGISTRATION: The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000040369)

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Uptake of Marasmius oreades agglutinin disrupts integrin-dependent cell adhesion

Background Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a lectin from the fairy ring mushroom with specificity for Galα1-3Gal containing carbohydrates. This lectin is composed of an N-terminal carbohydrate-binding domain and a C-terminal dimerization domain. The dimerization domain of MOA shows in addition calcium-dependent cysteine protease activity, similar to the calpain family. Methods Cell detachment assay, cell viability assay, immunofluorescence, live cell imaging and Western blot using MDCKII cell line. Results In this study, we demonstrate in MDCKII cells that after internalization, MOA protease activity induces profound physiological cellular responses, like cytoskeleton rearrangement, cell detachment and cell death. These changes are preceded by a decrease in FAK phosphorylation and an internalization and degradation of β1-integrin, consistent with a disruption of integrin-dependent cell adhesion signaling. Once internalized, MOA accumulates in late endosomal compartments. Conclusion Our results suggest a possible toxic mechanism of MOA, which consists of disturbing the cell adhesion and the cell viability. General significance After being ingested by a predator, MOA might exert a protective role by diminishing host cell integrity.ISSN:0304-4165ISSN:1872-800