Annexin-A5 and annexin-A6 silencing prevents metastasis of breast cancer cells in zebrafish.

BACKGROUND INFORMATION: During tumor invasion and metastasis processes, cancer cells are exposed to major compressive and shearing forces, due to their migration through extracellular matrix, dense cell areas, and complex fluids, which may lead to numerous plasma membrane damages. Cancer cells may survive to these mechanical stresses thanks to an efficient membrane repair machinery. Consequently, this machinery may constitute a relevant target to inhibit cancer cell dissemination. RESULTS: We show here that annexin-A5 (ANXA5) and ANXA6 participate in membrane repair of MDA-MB-231 cells, a highly invasive triple-negative breast cancer cell line. These crucial components of the membrane repair machinery are substantially expressed in breast cancer cells in correlation with their invasive properties. In addition, high expression of ANXA5 and ANXA6 predict poor prognosis in high-grade lung, gastric, and breast cancers. In zebrafish, the genetic inhibition of ANXA5 and ANXA6 leads to drastic reduction of tumor cell dissemination. CONCLUSION: We conclude that the inhibition of ANXA5 and ANXA6 prevents membrane repair in cancer cells, which are thus unable to survive to membrane damage during metastasis. SIGNIFICANCE: This result opens a new therapeutic strategy based on targeting membrane repair machinery to inhibit tumor invasion and metastasis.

Plasmon Waveguide Resonance: Principles, Applications and Historical Perspectives on Instrument Development.

Plasmon waveguide resonance (PWR) is a variant of surface plasmon resonance (SPR) that was invented about two decades ago at the University of Arizona. In addition to the characterization of the kinetics and affinity of molecular interactions, PWR possesses several advantages relative to SPR, namely, the ability to monitor both mass and structural changes. PWR allows anisotropy information to be obtained and is ideal for the investigation of molecular interactions occurring in anisotropic-oriented thin films. In this review, we will revisit main PWR applications, aiming at characterizing molecular interactions occurring (1) at lipid membranes deposited in the sensor and (2) in chemically modified sensors. Among the most widely used applications is the investigation of G-protein coupled receptor (GPCR) ligand activation and the study of the lipid environment's impact on this process. Pioneering PWR studies on GPCRs were carried out thanks to the strong and effective collaboration between two laboratories in the University of Arizona leaded by Dr. Gordon Tollin and Dr. Victor J. Hruby. This review provides an overview of the main applications of PWR and provides a historical perspective on the development of instruments since the first prototype and continuous technological improvements to ongoing and future developments, aiming at broadening the information obtained and expanding the application portfolio.

Improved Detection of Plasmon Waveguide Resonance Using Diverging Beam, Liquid Crystal Retarder, and Application to Lipid Orientation Determination.

Plasmon waveguide resonance (PWR) sensors exhibit narrow resonances at the two orthogonal polarizations, transverse electric (TE) and transverse magnetic (TM), which are narrower by almost an order of a magnitude than the standard surface plasmon resonance (SPR), and thus the figure of merit is enhanced. This fact is useful for measuring optical anisotropy of materials on the surface and determining the orientation of molecules with high resolution. Using the diverging beam approach and a liquid crystal retarder, we present experimental results by simultaneous detection of TE and TM polarized resonances as well as using fast higher contrast serial detection with a variable liquid crystal retarder. While simultaneous detection makes the system simpler, a serial one has the advantage of obtaining a larger contrast of the resonances and thus an improved signal-to-noise ratio. Although the sensitivity of the PWR resonances is smaller than the standard SPR, the angular width is much smaller, and thus the figure of merit is improved. When the measurement methodology has a high enough angular resolution, as is the one presented here, the PWR becomes advantageous over other SPR modes. The possibility of carrying out exact numerical simulations for anisotropic molecules using the 4 × 4 matrix approach brings another advantage of the PWR over SPR on the possibility of extracting the orientation of molecules adsorbed to the surface. High sensitivity of the TE and TM signals to the anisotropic molecules orientation is found here, and comparison to the experimental data allowed detection of the orientation of lipids on the sensor surface. The molecular orientations cannot be fully determined from the TM polarization alone as in standard SPR, which underlines the additional advantage of the PWR technique.

Thickness determination in anisotropic media with plasmon waveguide resonance imaging.

This paper describes a simple procedure to determine the local thickness of a thin anisotropic layer. It also discriminates between isotropic and anisotropic regions, provided a smoothness hypothesis on the refractive index distribution is satisfied. The procedure is based on the analysis of surface plasmon resonance (SPR) data acquired in an imaging mode. The general arrangement of the setup is the Kretschmann configuration. We show, on an azobenzene modified polymer layer, good agreement between atomic force microscopy and optical measurements of thickness variation.

Influence of Implants Composition on Melatonin Release from Ethylcellulose Matrix.

BACKGROUND: Melatonin release from Ethylcellulose matrix has never been studied on the whole range of compositions. OBJECTIVE: To perform a comprehensive study about the influence of the melatonin loading on its release from solid ethylcellulose implants, from both a kinetic and structural point of view. METHOD: Cylindrical implants differing in their Melatonin:Ethylcellulose ratio were fabricated to cover a large range of compositions. Drug release was assayed by in vitro dissolution tests in CTAB micellar solutions. The 2D imaging of implant chemical composition during Melatonin release was performed by confocal Raman spectroscopy. FT-IR spectroscopy and Karl-Fisher technique were employed to study implants hydration. RESULTS: A drug radial leakage, whatever the implant composition, is imaged. The apparent diffusion coefficient, D of melatonin was evaluated considering Fickian radial diffusion: its value ranges from 2 to 6 10-12 cm2/s depending on the EC content. The variation of the characteristic drug delivery time with composition was non-monotonous and two different regimes were identified. CONCLUSION: A micellar transport of Melatonin was found. The two regimes in drug release were interpreted considering the polymer barrier effect, the initial porosity and M domains connectivity.

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids.

G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the drugs on the market exert their action through these proteins. To decipher the molecular mechanisms of activation and signaling, GPCRs often need to be isolated and reconstituted from a detergent-solubilized state into a well-defined and controllable lipid model system. Several methods exist to reconstitute membrane proteins in lipid systems but usually the reconstitution success is tested at the end of the experiment and often by an additional and indirect method. Irrespective of the method used, the reconstitution process is often an intractable and time-consuming trial-and-error procedure. Herein, we present a method that allows directly monitoring the reconstitution of GPCRs in model planar lipid membranes. Plasmon waveguide resonance (PWR) allows following GPCR lipid reconstitution process without any labeling and with high sensitivity. Additionally, the method is ideal to probe the lipid effect on receptor ligand binding as demonstrated by antagonist binding to the chemokine CCR5 receptor.

Specific Anti-Leukemic Activity of the Peptide Warnericin RK and Analogues and Visualization of Their Effect on Cancer Cells by Chemical Raman Imaging.

Antimicrobial peptides can be used as therapeutic agents against cancer cells. Warnericin RK and derivatives (WarnG20D and WarnF14V) were tested on various, solid tumor or leukemia, cancer cells. These peptides appeared to be cytotoxic on all the cell types tested, cancerous as well healthy, but very interestingly displayed no deleterious effect on healthy mononuclear cells. The mode of action of the peptide was proposed to be membranolytic, using chemical Raman imaging. Addition of peptide induced a large disorganization of the membrane leading to the loss of the content of inner compartments of Jurkat cell, whereas no effect was observed on the healthy mononuclear cells. The less hemolytic peptides WarnG20D and WarnF14V could be good candidates for the leukemia treatment.

Plasmon waveguide resonance for sensing glycan-lectin interactions.

Carbohydrate-modified interfaces have been shown to be valuable tools for the study of protein-glycan recognition events. Label-free approache such as plasmonic based techniques are particularly attractive. This paper describes a new analytical platform for the sensitive and selective screening of carbohydrate-lectin interactions using plasmon waveguide resonance. Planar optical waveguides (POW), consisting of glass prisms coated with silver (50 nm) and silica (460 nm) layers were derivatized with mannose or lactose moieties. The specific association of the resulting interface with selected lectins was assessed by following the changes in its plasmonic response. The immobilization strategy investigated in this work is based on the formation of a covalent bond between propargyl-functionalized glycans and surface-linked azide groups via a Cu(I) "click" chemistry. Optimization of the surface architecture through the introduction of an oligo(ethylene glycol) spacer between the plasmonic surface and the glycan ligands provided an interface which allowed screening of glycan-lectin interactions in a highly selective manner. The limit of detection (LOD) of this method for this particular application was found to be in the subnanomolar range (0.5 nM), showing it to constitute a promising analytical platform for future development and use in a pharmaceutical or biomedical setting.

Interaction of Aß(1-42) amyloids with lipids promotes "off-pathway" oligomerization and membrane damage.

The toxicity of amyloids, as Aß(1-42) involved in Alzheimer disease, is a subject under intense scrutiny. Many studies link their toxicity to the existence of various intermediate structures prior to fiber formation and/or their specific interaction with membranes. In this study we focused on the interaction between membrane models and Aß(1-42) peptides and variants (L34T, mG37C) produced in E. coli and purified in monomeric form. We evaluated the interaction of a toxic stable oligomeric form (oG37C) with membranes as comparison. Using various biophysical techniques as fluorescence and plasmon waveguide resonance, we clearly established that the oG37C interacts strongly with membranes leading to its disruption. All the studied peptides destabilized liposomes and accumulated slowly on the membrane (rate constant 0.02 min(-1)). Only the oG37C exhibited a particular pattern of interaction, comprising two steps: the initial binding followed by membrane reorganization. Cryo-TEM was used to visualize the peptide effect on liposome morphologies. Both oG37C and mG37C lead to PG membrane fragmentation. The PG membrane promotes peptide oligomerization, implicated in membrane disruption. WT (Aß(1-42)) also perturbs liposome organization with membrane deformation rather than disruption. For all the peptides studied, their interaction with the membranes changes their fibrillization process, with less fibers and more small aggregates being formed. These studies allowed to establish, a correlation between toxicity, fiber formation, and membrane disruption.

Detection of an estrogen derivative in two breast cancer cell lines using a single core multimodal probe for imaging (SCoMPI) imaged by a panel of luminescent and vibrational techniques.

3-Methoxy-17α-ethynylestradiol or mestranol is a prodrug for ethynylestradiol and the estrogen component of some oral contraceptive formulations. We demonstrate here that a single core multimodal probe for imaging - SCoMPI - can be efficiently grafted onto mestranol allowing its tracking in two breast cancer cell lines, MDA-MB-231 and MCF-7 fixed cells. Correlative imaging studies based on luminescence (synchrotron UV spectromicroscopy, wide field and confocal fluorescence microscopies) and vibrational (AFMIR, synchrotron FTIR spectromicroscopy, synchrotron-based multiple beam FTIR imaging, confocal Raman microspectroscopy) spectroscopies were consistent with one another and showed a Golgi apparatus distribution of the SCoMPI-mestranol conjugate in both cell lines.

The role of asbestos morphology on their cellular toxicity: an in vitro 3D Raman/Rayleigh imaging study.

Amphiboles caused cohorts of deaths in exposed workers, leading to some of the largest class actions in the industry. Once inhaled, these inorganic fibers are thought to be both chemically and morphologically toxic, and their biopersistence in the lungs over decades lead to progressive pathologies, mesothelioma, and asbestosis. However, this exceptionally long chronicity for human pathologies suggests that chemical toxicity is certainly low, suggesting that morphological parameters could be more relevant in the pathology. Here, we developed a 3D Raman/optical imaging methodology in vitro to characterize both morphological and chemical parameters of cell/fiber interactions. We determined that lung cells could vesiculate amphiboles with length below 5 µm or could embed those not exceeding 15 µm in their fibrous extracellular matrix. Lung cells can thus develop defense strategies for handling the biopersistence of inorganic species, which may thus have major impact for biosafety issues related to nanomaterials.

Plasmonic properties of Fischer's patterns: polarization effects.

We report the fabrication and the optical study of Fisher's patterns inscribed on glass slides. Such structures, fabricated by electron beam lithography, consist of gold nanotriangles, organized in a hexagonal arrangement. By changing the fabrication conditions, it is possible to control precisely the size of the structures and the gap distance between facing triangles but most importantly, to finely tune their localized surface plasmon resonance. In addition to the experimental studies, the plasmonic properties of the Fischer's patterns were characterized as a function of the polarization of the incoming light. Finite difference time domain (FDTD) method was used to support the experimental results and to investigate the electromagnetic field enhancement on a Fischer's pattern lattice unit for different wavelengths and polarization of the irradiation source.

New concepts in molecular imaging: non-invasive MRI spotting of proteolysis using an Overhauser effect switch.

BACKGROUND: Proteolysis, involved in many processes in living organisms, is tightly regulated in space and time under physiological conditions. However deregulation can occur with local persistent proteolytic activities, e.g. in inflammation, cystic fibrosis, tumors, or pancreatitis. Furthermore, little is known about the role of many proteases, hence there is a need of new imaging methods to visualize specifically normal or disease-related proteolysis in intact bodies. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, a new concept for non invasive proteolysis imaging is proposed. Overhauser-enhanced Magnetic Resonance Imaging (OMRI) at 0.2 Tesla was used to monitor the enzymatic hydrolysis of a nitroxide-labeled protein. In vitro, image intensity switched from 1 to 25 upon proteolysis due to the associated decrease in the motional correlation time of the substrate. The OMRI experimental device used in this study is consistent with protease imaging in mice at 0.2 T without significant heating. Simulations show that this enzymatic-driven OMRI signal switch can be obtained at lower frequencies suitable for larger animals or humans. CONCLUSIONS/SIGNIFICANCE: The method is highly sensitive and makes possible proteolysis imaging in three dimensions with a good spatial resolution. Any protease could be targeted specifically through the use of taylor-made cleavable macromolecules. At short term OMRI of proteolysis may be applied to basic research as well as to evaluate therapeutic treatments in small animal models of experimental diseases.

Asymmetric spin crossover behaviour and evidence of light-induced excited spin state trapping in a dinuclear iron(II) helicate.

Reported herein are the synthesis, structural and magnetic characterisation of a dinuclear FeII triple helicate that displays an unprecedented reversible asymmetric high spin to low spin crossover characterised by a thermal hysteresis: indeed the high spin state can be recovered by white light irradiation at 10 K.

Magnetic and optical bistability driven by thermally and photoinduced intramolecular electron transfer in a molecular cobalt-iron prussian blue analogue.

A soluble molecular analogue of photoresponsive Co/Fe Prussian blues is described within this report. As judged via a variety of spectroscopic, magnetic, and crystallographic methods, electron transfer within the octanuclear complex (below 250 K) converts paramagnetic red crystals into green diamagnetic ones. The color and magnetic changes are associated with the transformation of FeIIILS-CN-CoIIHS units into FeIILS-CN-CoIIILS fragments in manner that is identical to that found for the An[Co(OH2)(6-6m)][Fe(CN)6]m.xH2O (An = alkali metal cation) family of three-dimensional Prussian blues. Moreover, this intramolecular electron transfer can be quantitatively circumvented via rapid thermal quenching and reversed via simple white light irradiation at low temperatures. Remarkably the data suggests that thermally or photoinduced paramagnetic metastable phases are identical and exhibit long relaxation times that approach 10 years at 120 K.

Balancing framework densification with charged, halogen-bonded-pi-conjugated linkages: [PPh4]2{[E-TTF-I2][Re6Se8(CN)6]} versus [PPh4]2[EDT-TTF-I]2{[EDT-TTF-I][Re6Se8(CN)6]}.

The ionic character of a set of two redox linkages and strong, directional halogen bonding at the organic-inorganic interface compromise to produce two materials sharing a common two-dimensional net, eventually extended in a third dimension, although two of the six symmetrical halogen bond acceptors ultimately remain uninvolved as a result of charge densification.