Integrating microfluidic generation, handling and analysis of biomimetic giant unilamellar vesicles

The key roles played by phospholipids in many cellular processes, has led to the development of model systems, to explore both lipid–lipid and lipid–peptide interactions. Biomimetic giant unilamellar vesicles represent close facsimiles of in vivo cellular membranes, although currently their widespread use in research is hindered by difficulties involving their integration into high-throughput techniques, for exploring membrane biology intensively in situ. This paper presents an integrated microfluidic device for the production, manipulation and high-throughput analysis of giant unilamellar vesicles. Its utility is demonstrated by exploring the lipid interaction dynamics of the pore-forming antimicrobial peptide melittin, assessed through the release of fluorescent dyes from within biomimetic vesicles, with membrane compositions similar to mammalian plasma membranes

Intracoronary Brachytherapy, a Promising Treatment Option for Diabetic Patients: Results from a European Multicenter Registry (RENO)

Despite advances in the interventional treatment of coronary disease, diabetics still have double the case fatality rate as nondiabetics. The purpose of this an

Endoluminal beta-radiation therapy for the prevention of coronary restenosis after balloon angioplasty.

BACKGROUND: Beta radiation is effective in reducing vascular neointimal proliferation in animals after injury caused by balloon angioplasty. However, the lowest dose that can prevent restenosis after coronary angioplasty has yet to be determined. METHODS: After successful balloon angioplasty of a previously untreated coronary stenosis, 181 patients were randomly assigned to receive 9, 12, 15, or 18 Gy of radiation delivered by a centered yttrium-90 source. Adjunctive stenting was required in 28 percent of the patients. The primary end point was the minimal luminal diameter six months after treatment, as a function of the delivered dose of radiation. RESULTS: At the time of follow-up coronary angiography, the mean minimal luminal diameter was 1.67 mm in the 9-Gy group, 1.76 mm in the 12-Gy group, 1.83 mm in the 15-Gy group, and 1.97 mm in the 18-Gy group (P=0.06 for the comparison of 9 Gy with 18 Gy), resulting in restenosis rates of 29 percent, 21 percent, 16 percent, and 15 percent, respectively (P=0.14 for the comparison of 9 Gy with 18 Gy). At that time, 86 percent of the patients had had no serious cardiac events. In 130 patients treated with balloon angioplasty alone, restenosis rates were 28 percent, 17 percent, 16 percent, and 4 percent, respectively (P=0.02 for the comparison of 9 Gy with 18 Gy). Among these patients, there was a dose-dependent enlargement of the lumen in 28 percent, 50 percent, 45 percent, and 74 percent of patients, respectively (P<0.001 for the comparison of 9 Gy with 18 Gy). The rate of repeated revascularization was 18 percent with 9 Gy and 6 percent with 18 Gy (P=0.26). CONCLUSIONS: Intracoronary beta radiation therapy produces a significant dose-dependent decrease in the rate of restenosis after angioplasty. An 18-Gy dose not only prevents the renarrowing of the lumen typically observed after successful balloon angioplasty, but actually induces luminal enlargement

Programmed Bending Reveals Dynamic Mechanochemical Coupling in Supported Lipid Bilayers

In living cells, mechanochemical coupling represents a dynamic means by which membrane components are spatially organized. An extra-ordinary example of such coupling involves curvature-dependent polar localization of chemically-distinct lipid domains at bacterial poles, which also undergo dramatic reequilibration upon subtle changes in their interfacial environment such as during sporulation. Here, we demonstrate that such interfacially-triggered mechanochemical coupling can be recapitulated in vitro by simultaneous, real-time introduction of mechanically-generated periodic curvatures and attendant strain-induced lateral forces in lipid bilayers supported on elastomeric substrates. In particular, we show that real-time wrinkling of the elastomeric substrate prompts a dynamic domain reorganization within the adhering bilayer, producing large, oriented liquid-ordered domains in regions of low curvature. Our results suggest a mechanism in which interfacial forces generated during surface wrinkling and the topographical deformation of the bilayer combine to facilitate dynamic reequilibration prompting the observed domain reorganization. We anticipate this curvature-generating model system will prove to be a simple and versatile tool for a broad range of studies of curvature-dependent dynamic reorganizations in membranes that are constrained by the interfacial elastic and dynamic frameworks such as the cell wall, glycocalyx, and cytoskeleton

Real-time intermembrane force measurements and imaging of lipid domain morphology during hemifusion

Membrane fusion is the core process in membrane trafficking and is essential for cellular transport of proteins and other biomacromolecules. During protein-mediated membrane fusion, membrane proteins are often excluded from the membrane-membrane contact, indicating that local structural transformations in lipid domains play a major role. However, the rearrangements of lipid domains during fusion have not been thoroughly examined. Here using a newly developed Fluorescence Surface Forces Apparatus (FL-SFA), migration of liquid-disordered clusters and depletion of liquid-ordered domains at the membrane-membrane contact are imaged in real time during hemifusion of model lipid membranes, together with simultaneous force-distance and lipid membrane thickness measurements. The load and contact time-dependent hemifusion results show that the domain rearrangements decrease the energy barrier to fusion, illustrating the significance of dynamic domain transformations in membrane fusion processes. Importantly, the FL-SFA can unambiguously correlate interaction forces and in situ imaging in many dynamic interfacial systems.open0

Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter

Modeling morphological instabilities in lipid membranes with anchored amphiphilic polymers

Anchoring molecules, like amphiphilic polymers, are able to dynamically regulate membrane morphology. Such molecules insert their hydrophobic groups into the bilayer, generating a local membrane curvature. In order to minimize the elastic energy penalty, a dynamic shape instability may occur, as in the case of the curvature-driven pearling instability or the polymer-induced tubulation of lipid vesicles. We review recent works on modeling of such instabilities by means of a mesoscopic dynamic model of the phase-field kind, which take into account the bending energy of lipid bilayers

Involking silvern voices in healthcare : transforming practice by engaging older adults in collaborative partnerships

Canada's population is aging. This growing trend will ultimately have an impact on nursing practice as older individuals continue to seek healthcare services. Nurses must be able to work in collaboration with the older population to provide quality care. This action research study explored participative healthcare from an older adult's perspective. This study revealed that older adults prefer to be active participants in their care. The major theme that emerged was true partnership. Three sub-themes that emerged were communication, respect, and trust. These three sub-themes work in unity to contribute to a healthcare experience that exemplifies true partnerships. This study proposes a definition of true partnership as being open to and inviting mutual communication in an atmosphere that encourages equity sharing of information contributing to respect and the development of trust that results in confident collaboration in care

Life-threatening infections in children in Europe (the EUCLIDS Project): a prospective cohort study

Background: Sepsis and severe focal infections represent a substantial disease burden in children admitted to hospital. We aimed to understand the burden of disease and outcomes in children with life-threatening bacterial infections in Europe. Methods: The European Union Childhood Life-threatening Infectious Disease Study (EUCLIDS) was a prospective, multicentre, cohort study done in six countries in Europe. Patients aged 1 month to 18 years with sepsis (or suspected sepsis) or severe focal infections, admitted to 98 participating hospitals in the UK, Austria, Germany, Lithuania, Spain, and the Netherlands were prospectively recruited between July 1, 2012, and Dec 31, 2015. To assess disease burden and outcomes, we collected demographic and clinical data using a secured web-based platform and obtained microbiological data using locally available clinical diagnostic procedures. Findings: 2844 patients were recruited and included in the analysis. 1512 (53·2%) of 2841 patients were male and median age was 39·1 months (IQR 12·4–93·9). 1229 (43·2%) patients had sepsis and 1615 (56·8%) had severe focal infections. Patients diagnosed with sepsis had a median age of 27·6 months (IQR 9·0–80·2), whereas those diagnosed with severe focal infections had a median age of 46·5 months (15·8–100·4; p<0·0001). Of 2844 patients in the entire cohort, the main clinical syndromes were pneumonia (511 [18·0%] patients), CNS infection (469 [16·5%]), and skin and soft tissue infection (247 [8·7%]). The causal microorganism was identified in 1359 (47·8%) children, with the most prevalent ones being Neisseria meningitidis (in 259 [9·1%] patients), followed by Staphylococcus aureus (in 222 [7·8%]), Streptococcus pneumoniae (in 219 [7·7%]), and group A streptococcus (in 162 [5·7%]). 1070 (37·6%) patients required admission to a paediatric intensive care unit. Of 2469 patients with outcome data, 57 (2·2%) deaths occurred: seven were in patients with severe focal infections and 50 in those with sepsis. Interpretation: Mortality in children admitted to hospital for sepsis or severe focal infections is low in Europe. The disease burden is mainly in children younger than 5 years and is largely due to vaccine-preventable meningococcal and pneumococcal infections. Despite the availability and application of clinical procedures for microbiological diagnosis, the causative organism remained unidentified in approximately 50% of patients