Enhanced Colorimetric Differentiation between Staphylococcus aureus and Pseudomonas aeruginosa Using a Shape-Encoded Sensor Hydrogel

Herein, we demonstrate a combined fluorescent probe/shape-encoded hydrogel strategy for the fast, sensitive, and selective detection of bacterial species via their characteristic enzymes. A poly(vinyl alcohol) (PVA) hydrogel loaded with the fluorescent probe N,N′-(3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′-diyl)bis(2,2,3,3,3-pentafluoropropanamide) (ACS-HNE) was designed for the detection of elastase, an enzyme produced by Pseudomonas aeruginosa. Likewise, a chitosan-derived hydrogel was loaded with the fluorescent probe 4-methylumbelliferyl-α-d-glucopyranoside (MUD) by entrapment for the selective detection of α-glucosidase, an enzyme produced by Staphylococcus aureus. For an observation time of 60 min, limits of detection (LODs) of ≤20 nM for elastase and ≤30 pM for α-glucosidase were obtained, which in the latter case is 3 orders of magnitude better than related chitosan systems with covalently coupled substrate. To illustrate the potential utility of these highly sensitive sensor hydrogels as a simple point-of-care test system, shaped hydrogel slabs representing the letters P and S were manufactured to detect P. aeruginosa and S. aureus, respectively. These shapes were shown to provide an additional unique color code under UV illumination corresponding to the characteristic enzyme produced by the corresponding bacteria. This study shows potential for the future development of an effective and simple point-of-care test for the rapid identification of bacterial species that can be operated by nonspecialists

Development of a high-throughput ex-vivo burn wound model using porcine skin, and its application to evaluate new approaches to control wound infection

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this mode

Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections

Altres ajuts: This work has been funded by the project PI13/01418 which is part of "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluacioón and "Fondo Europeo de Desarrollo Regional"(FEDER).D. Domínguez-Villanueva is funded by "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluación and "Fondo Europeo de Desarrollo Regional"(FEDER). M. Gomes-Fernandes is funded by CAPES Foundation, Ministry of Education of Brazil (Brasılia, Brazil). Maisem Laabei was supported by a joint ERS/SEPAR fellowship (LTRF2015).This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR054/2011).Objective. Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. Methods. S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse- transcriptase real-time PCR (qRT-PCR). Results. Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. Conclusions. Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay

Rapid dynamic activation of a marine-based Arctic ice cap

We use satellite observations to document rapid acceleration and ice loss from a formerly slow-flowing, marine-based sector of Austfonna, the largest ice cap in the Eurasian Arctic. During the past two decades, the sector ice discharge has increased 45-fold, the velocity regime has switched from predominantly slow (~ 101 m/yr) to fast (~ 103 m/yr) flow, and rates of ice thinning have exceeded 25 m/yr. At the time of widespread dynamic activation, parts of the terminus may have been near floatation. Subsequently, the imbalance has propagated 50 km inland to within 8 km of the ice cap summit. Our observations demonstrate the ability of slow-flowing ice to mobilize and quickly transmit the dynamic imbalance inland; a process that we show has initiated rapid ice loss to the ocean and redistribution of ice mass to locations more susceptible to melt, yet which remains poorly understood.This work was supported by the UK Natural Environment Research Council.This article was originally published in Geophysical Research Letters (M McMillan, A Shepherd, N Gourmelen, A Dehecq, A Leeson, A Ridout, T Flament, A Hogg, L Gilbert, T Benham, M van den Broeke, JA Dowdeswell, X Fettweis, B Noël, T Strozzi, Geophysical Research Letters 2014, 41, 8902–8909)

Translational pharmacology of an inhaled small molecule αvβ6 integrin inhibitor for idiopathic pulmonary fibrosis

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and diseaserelated end points. Here we report, GSK3008348 binds to αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy

PCSK6 and Survival in Idiopathic Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed. Objectives: To identify and validate molecular determinants of IPF survival. Methods: A staged genome-wide association study was performed using paired genomic and survival data. Stage I cases were drawn from centers across the United States and Europe and stage II cases from Vanderbilt University. Cox proportional hazards regression was used to identify gene variants associated with differential transplantation-free survival (TFS). Stage I variants with nominal significance (P < 5 x 10(-5)) were advanced for stage II testing and meta-analyzed to identify those reaching genome-wide significance (P < 5 x 10(-8)). Downstream analyses were performed for genes and proteins associated with variants reaching genome-wide significance. Measurements and Main Results: After quality controls, 1,481 stage I cases and 397 stage II cases were included in the analysis. After filtering, 9,075,629 variants were tested in stage I, with 158 meeting advancement criteria. Four variants associated with TFS with consistent effect direction were identified in stage II, including one in an intron of PCSK6 (proprotein convertase subtilisin/kexin type 6) reaching genome-wide significance (hazard ratio, 4.11 [95% confidence interval, 2.54-6.67]; P = 9.45 x 10(-9)). PCSK6 protein was highly expressed in IPF lung parenchyma. PCSK6 lung staining intensity, peripheral blood gene expression, and plasma concentration were associated with reduced TFS. Conclusions: We identified four novel variants associated with IPF survival, including one in PCSK6 that reached genome-wide significance. Downstream analyses suggested that PCSK6 protein plays a potentially important role in IPF progression

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Genome-Wide Association Study of Susceptibility to Idiopathic Pulmonary Fibrosis

Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterised by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide association studies have reported signals of association implicating multiple pathways including host defence, telomere maintenance, signalling and cell-cell adhesion. Objectives: To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations. Methods and measurements: We conducted genome-wide analyses across three independent studies and meta-analysed these results to generate the largest genome-wide association study of IPF to date (2,668 IPF cases and 8,591 controls). We performed replication in two independent studies (1,456 IPF cases and 11,874 controls) and functional analyses (including statistical fine-mapping, investigations into gene expression and testing for enrichment of IPF susceptibility signals in regulatory regions) to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF. Main results: We identified and replicated three new genome-wide significant (P<5×10−8) signals of association with IPF susceptibility (associated with altered gene expression of KIF15, MAD1L1 and DEPTOR) and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as-yet unreported IPF susceptibility variants contribute to IPF susceptibility. Conclusions: The observation that decreased DEPTOR expression associates with increased susceptibility to IPF, supports recent studies demonstrating the importance of mTOR signalling in lung fibrosis. New signals of association implicating KIF15 and MAD1L1 suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility