Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Heat Dissipation of Hybrid Iron Oxide-Gold Nanoparticles in an Agar Phantom

Hybrid iron oxide-gold nanoparticles (HNPs) have shown potential in cancer therapy as agents for tumour ablation
and thermal switches for targeted drug release. Heat generation occurs by exploitation of the surface plasmon
resonance of the gold coating, which usually occurs at the maximum UV absorption wavelength. However, lasers
at such wavelength are often expensive and highly specialised. Here, we report the heating and monitoring of heat
dissipation of HNPs suspended in agar phantoms using a relatively inexpensive Ng: YAG pulsed 1064 nm laser source.
The particles experience heating of up to 40°C with a total area of heat dissipation up to 132.73 mm2 from the 1 mm
diameter irradiation point after 60 seconds. This work reports the potential and possible drawbacks of these particles
for translation into cancer therapy based on our findings.

Immunoassays, optical and electrochemical immunosensorsfor tetrodotoxin determination in puffer fish samples

Tetrodotoxin (TTX) is a low molecular weight and potent marine neurotoxin which is usually present in some species of puffer fish. TTX selectively binds to voltage-sensitive sodium channels (VSGCs), blocking the influx of sodium into the cell and affecting neural transmission. The bioaccumulation of this toxin in seafood can poses a risk to human safety. With the purpose of achieving cheap, specific and reliable tools to determine TTX in puffer fish samples, a self-assembled dithiol-based immunoassay, an electrochemical immunosensor and an optical Surface Plasmon Resonance (SPR) immunosensor are proposed. The immunoassay for TTX based on the use of dithiols self-assembled on maleimide-plates (mELISA) has been able to detect as low as 2.28 μg/L of TTX. The effect of different puffer fish matrixes on this mELISA has been quantified and the corresponding correction factors have been established. This
mELISA has enabled to establish the cross-reactivity factors for four TTX analogues: 5,6,11-trideoxy-TTX, 5,6,11-trideoxy-4-anhydro-TTX, 11-nor-TTX-6-ol and 5,11-deoxy-TTX. The crossreactivity factors have also been established by the optical SPR immunosensor previously reported, which had a limit of detection (LOD) of 4.27 μg/L. The mELISA and the SPR immunosensor have then been tested with spiked-puffer fish matrixes, providing an effective
LOD of 0.23 and 0.43 mg/kg respectively, well below the limit set in Japan (2 mg/kg). The mELISA and the SPR immunosensor have also been applied to the analysis of naturally contaminated puffer fish samples, providing similar TTXs contents between techniques and also compared to LC-MS/MS. The suitability of these immunochemical techniques has been demonstrated not only for screening purposes, but also for research activities. Currently, given that dithiols could improve the electron transfer and the sensitivity of an electrochemical assay, the mELISA strategy is being transferred to gold electrodes for the electrochemical detection of TTX and the subsequent development of the multiplexed electrochemical immunosensor.

Conception, fabrication et caractérisation d'un biocapteur SPR à base de guides d'ondes photoniques sur substrat de verre

Résumé : Malgré le nombre croissant de capteurs dans les domaines de la chimie et la biologie, il reste encore à étudier en profondeur la complexité des interactions entre les différentes molécules présentes lors d’une détection à l’interface solide-liquide. Dans ce cadre, il est de tout intérêt de croiser différentes méthodes de détection afin d’obtenir des informations complémentaires. Le principal objectif de cette étude est de dimensionner, fabriquer et caractériser un détecteur optique intégré sur verre basé sur la résonance plasmonique de surface, destiné à terme à être combiné avec d’autres techniques de détection, dont un microcalorimètre. La résonance plasmonique de surface est une technique reconnue pour sa sensibilité adaptée à la détection de surface, qui a l’avantage d’être sans marquage et permet de fournir un suivi en temps réel de la cinétique d’une réaction. L’avantage principal de ce capteur est qu’il a été dimensionné pour une large gamme d’indice de réfraction de l’analyte, allant de 1,33 à 1,48. Ces valeurs correspondent à la plupart des entités biologiques associées à leurs couches d’accroche dont les matrices de polymères, présentés dans ce travail. Étant donné que beaucoup d’études biologiques nécessitent la comparaison de la mesure à une référence ou à une autre mesure, le second objectif du projet est d’étudier le potentiel du système SPR intégré sur verre pour la détection multi-analyte. Les trois premiers chapitres se concentrent sur l’objectif principal du projet. Le dimensionnement du dispositif est ainsi présenté, basé sur deux modélisations différentes, associées à plusieurs outils de calcul analytique et numérique. La première modélisation, basée sur l’approximation des interactions faibles, permet d’obtenir la plupart des informations nécessaires au dimensionnement du dispositif. La seconde modélisation, sans approximation, permet de valider le premier modèle approché et de compléter et affiner le dimensionnement. Le procédé de fabrication de la puce optique sur verre est ensuite décrit, ainsi que les instruments et protocoles de caractérisation. Un dispositif est obtenu présentant des sensibilités volumiques entre 1000 nm/RIU et 6000 nm/RIU suivant l’indice de réfraction de l’analyte. L’intégration 3D du guide grâce à son enterrage sélectif dans le verre confère au dispositif une grande compacité, le rendant adapté à la cointégration avec un microcalorimètre en particulier. Le dernier chapitre de la thèse présente l’étude de plusieurs techniques de multiplexage spectral adaptées à un système SPR intégré, exploitant en particulier la technologie sur verre. L’objectif est de fournir au moins deux détections simultanées. Dans ce cadre, plusieurs solutions sont proposées et les dispositifs associés sont dimensionnés, fabriqués et testés.

Storage stability of bevacizumab in polycarbonate and polypropylene syringes

Purpose To compare and examine the storage stability of compounded bevacizumab in polycarbonate (PC) and polypropylene (PP) syringes over a 6-month period. PC syringes have been used in a recent clinical study and bevacizumab stability has not been reported for this type of syringe. Methods Repackaged bevacizumab was obtained from Moorfields Pharmaceuticals in polycarbonate (PC) and polypropylene (PP) syringes. Bevacizumab from the stored syringes was analysed at monthly time points for a 6-month period and compared with bevacizumab from a freshly opened vial at each time point. SDS-PAGE electrophoresis and size-exclusion chromatography (SEC) was used to observe aggregation and degradation. Dynamic light scattering (DLS) provided information about the hydrodynamic size and particle size distribution of bevacizumab in solution. VEGF binding and the active concentration of bevacizumab was determined by surface plasmon resonance (SPR) using Biacore. Results SDS-PAGE and SEC analysis did not show any changes in the presence of higher molecular species (HMWS) or degradation products in PC and PP syringes from T0 to T6 compared to bevacizumab sampled from a freshly opened vial. The hydrodynamic diameter of bevacizumab in the PC syringe after six months of storage was not significantly different to bevacizumab taken from a freshly opened vial. Using SPR, the VEGF binding activity of bevacizumab in the PC syringe was comparable with bevacizumab taken from a freshly opened vial. Conclusion No significant difference over a 6-month period was observed in the quality of bevacizumab repackaged into prefilled PC polycarbonate and PP polypropylene syringes when compared to bevacizumab that is supplied from the vial.

An anti-TNF--α antibody mimetic to treat ocular inflammation

Infliximab is an antibody that neutralizes TNF-α and is used principally by systemic administration to treat many inflammatory disorders. We prepared the antibody mimetic Fab-PEG-Fab (FpFinfliximab) for direct intravitreal injection to assess whether such formulations have biological activity and potential utility for ocular use. FpFinfliximab was designed to address side effects caused by antibody degradation and the presence of the Fc region. Surface plasmon resonance analysis indicated that infliximab and FpFinfliximab maintained binding affinity for both human and murine recombinant TNF-α. No Fc mediated RPE cellular uptake was observed for FpFinfliximab. Both Infliximab and FpFinfliximab suppressed ocular inflammation by reducing the number of CD45+ infiltrate cells in the EAU mice model after a single intravitreal injection at the onset of peak disease. These results offer an opportunity to develop and formulate for ocular use, FpF molecules designed for single and potentially multiple targets using bi-specific FpFs.

Design of passive methane oxidation biosystems considering their response to the presence of capillary barrier effect

La construction des biosystèmes d’oxydation passive du méthane (BOPM) est une option économique et durable pour réduire les émissions de méthane des sites d’enfouissement de déchets et des effets subséquents du réchauffement climatique. Les BOPM sont constitués de deux couches principales: la couche d'oxydation du méthane (MOL) et la couche de distribution du gaz (GDL). L'oxydation du méthane se produit dans la MOL par les réactions biochimiques des bactéries méthanotrophes, et la GDL est construite sous la MOL pour intercepter et distribuer les émissions fugitives de biogaz à la base de la MOL. Fondamentalement, l'efficacité d'un BOPM est définie en fonction de l'efficacité d'oxydation du méthane dans la MOL. Par conséquent, il est indispensable de fournir des conditions adéquates pour les activités bactériennes des méthanotrophes. En plus des paramètres environnementaux, l'intensité et la distribution du biogaz influencent l'efficacité des BOPM, et ils peuvent rendre le matériau de la MOL - avec une grande capacité d'accueillir les activités bactériennes - inutilisables en termes d'oxydation du méthane sur place. L'effet de barrière capillaire le long de l'interface entre la GDL et la MOL peut provoquer des émissions localisées de méthane, due à la restriction ou la distribution non uniforme de l’écoulement ascendant du biogaz à la base de la MOL. L'objectif principal de cette étude est d'incorporer le comportement hydraulique non saturé des BOPM dans la conception des BOPM, afin d’assurer la facilité et la distribution adéquates de l'écoulement du biogaz à la base de la MOL. Les fonctions de perméabilité à l'air des matériaux utilisés pour construire la MOL des BOPM expérimentaux au site d’enfouissement des déchets de St Nicéphore (Québec, Canada), ainsi que celles d'autres de la littérature technique, ont été étudiés pour évaluer le comportement d'écoulement non saturé du gaz dans les matériaux et pour identifier le seuil de migration sans restriction du gaz. Ce dernier seuil a été introduit en tant que un paramètre de conception avec lequel le critère de conception recommandé ici, c’est à dire la longueur de la migration sans restriction de gaz (LMSG), a été défini. La LMSG est considérée comme la longueur le long de l'interface entre la GDL et la MOL où le biogaz peut migrer à travers la MOL sans restriction. En réalisant des simulations numériques avec SEEP/W, les effets de la pente de l'interface, des paramètres définissant la courbe de rétention d'eau, de la fonction de la conductivité hydraulique du matériau de la MOL sur la valeur de la LMSG (représentant la facilité d'écoulement du biogaz à l'interface) et de la distribution de l'humidité (et par conséquent celle du biogaz) ont été évalués. Selon les résultats des simulations, la conductivité hydraulique saturée et la distribution des tailles de pores du matériau de la MOL sont les paramètres les plus importants sur la distribution de l'humidité le long de l'interface. Ce dernier paramètre influe également sur la valeur du degré de saturation et donc la facilité du biogaz à la base de la MOL. La densité sèche du matériau de MOL est un autre paramètre qui contrôle la facilité d'écoulement ascendant du biogaz. Les limitations principales de la présente étude sont associées au nombre de matériaux de MOL testés et à l'incapacité de SEEP/W de considérer l'évapotranspiration. Toutefois, compte tenu des hypothèses raisonnables dans les simulations et en utilisant les données de la littérature, on a essayé de réduire ces limitations. En utilisant les résultats des expériences et des simulations numériques, des étapes et des considérations de conception pour la sélection du matériau de MOL et de la pente d'interface ont été proposées. En effet,le comportement hydraulique non saturé des matériaux serait intégré dans les nécessités de conception pour un BOPM efficace, de sorte que la capacité maximale possible d'oxydation du méthane du matériau de la MOL soit exploitée.

Structural Basis for Clonal Diversity of the Public T Cell Response to Dominant Epitopes from Cytomegalovirus and Influenza

A diverse T cell receptor (TCR) repertoire is a prerequisite for effective viral clearance. However, knowledge of human TCR repertoire to defined viral antigens is limited. Recent advances in high-throughput sequencing (HTS) and single-cell sorting have revolutionized the study of human TCR repertoires to different types of viruses. In collaboration with the laboratory of Dr. Nan-ping Weng (National Institute on Aging, NIH), we applied unique molecular identifier (UMI)-labelled HTS, single-cell paired TCR analysis, surface plasmon resonance, and X-ray crystallography to exhaustively interrogate CD8+ TCR repertoires specific for cytomegalovirus (CMV) and influenza A (Flu) in HLA-A2+ humans. Our two CMV-specific TCR-pMHC structures and two Flu-specific TCR-pMHC structures provide a plausible explanation for the much higher diversity of CMV-specific than Flu-specific TCR repertoires in humans. Our comprehensive biochemical and structural portrait of two different anti-viral T cell responses may contribute to the future development of predictors of immunity or disease at the individual level.

Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

International audience

Mesoscale Optoelectronic Design of Wire-Based Photovoltaic and Photoelectrochemical Devices

The overarching theme of this thesis is mesoscale optical and optoelectronic design of photovoltaic and photoelectrochemical devices. In a photovoltaic device, light absorption and charge carrier transport are coupled together on the mesoscale, and in a photoelectrochemical device, light absorption, charge carrier transport, catalysis, and solution species transport are all coupled together on the mesoscale. The work discussed herein demonstrates that simulation-based mesoscale optical and optoelectronic modeling can lead to detailed understanding of the operation and performance of these complex mesostructured devices, serve as a powerful tool for device optimization, and efficiently guide device design and experimental fabrication efforts. In-depth studies of two mesoscale wire-based device designs illustrate these principles—(i) an optoelectronic study of a tandem Si|WO3 microwire photoelectrochemical device, and (ii) an optical study of III-V nanowire arrays.

The study of the monolithic, tandem, Si|WO3 microwire photoelectrochemical device begins with development and validation of an optoelectronic model with experiment. This study capitalizes on synergy between experiment and simulation to demonstrate the model’s predictive power for extractable device voltage and light-limited current density. The developed model is then used to understand the limiting factors of the device and optimize its optoelectronic performance. The results of this work reveal that high fidelity modeling can facilitate unequivocal identification of limiting phenomena, such as parasitic absorption via excitation of a surface plasmon-polariton mode, and quick design optimization, achieving over a 300% enhancement in optoelectronic performance over a nominal design for this device architecture, which would be time-consuming and challenging to do via experiment.

The work on III-V nanowire arrays also starts as a collaboration of experiment and simulation aimed at gaining understanding of unprecedented, experimentally observed absorption enhancements in sparse arrays of vertically-oriented GaAs nanowires. To explain this resonant absorption in periodic arrays of high index semiconductor nanowires, a unified framework that combines a leaky waveguide theory perspective and that of photonic crystals supporting Bloch modes is developed in the context of silicon, using both analytic theory and electromagnetic simulations. This detailed theoretical understanding is then applied to a simulation-based optimization of light absorption in sparse arrays of GaAs nanowires. Near-unity absorption in sparse, 5% fill fraction arrays is demonstrated via tapering of nanowires and multiple wire radii in a single array. Finally, experimental efforts are presented towards fabrication of the optimized array geometries. A hybrid self-catalyzed and selective area MOCVD growth method is used to establish morphology control of GaP nanowire arrays. Similarly, morphology and pattern control of nanowires is demonstrated with ICP-RIE of InP. Optical characterization of the InP nanowire arrays gives proof of principle that tapering and multiple wire radii can lead to near-unity absorption in sparse arrays of InP nanowires.

Comment on the plasmon model for image-potential-induced surface states

Calculations of the binding energy of bound positron states in metal surfaces, with explicit inclusion of plasmon dispersion and single-particle effects, are presented. The binding energy is greatly reduced with respect to the undispersed case.

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Na+/Cl−-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [35S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na+/Cl−-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

In-field Mossbauer study of disordered surface spins in core/shell ferrite nanoparticles

Magnetization and Mossbauer spectroscopy measurements are performed at low temperature under high field, on nanoparticles with a nickel ferrite core and a maghemite shell. These nanoparticles present finite size and surface effects, together with exchange anisotropy. High field magnetization brings the evidences of a monodomain ordered core and surface spins freezing in disorder at low temperature. Mossbauer spectra at 4.2 K present an extra contribution from the disordered surface which is field dependent. Field and size dependences of this latter show a progressive spin alignment along the ferrite core which is size dependent. The weak surface pinning condition of the nanoparticles confirms that the spin disorder is localized in the external shell. The underfield decrease in the mean canting angle in the superficial shell is then directly related to the unidirectional exchange anisotropy through the interface between the ordered core and the disordered shell. The obtained anisotropy field H(Ea) scales as the inverse of the nanoparticle diameter, validating its interfacial origin. The associated anisotropy constant K(Ea) equals 2.5 x 10(-4) J/m(2). (C) 2009 American Institute qf Physics. [doi: 10.1063/1.3245326]

Pharmacokinetics and pharmacodynamics of melphalan in isolated limb infusion for recurrent localized limb malignancy

Isolated limb infusion (ILI) is an attractive, less complex alternative to Isolated limb perfusion (ILP). It has a lower morbidity in treating localized recurrences and in transit metastases of the limb for tumours such as melanoma, Merkel cell tumour and Kaposi's sarcoma, allowing administration of high concentrations of cytotoxic agent to the affected limb under hypoxic conditions. Melphalan is the preferred cytotoxic agent for the treatment of melanoma by ILP or ILI. We report pharmacokinetic data from 12 patients treated by ILI for tumours of the limb in Brisbane. The kinetics of drug distribution in the limb was calculated using a two-compartment vascular model, where both tissue and infusate act as well-stirred compartments. Analysis of melphalan concentrations in the perfusate during ILI showed good agreement between the values measured and the concentrations predicted by the model. Recirculation and wash-out flow rates, tissue concentrations and the permeability surface area product (PS) were calculated. Correlations between the PS value and the drug concentrations In the perfusate and tissue were supported by the results. These data contribute to a better understanding of the distribution of melphalan during ILI in the limb, and offer the opportunity to optimize the drug regimen for patients undergoing ILI. (C) 2001 Lippincott Williams & Wilkins.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.