The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

Quantification of nanoparticle endocytosis based on double fluorescent pH-sensitive nanoparticles.

Amorphous silica is a particularly interesting material because of its inertness and chemical stability. Silica nanoparticles have been recently developed for biomedical purposes but their innocuousness must be carefully investigated before clinical use. The relationship between nanoparticles physicochemical features, their uptake by cells and their biological activity represents a crucial issue, especially for the development of nanomedicine. This work aimed at adapting a method for the quantification of nanoparticle endocytosis based on pH-sensitive and double fluorescent particles. For that purpose, silica nanoparticles containing two fluorophores: FITC and pHrodo(TM) were developed, their respective fluorescence emission depends on the external pH. Indeed, FITC emits a green fluorescence at physiological pH and pHrodo(TM) emits a red fluorescence which intensity increased with acidification. Therefore, nanoparticles remained outside the cells could be clearly distinguished from nanoparticles uptaken by cells as these latter could be spotted inside cellular acidic compartments (such as phagolysosomes, micropinosomes…). Using this model, the endocytosis of 60 nm nanoparticles incubated with the RAW 264.7 macrophages was quantified using time-lapse microscopy and compared to that of 130 nm submicronic particles. The amount of internalized particles was also evaluated by fluorimetry. The biological impact of the particles was also investigated in terms of cytotoxicity, pro-inflammatory response and oxidative stress. Results clearly demonstrated that nanoparticles were more uptaken and more reactive than submicronic particles. Moreover, we validated a method of endocytosis quantification.

Assembly of Poly(vinylphosphonic acid)-Based Double Hydrophilic Block Copolymers by Gadolinium Ions for the Formation of Highly Stable MRI Contrast Agents.

Mixing double-hydrophilic block copolymers containing a poly(vinylphosphonic acid) block with gadolinium ions in water leads to the spontaneous formation of polymeric nanoparticles. With an average diameter near 20 nm, the nanoparticles are stable after dilution or change of pH and ionic strength. High magnetic relaxivities were measured in vitro, and in vivo magnetic resonance imaging on rats demonstrates the high potential of such polymeric assemblies.

Biodistribution study of nanometric hybrid gadolinium oxide particles as a multimodal SPECT/MR/optical imaging and theragnostic agent.

Nanometric hybrid gadolinium oxide particles (Gado-6Si-NP) for diagnostic and therapeutic applications (mean diameter 3-4 nm) were obtained by encapsulating Gd(2)O(3) cores within a polysiloxane shell, which carries organic fluorophore (Cy 5) and is derivatized by a hydrophilic carboxylic layer. As residency time in the living body and methods of waste elimination are crucial to defining a good nanoparticle candidate and moving forward with steps for validation, this study was aimed at evaluating the biodistribution of these multimodal Gado-6Si-NP in rodents. Gado-6Si-NP were imaged following intravenous injection in control Wistar rats and mice using MRI (7 T), optical fluorescent imaging, and SPECT. A clear correlation was observed among MRI, optical imaging, and SPECT regarding the renal elimination. Quantitative biodistribution using gamma-counting of each sampled organ confirmed that these nanoparticles circulated freely in the blood pool and were rapidly cleared by renal excretion without accumulation in liver and RES uptake. These results demonstrate that Gado-6Si-NP display optimal biodistribution properties, enabling them to be developed as multimodal agents for in vivo imaging and theragnostics, especially in oncological applications.

Chromogenic substrates for feruloyl esterases.

Chromogenic mono- and diferuloyl-butanetriol analogs were prepared by chemical syntheses and their efficiency was evaluated as substrates for feruloyl esterases from Aspergillus niger.

Synthesis of triazole-linked pseudo-starch fragments.

Rapid assembly of starch fragment analogues was achieved using 'click chemistry'. Specifically, a pentadecasaccharide and two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to glucose or maltose core were synthesised using Cu(I)-catalyzed [3+2] dipolar cycloaddition of azidosaccharides and 4,6-di-O-propargylated methyl alpha-d-glucopyranoside and 6,6'- and 4',6'-di-O-propargylated p-methoxyphenyl beta-maltoside.

Respective importance of protein folding and glycosylation in the thermal stability of recombinant feruloyl esterase A.

The thermal stability of four molecular forms (native, refolded, glycosylated, non-glycosylated) of feruloyl esterase A (FAEA) was studied. From the most to the least thermo-resistant, the four molecular species ranked as follows: (i) glycosylated form produced native, (ii) non-glycosylated form produced native, (iii) non-glycosylated form produced as inclusion bodies and refolded, and (iv) glycosylated form produced native chemically denatured and then refolded. On the basis of these results and of crystal structure data, we discuss the respective importance of protein folding and glycosylation in the thermal stability of recombinant FAEA.

Lipo-chitin oligosaccharides, plant symbiosis signalling molecules that modulate mammalian angiogenesis in vitro.

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.

Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer.

Nowadays, the easy access of tetra-N-acetyl-chitopentaose and its counterparts is highly interesting since such chemical compounds are precursors of biological signal molecules with a strong agro-economic impact. The chemical synthesis of tetra-N-acetyl-chitopentaose by controlled N-acetylation of the glucosamine pentamer hydrochloride under mild conditions is described herein. A systematic study on the influence of the different parameters involved in this reaction, such as the solvent, the acetylating agent, and the base used for the deprotonation of ammonium groups of the starting material was carried out. The characterization of final reaction products by HPLC and MALDI-TOF mass spectrometry showed that each of these parameters affects differently the acetylation reaction. Whereas the solvent plays an important role in the N- or O-acetylation selectivity, the acetylating agent and the base were found to influence both the degree of N-acetylation and the distribution of the partially N-acetylated derivatives in the product mixtures. Based on these results, optimized reaction conditions have been established allowing tetra-N-acetyl-chitopentaose to be synthesized in a one-pot deprotonation/N-acetylation of the glucosamine pentamer hydrochloride in a moderate yield (ca 30%).

New chromogenic substrates for feruloyl esterases.

Indolyl and nitrophenyl 5-O-hydroxycinnamoyl-alpha-L-arabinofuranosides were prepared by chemo-enzymatic syntheses. These probes were designed as substrates to be used in assays of feruloyl esterase activity (EC 3.1.1.77). Color development in the assays only occurs when feruloyl esterase activity releases an intermediate chromogenic arabinoside that is a suitable substrate for alpha-L-arabinofuranosidase (EC 3.2.1.55), which in turn releases the free chromogenic group. The usefulness of these compounds was evaluated in both qualitative solid media-based assays and quantitative liquid assays that can be performed in microtiter plates using feruloyl esterases and arabinofuranosidases from various origins.

"Click chemistry"en route to pseudo-starch.

Rapid assembly of starch fragment analogues was achieved using "click chemistry". Specifically, two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to a maltose core were synthesized using Cu(i)-catalyzed [3 + 2] dipolar cycloaddition of azido saccharides and 6,6'- and 4',6'-dipropargylated p-methoxyphenyl maltoside.