Anti-Metastatic Properties of a Marine Bacterial Exopolysaccharide-Based Derivative Designed to Mimic Glycosaminoglycans

Osteosarcoma is the most frequent malignant primary bone tumor characterized by a high potency to form lung metastases. In this study, the effect of three oversulfated low molecular weight marine bacterial exopolysaccharides (OS-EPS) with different molecular weights (4, 8 and 15 kDa) were first evaluated in vitro on human and murine osteosarcoma cell lines. Different biological activities were studied: cell proliferation, cell adhesion and migration, matrix metalloproteinase expression. This in vitro study showed that only the OS-EPS 15 kDa derivative could inhibit the invasiveness of osteosarcoma cells with an inhibition rate close to 90%. Moreover, this derivative was potent to inhibit both migration and invasiveness of osteosarcoma cell lines; had no significant effect on their cell cycle; and increased slightly the expression of MMP-9, and more highly the expression of its physiological specific tissue inhibitor TIMP-1. Then, the in vivo experiments showed that the OS-EPS 15 kDa derivative had no effect on the primary osteosarcoma tumor induced by osteosarcoma cell lines but was very efficient to inhibit the establishment of lung metastases in vivo. These results can help to better understand the mechanisms of GAGs and GAG-like derivatives in the biology of the tumor cells and their interactions with the bone environment to develop new therapeutic strategies

Progress toward developing the TMT adaptive optical systems and their components

Atmospheric turbulence compensation via adaptive optics (AO) will be essential for achieving most objectives of the TMT science case. The performance requirements for the initial implementation of the observatory's facility AO system include diffraction-limited performance in the near IR with 50 per cent sky coverage at the galactic pole. This capability will be achieved via an order 60x60 multi-conjugate AO system (NFIRAOS) with two deformable mirrors optically conjugate to ranges of 0 and 12 km, six high-order wavefront sensors observing laser guide stars in the mesospheric sodium layer, and up to three low-order, IR, natural guide star wavefront sensors located within each client instrument. The associated laser guide star facility (LGSF) will consist of 3 50W class, solid state, sum frequency lasers, conventional beam transport optics, and a launch telescope located behind the TMT secondary mirror. In this paper, we report on the progress made in designing, modeling, and validating these systems and their components over the last two years. This includes work on the overall layout and detailed opto-mechanical designs of NFIRAOS and the LGSF; reliable wavefront sensing methods for use with elongated and time-varying sodium laser guide stars; developing and validating a robust tip/tilt control architecture and its components; computationally efficient algorithms for very high order wavefront control; detailed AO system modeling and performance optimization incorporating all of these effects; and a range of supporting lab/field tests and component prototyping activities at TMT partners. Further details may be found in the additional papers on each of the above topics

TMT DMs final design and advanced prototyping results at Cilas

In order to prepare for the construction phase of the two Deformable Mirrors (DMs), which will be used in the Thirty Meter Telescope (TMT) first light Adaptive Optics (AO) system, Cilas has advanced the design of these two large size piezo DMs and has manufactured and tested a scaled demonstration prototype. The work done allowed significant reduction of the risks related to the demanding specifications of the TMT DMs; the main issues were: (i) Large pupil (up to 370 mm) and high order (up to 74x74); (ii) Relatively low operational temperature (DMs working at -30°C); (iii) New piezo material. It is important to develop such a prototype to take into account these three specifications all together (dimension, low temperature and new piezo material). The new prototype is a 6x60 actuators and has the same characteristics as the future TMT DMs. In this paper, we give the conclusions of the work through the presentation of the following items: (i) Design and finite element analysis of the two DMs and prototype; (ii) Test results obtained with the prototype with validation of the finite element analysis and compliance with the TMT AO specifications; (iii) Special focus on thermal behavior, actuator reliability and shape at rest stability

Progress toward developing the TMT adaptive optical systems and their components

Atmospheric turbulence compensation via adaptive optics (AO) will be essential for achieving most objectives of the TMT science case. The performance requirements for the initial implementation of the observatory's facility AO system include diffraction-limited performance in the near IR with 50 per cent sky coverage at the galactic pole. This capability will be achieved via an order 60x60 multi-conjugate AO system (NFIRAOS) with two deformable mirrors optically conjugate to ranges of 0 and 12 km, six high-order wavefront sensors observing laser guide stars in the mesospheric sodium layer, and up to three low-order, IR, natural guide star wavefront sensors located within each client instrument. The associated laser guide star facility (LGSF) will consist of 3 50W class, solid state, sum frequency lasers, conventional beam transport optics, and a launch telescope located behind the TMT secondary mirror. In this paper, we report on the progress made in designing, modeling, and validating these systems and their components over the last two years. This includes work on the overall layout and detailed opto-mechanical designs of NFIRAOS and the LGSF; reliable wavefront sensing methods for use with elongated and time-varying sodium laser guide stars; developing and validating a robust tip/tilt control architecture and its components; computationally efficient algorithms for very high order wavefront control; detailed AO system modeling and performance optimization incorporating all of these effects; and a range of supporting lab/field tests and component prototyping activities at TMT partners. Further details may be found in the additional papers on each of the above topics

Sterilization of Exopolysaccharides Produced by Deep-Sea Bacteria: Impact on Their Stability and Degradation

Polysaccharides are highly heat-sensitive macromolecules, so high temperature treatments are greatly destructive and cause considerable damage, such as a great decrease in both viscosity and molecular weight of the polymer. The technical feasibility of the production of exopolysaccharides by deep-sea bacteria Vibrio diabolicus and Alteromonas infernus was previously demonstrated using a bioproduct manufacturing process. The objective of this study was to determine which sterilization method, other than heat sterilization, was the most appropriate for these marine exopolysaccharides and was in accordance with bioprocess engineering requirements. Chemical sterilization using low-temperature ethylene oxide and a mixture of ionized gases (plasmas) was compared to the sterilization methods using gamma and beta radiations. The changes to both the physical and chemical properties of the sterilized exopolysaccharides were analyzed. The use of ethylene oxide can be recommended for the sterilization of polysaccharides as a weak effect on both rheological and structural properties was observed. This low-temperature gas sterilizing process is very efficient, giving a good Sterility Assurance Level (SAL), and is also well suited to large-scale compound manufacturing in the pharmaceutical industry

Antiviral Activities of Sulfated Polysaccharides Isolated from Sphaerococcus coronopifolius (Rhodophytha, Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales)

Water-soluble sulfated polysaccharides isolated from two red algae Sphaerococcus coronopifolius (Gigartinales, Sphaerococcaceae) and Boergeseniella thuyoides (Ceramiales, Rhodomelaceae) collected on the coast of Morocco inhibited in vitro replication of the Human Immunodeficiency Virus (HIV) at 12.5 μg/mL. In addition, polysaccharides were capable of inhibiting the in vitro replication of Herpes simplex virus type 1 (HSV-1) on Vero cells values of EC50 of 4.1 and 17.2 μg/mL, respectively. The adsorption step of HSV-1 to the host cell seems to be the specific target for polysaccharide action. While for HIV-1, these results suggest a direct inhibitory effect on HIV-1 replication by controlling the appearance of the new generations of virus and potential virucidal effect. The polysaccharides from S. coronopifolius (PSC) and B. thuyoides (PBT) were composed of galactose, 3,6-anhydrogalactose, uronics acids, sulfate in ratios of 33.1, 11.0, 7.7 and 24.0% (w/w) and 25.4, 16.0, 3.2, 7.6% (w/w), respectively

RB818 and RB819 antibodies recognize a low-molecular weight highly sulfated marine exopolysaccharide derivative by ELISA

The recombinant antibodies RB818 and RB819 detect by ELISA a low-molecular weight highly sulfated marine exopolysaccharide derivative

Flow Field-Flow fractionation for an accurate characterization of polysaccharides

As size exclusion chromatography (SEC), Flow Field Flow Fractionation is a separation technique that assesses the size distribution of the molecules via theoretical equations or representative standards. In FFF technique, high-resolution separation is achieved within a very thin flow against which a perpendicular force field is applied. It takes place in an asymmetrical flat channel (A4F) or in a hollow fiber (HF5). Separation depends on the Brownian diffusion coefficient of the molecule / particle, and thus on its size. Multi Angle Light Scattering detector (MALS) is used for the determination of the absolute molar mass and size determination of macromolecules / nanoparticles. In FFF separation, no stationary phase is required that avoids interferences with the samples. Consequently, for very high molar weight polymers, shearing forces do not apply. The entire separation is smooth, fast, and non-destructive without a stationary phase that may interact, degrade, or alter the sample.   Field-Flow Fractionation-based techniques are powerful tools for characterizing polysaccharides and for monitoring the potential polydispersity and/or polymodality. They are powerful tools especially in the case of drug delivery. In a Pharmacopeia approach, these techniques must be coupled to complementary techniques for a full characterization of products. Viscosity measurements have been conducted together with A4F-UV-MALS and to study the size and size distribution of exopolysaccharides.

Assembly of HE800 exopolysaccharide produced by a deep-sea hydrothermal bacterium into microgels for protein delivery applications

Assembly of biopolymers into microgels is an elegant strategy for bioencapsulation with various potential biomedical applications. Such biocompatible and biodegradable microassemblies are developed not only to protect the encapsulated molecule but also to ensure its sustained local delivery. The present study describes the fabrication of microassemblies from a marine HE800 exopolysaccharide (EPS), which displays a glycosaminoglycan (GAG)-like structure and biological properties. HE800 EPS was assembled, through physical cross-linking with divalent ions, into microgel particles and microfibers using microfluidics. The microparticle morphology was highly affected by the polysaccharide concentration and its molecular weight. A model protein, namely Bovine Serum Albumin (BSA) was subsequently encapsulated within HE800 microparticles in one-step process using microfluidics. The protein release was tuned by the microparticle morphology with a lower protein amount released from the most homogeneous structures. Our findings demonstrate the high potential of HE800 EPS based microassemblies as innovative protein microcarriers for further biomedical applications

Mechanical relaxations of hydrogels governed by their physical or chemical crosslinks

In the field of tissue engineering, in order to restore tissue functionality hydrogels that closely mimic biological and mechanical properties of the extracellular matrix are intensely developed. Mechanical properties including relaxation of the surrounding microenvironment regulate essential cellular processes. However, the mechanical properties of engineered hydrogels are particularly complex since they involve not only a nonlinear elastic behavior but also time-dependent responses. An accurate determination of these properties at microscale, i.e. as probed by cells, becomes an essential step to further design hydrogel-based biomaterials able to induce specific cellular responses. Atomic Force Microscopy (AFM) with contact sizes of the order of few micrometers constitutes an appropriate technique to determine the origin of relaxation mechanisms occurring in hydrogels. In the present study, AFM force relaxation experiments are conducted on chemically and physically crosslinked hydrogels respectively based on a synthetic polymer, polyacrylamide and a natural polymer, a bacterial exopolysaccharide infernan, produced by the deep-sea hydrothermal vent bacterium, Alteromonas infernus. Two distinct relaxation mechanisms are clearly evidenced depending on the nature of hydrogel network crosslinks. Chemically crosslinked hydrogel exhibits poroelastic relaxations, whereas physically crosslinked hydrogel shows time-dependent responses arising from viscoelastic effects. In addition, two relaxation processes are revealed in ionic physical hydrogel originating from chain rearrangement and breaking/reforming of the ionic crosslinks. The effect of the ionic strength on both the long-term elastic modulus and relaxation times of physical hydrogels was also shown. These findings highlight that physical hydrogels with well-defined time-dependent mechanical properties could be tuned for an optimized response of cells