Magnetically localized and wash-free fluorescence immunoassay (MLFIA): proof of concept and clinical applications.

Immunoassays are used for many applications in various markets, from clinical diagnostics to the food industry, generally relying on gold-standard ELISAs that are sensitive, robust, and cheap but also time-consuming and labour intensive. As an alternative, we propose here the magnetically localized and wash-free fluorescence immunoassay (MLFIA): a no-wash assay to directly measure a biomolecule concentration, without mixing nor washing steps. To do so, a fluorescence no-wash measurement is performed to generate a detectable signal. It consists of a differential measurement between the fluorescence of fluorophores bound to magnetic nanoparticles specifically captured by micro-magnets against the residual background fluorescence of unbound fluorophores. Targeted biomolecules (antibodies or antigens) are locally concentrated on micro-magnet lines, with the number of captured biomolecules quantitatively measured without any washing step. The performance of the MLFIA platform is assessed and its use is demonstrated with several biological models as well as clinical blood samples for HIV, HCV and HBV detection, with benchmarking to standard analyzers of healthcare laboratories. Thus, we demonstrated for the first time the versatility of the innovative MLFIA platform. We highlighted promising performances with the successful quantitative detection of various targets (antigens and antibodies), in different biological samples (serum and plasma), for different clinical tests (HCV, HBV, HIV).

Effect of the mechanical properties of carbon-based coatings on the mechanics of cell-material interactions.

The paper presents an influence of the surface mechanical properties of thin-film materials on blood cell adhesion under shear stress conditions. Physical vapour deposited (PVD) coatings i.e. hydrogenated amorphous carbon (a-C:H) doped with nitrogen or silicon have been investigated. The mechanical properties of materials, namely their microhardness and Young's modulus were measured using indentation test with Rockwell indenter. The adhesion efficiency of blood cells in dynamic conditions were analysed using a radial flow chamber. Red blood cells (RBC) were used as representative cells to analyse cell-material interactions. The biomaterial examinations were performed under physiological flow conditions at the single-cell level. The 3D FVM (finite volume method) model of multi-phase radial flow test was developed to reproduce the physical test and to predict distributions of shear stresses and velocity during blood washout with PBS. Cell-material interactions were found to be strongly associated with the mechanical properties of the thin-film material. The decrease in the hardness of the coatings translated into a weaker cell - material interactions.

Magnetophoretic induced convective capture of highly diffusive superparamagnetic nanoparticles.

Micro-magnets producing magnetic field gradients as high as 106 T m-1 have been used to efficiently trap nanoparticles with a magnetic core of just 12 nm in diameter. Particle capture efficiency increases with increasing particle concentration. Comparison of measured capture kinetics with numerical modelling reveals that a threshold concentration exists below which capture is diffusion-driven and above which it is convectively-driven. This comparison also shows that two-way fluid-particle coupling is responsible for the formation of convective cells, the size of which is governed by the height of the droplet. Our results indicate that for a suspension with a nanoparticle concentration suitable for bioassays (around 0.25 mg ml-1), all particles can be captured in less than 10 minutes. Since nanoparticles have a significantly higher surface-to-volume ratio than the more widely used microparticles, their efficient capture should contribute to the development of next generation digital microfluidic lab-on-chip immunoassays.

Understanding the mechanisms leading to failure in metallic nanowire-based transparent heaters, and solution for stability enhancement.

Silver nanowire (AgNW) networks are emerging as one of the most promising alternatives to indium tin oxide (ITO) for transparent electrodes in flexible electronic devices. They can be used in a variety of optoelectronic applications such as solar cells, touch panels and organic light-emitting diodes. Recently they have also proven to be very efficient when used as transparent heaters (THs). In addition to the study of AgNW networks acting as THs in regular use, i.e. at low voltage and moderate temperature, their stability and physical behavior at higher voltages and for longer durations should be studied in view of their integration into real devices. The properties of AgNW networks deposited by spray coating on glass or flexible transparent substrates are thoroughly studied via in situ measurements. The AgNW networks' behavior at different voltages for different durations and under different atmospheric conditions, both in air and under vacuum, has been examined. At low voltage, a reversible electrical response is observed while irreversibility and even failure are observed at higher voltages. In order to gain a deeper insight into the behavior of AgNW networks used as THs, simple but realistic physical models are proposed and are found to be in fair agreement with the experimental data. Finally, as the stability of AgNW networks is a key issue, we demonstrate that coating AgNW networks with a very thin layer of TiO2 using atomic layer deposition (ALD) improves the material's resistance against electrical and thermal instabilities without altering optical transmittance. We show that the critical annealing temperature associated to network breakdown increases from 270 °C for the as-deposited AgNW networks to 420 °C for AgNW networks coated with TiO2. Similarly, the electrical failure which occurs at 7 V for the as-deposited networks increases to 13 V for TiO2-coated networks. TiO2 is also proved to stabilize AgNW networks during long duration operation and at high voltage. Temperature higher than 235 °C was achieved at 7 V without failure.

Physical model for membrane protrusions during spreading.

During cell spreading onto a substrate, the kinetics of the contact area is an observable quantity. This paper is concerned with a physical approach to modeling this process in the case of ameboid motility where the membrane detaches itself from the underlying cytoskeleton at the leading edge. The physical model we propose is based on previous reports which highlight that membrane tension regulates cell spreading. Using a phenomenological feedback loop to mimic stress-dependent biochemistry, we show that the actin polymerization rate can be coupled to the stress which builds up at the margin of the contact area between the cell and the substrate. In the limit of small variation of membrane tension, we show that the actin polymerization rate can be written in a closed form. Our analysis defines characteristic lengths which depend on elastic properties of the membrane-cytoskeleton complex, such as the membrane-cytoskeleton interaction, and on molecular parameters, the rate of actin polymerization. We discuss our model in the case of axi-symmetric and non-axi-symmetric spreading and we compute the characteristic time scales as a function of fundamental elastic constants such as the strength of membrane-cytoskeleton adherence.

Kinetics of cell spreading.

Cell spreading is a fundamental event where the contact area with a solid substrate increases because of actin polymerization. We propose in this Letter a physical model to study the growth of the contact area with time. This analysis is compared with experimental data using the ameoba Dictyostelium discoideum. Our model couples the stress, which builds up at the margin of the contact area when the cell spreads, to the biochemical processes of actin polymerization. This leads to a scaling analysis of experimental data with a characteristic time whose order of magnitude compares well with our experimental results.

Peeling model for cell detachment.

In many experimental situations, the adhesion of cells to solid substrates is due to non-covalent chemical bonds. It is the thesis of this paper that many phenomena occurring in cell detachment experiments, such as in I (E. Decavé, G. Garriver, Y. Brechet, B. Fourcade, F. Bruckert, Biophys. J. 82, 2383 (2002)), result from the static and dynamic properties of the adhesive bridges at the extreme margin of the cell. This region defines the adhesive belt where the distribution of connected bonds crosses over to zero where the membrane leaves the substrate. The theoretical model we introduce in this paper discusses the threshold force together with the peeling velocity in the same theoretical framework. In this one-dimensional model, the threshold force results from a non-homogeneous distribution of anchor proteins along the membrane so that the adhesive belt increases its capacity to resist motion with increasing the external force. Analyzing the kinetics of the the contact line motion, we derive the characteristic relationship speed versus external force and we describe the non-equilibrium state of the adhesive belt as a function of the speed. We discuss our model in view of the experimental results obtained with D. discoideum for hydrodynamic shear experiments. Our results could be also confronted to single-cell observations.

Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family.

The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices. A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion. Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif. In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes.

New CT index to quantify arterial obstruction in pulmonary embolism: comparison with angiographic index and echocardiography.

OBJECTIVE: This study was designed to define and evaluate a specific index to quantify arterial obstruction with helical CT in acute pulmonary embolism. MATERIALS AND METHODS: Fifty-four patients (mean age, 56 years) with proven pulmonary emboli among 158 consecutive patients, who had undergone both CT and pulmonary angiography for clinically suspected pulmonary embolism, were eligible for the study. The CT obstruction index was defined as (n. d) (n, value of the proximal clot site, equal to the number of segmental branches arising distally; d, degree of obstruction scored as partial obstruction [value of 1] or total obstruction [value of 2]). We compared the CT obstruction index with pulmonary arterial obstruction on angiography (assessed by the Miller index), using linear regression, and correlated it with findings on echocardiography. Interobserver variability was determined for both CT and pulmonary angiography indexes. RESULTS: The CT obstruction index (29% +/- 17%) and the Miller index (43% +/- 25%) were well correlated (r = 0.867, p < 0.0001) with an excellent concordance between investigators for both the CT index (r = 0.944, p < 0.0001) and the Miller index (r = 0.904, p < 0.0001). A CT obstruction index greater than 40% identified more than 90% of patients with right ventricular dilatation. CONCLUSION: The degree of arterial obstruction in pulmonary embolism may be quantified by a specific CT index that appears reproducible and highly correlated to the previously described index with pulmonary angiography. Further evaluations are needed to investigate the usefulness of the CT obstruction index for stratification of patient risk and determining therapeutic options.

Pulmonary embolism detection: prospective evaluation of dual-section helical CT versus selective pulmonary arteriography in 157 patients.

PURPOSE: To evaluate the accuracy of dual-section helical computed tomography (CT) in acute pulmonary embolism (PE) diagnosis. MATERIALS AND METHODS: Of 204 consecutive patients with clinically suspected acute PE (mean age, 58 years +/- 14 [SD]), 158 were enrolled. All patients underwent dual-section helical CT (2.7-mm effective section thickness) and selective pulmonary arteriography within 12 hours of each other. Each image was analyzed independently by two observers, who determined image quality and presence of PE among arterial segments, including at the subsegmental level. The final diagnosis was made with consensus. RESULTS: Selective pulmonary arteriography was considered optimal in 147 (93%), suboptimal in 10 (6%), and inconclusive in one (0.6%) of 158 patients. Dual-section helical CT findings were considered technically optimal in 140 (89%), suboptimal in 11 (7%), and inconclusive in six (4%). Selective pulmonary arteriography demonstrated PE in 62 patients. Four (6%) of 62 patients had isolated subsegmental PE. The sensitivity of dual-section helical CT was 90%, and the specificity was 94%. The positive and negative predictive values were 90% and 94%, respectively. CONCLUSION: Dual-section helical CT is an improvement in helical CT that offers a high sensitivity and specificity for the depiction of PE, including at the subsegmental level. Dual-section helical CT can replace pulmonary arteriography for the direct demonstration of PE in a majority of patients.

A syntaxin 7 homologue is present in Dictyostelium discoideum endosomes and controls their homotypic fusion.

Endo-phagocytic activity is prominent in Dictyostelium discoideum and makes it a good model organism to study the molecular organization of membrane traffic in this pathway. We have identified a syntaxin 7 homologue (26% identity and 54% similarity to human syntaxin 7) in Dictyostelium cDNA and genomic data banks. In addition to the Habc and H3 helices and the C-terminal transmembrane domain characteristic of syntaxins, this protein contains a repetitive N-terminal extension of 68 amino acids. We first showed that Dictyostelium syntaxin 7 was able to form a complex with N-ethylmaleimide-sensitive fusion protein and alpha- and gamma-soluble N-ethylmaleimide-sensitive fusion protein attachment protein. Its intracellular localization was then studied by cell fractionation techniques and magnetic purification of the endocytic compartments. Most of D. discoideum syntaxin 7 is contained in endosomes. Finally, an in vitro endosome homotypic fusion assay (Laurent, O., Bruckert, F., Adessi, C., and Satre, M. (1998) J. Biol. Chem. 273, 793-799) was used to study a possible role for syntaxin 7 in this process. Purified anti-syntaxin 7 antibodies and a recombinant soluble fragment of syntaxin 7 both strongly inhibited fusion activity, indicating that this protein was necessary for endosome-endosome fusion. These results demonstrate the importance of this syntaxin 7 homologue in the early phases of Dictyostelium endo-phagocytic pathway.

Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis.

To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants of Dictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, named PHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species; however, their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and Escherichia coli, but not Klebsiella aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyostelium to various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process.

Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment proteins) families in Dictyostelium discoideum.

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.

[Arterial lipiodol chemoembolization of a left liver metastasis through the right internal thoracic artery]. / Chimioembolisation artérielle lipiodolée d'une métastase du foie gauche via l'artère thoracique interne droite.

The purpose of this article is to illustrate the efficacy of the chemoembolization in patients with hypervascular metastases and to describe the post-embolization change in vascularization pattern. Unusual collaterals may develop following embolization. A 59-year-old woman, followed for unresectable small bowel carcinoid tumor since 1991, underwent successful chemoembolization of several liver metastases. Only one liver lesion, located in segment IV, showed interval increase in size. This lesion was supplied by the right internal mammary artery. A branch of the right internal mammary artery was catheterized using a microcatheter and embolization was performed using doxorubicine-Lipiodol (Adriblastine, Lipiodol) and gelfoam (Spongel). No complications occurred after the procedure. The right internal mammary artery should be considered as a possible source of collateral arterial supply to the liver and should be evaluated in patients with local progression of disease.

[Post-traumatic aneurysmal dissection of the extracranial internal carotid artery; helical computed tomographic and angiographic aspects: a case]. / Dissection post-traumatique anévrismale de la carotide interne extracrânienne; aspect tomodensiométrique hélicoïdal et angiographique: un cas.

A 20-year-old man presented with mild intracranial bleeding, a Horner's syndrome, and left neck swelling following head injury. Following noncontrast CT of the brain, a contrast-enhanced helical CT was performed through the neck that showed a hematoma in the poststyloid space (carotid sheath) with irregular diameter of the ICA. Selective digital subtraction angiography confirmed the presence of left cervical ICA dissection with pseudoaneurysm formation. The aneurysm was resected and an end-to-end anastomosis was done using an inverted saphenous graft. Histology confirmed a diagnosis of traumatic ICA dissection with pseudoaneurysm formation and there was no evidence of pre-existing pathology. Helical CT is a simple, widely available, and relatively non-invasive imaging technique that correlates well with angiography. It should be considered in the evaluation of patients with suspected cervical ICA dissection.

Life-threatening spontaneous psoas haematoma treated by transcatheter arterial embolization.

We report two cases of large spontaneous psoas haematoma in patients on anticoagulant therapy. Active bleeding was well demonstrated on spiral CT. Transcatheter arterial embolization was used successfully to stop bleeding. The diagnostic strategy, the relevance of spiral computed tomography and our therapeutic approach to this complication are discussed.

Helical CT phlebography of the superior vena cava: diagnosis and evaluation of venous obstruction.

OBJECTIVE: The purpose of this study was to determine the feasibility of helical CT phlebography of the superior vena cava (SVC) and to evaluate the role of this imaging technique in the diagnosis and treatment of SVC obstruction. SUBJECTS AND METHODS: Twenty-three helical CT phlebograms were obtained of patients with clinical findings that were suggestive of SVC obstruction (n = 19) and of patients undergoing posttherapeutic evaluation for SVC obstruction (n = 4). CT examinations consisted of helical acquisitions obtained in the craniocaudal direction with simultaneous bilateral antecubital vein injection of 2 x 90 ml of 12% iodinated contrast material at 2 ml/sec. Combined analysis of axial, multiplanar, and maximum-intensity-projection reformatted images was used for all patients. Image quality, venous stenosis or obstruction, intraluminal thrombus, and collateral pathways were evaluated. Comparison with digital phlebographic data was available for 16 patients; this comparison was performed in a nonblinded manner. RESULTS: CT phlebograms were considered technically optimal in 91% of the patients. In all these patients, helical CT phlebograms showed the venous obstruction: the site, extent, cause, and collateral pathways. CT phlebography appeared to be well correlated with digital phlebography in 16 patients regarding the degree of obstruction, the presence of collateral pathways, and the presence of thrombus. CONCLUSION: Helical CT phlebography may be a useful technique for imaging the SVC and its tributaries. This imaging technique is simple to perform and can provide all the information necessary to diagnose and treat SVC obstruction.

[Positional occlusion of the descending scapular artery in transverse thoraco-brachial syndrome with neurologic symptoms]. / Occlusion positionnelle de l'artère scapulaire descendante dans un syndrome de la traversée thoraco-brachiale à traduction neurologique.

We report a thoracic outlet syndrome revealed by neurological complications. Angiography of the subclavian artery depicted an isolated positional occlusion of the descending scapular artery. This side branch of the subclavian artery is anatomically located close to and supplies the brachial plexus. Surgical treatment led to improvement of most symptoms and post-operative control angiography was normal. Not previously described, this sign illustrates the objective compression of the brachial plexus. Ischemia is perhaps intricated with compression, a well-known pathophysiological mechanism of neurological complications in this syndrome. This artery feeding the brachial plexus is usually ligated during surgical neurolysis but must be preserved in order to improve recovery of neurological function and prevent surgical failures.

[Bronchial and pulmonary vaso-occlusions]. / Vaso-occlusions bronchiques et pulmonaires.

The technique of vaso-occlusion (or embolisation) consists in occluding one or several vessels which are causing haemoptysis. Either of the pulmonary circulations (systemic bronchial or pulmonary) may be the cause and sometimes both are. In systemic pulmonary hypervascularization the pathological issues are affected by high pressure vascularisation at the expense of functional vascularisation and are the source of frequent episodes of bleeding. Bronchial and systemic vaso-occlusion has proven efficacy and safety on condition that strict rules are respected to avoid complications; the detection of potentially dangerous arteries (spinal, coronary and visceral) and a choice of material for embolisation which is adapted to the therapeutic strategy and the clinical and angiographic presentation. The indications for the procedure are dictated by severe or recurrent haemoptysis. Vaso-occlusion of pulmonary arteries which is a rarer indication dominated by conditions such as arteriovenous malformation (MAVP). The technique is adapted to the number, to the size and to the diffusion of MAVP which is characterised precisely by computed tomography. Vaso-occlusion is the treatment of first choice for MAVP only leaving failures of vaso-occlusion to surgery and certain pedicular forms have a very short afferent of large calibre.