A methodological approach for the thermal stability and stress exposure studies of a model antibody.

The anti-horseradish peroxidase (HRP) antibody is conventionally used in immunohistochemistry. More recently, it has been used as the key element in a gold standard method to evaluate the functionality of antibody-based materials. However, few information are available about its melting temperature and its stability after exposition to laboratory stress conditions including freeze-drying and freeze-thawing cycles. The aim of this study was to evaluate the effects of these environmental constraints on the anti-HRP antibody in order to further use it as a reference in quality control and in the development of new antibody-based materials. In the developed method, the anti-HRP antibody is covalently immobilized onto a solid surface. After the direct recognition of its antigen HRP, the signal is proportional to the number of antibody active binding sites. The method was successfully utilized to accurately evaluate the anti-HRP antibody melting temperature (Tm was 73.5 ±â€¯0.2 °C). The method is a rapid and reliable tool with minimal cost for studying the anti-HRP antibody stability to solvent stress, freeze-thawing cycles, and freeze-drying process. The obtained information may be useful for routine analysis or in the development of antibody-based materials. This can be also proposed as an easy way to control antibody freeze-drying process.

ANGPTL4-αvß3 interaction counteracts hypoxia-induced vascular permeability by modulating Src signalling downstream of vascular endothelial growth factor receptor 2.

Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvß3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvß3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvß3 interaction enhances Src recruitment to integrin αvß3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvß3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvß3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvß3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extended interaction network of procollagen C-proteinase enhancer-1 in the extracellular matrix.

PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases such as BMP-1 (bone morphogenetic protein 1). PCPE-1 interacts with several proteins in addition to procollagens and BMP-1, suggesting that it could be involved in biological processes other than collagen maturation. We thus searched for additional partners of PCPE-1 in the extracellular matrix, which could provide new insights into its biological roles. We identified 17 new partners of PCPE-1 by SPR (surface plasmon resonance) imaging. PCPE-1 forms a transient complex with the ß-amyloid peptide, whereas it forms high or very high affinity complexes with laminin-111 (KD=58.8 pM), collagen VI (KD=9.5 nM), TSP-1 (thrombospondin-1) (KD1=19.9 pM, KD2=14.5 nM), collagen IV (KD=49.4 nM) and endostatin, a fragment of collagen XVIII (KD1=0.30 nM, KD2=1.1 nM). Endostatin binds to the NTR (netrin-like) domain of PCPE-1 and decreases the degree of superstimulation of PCPE-1 enhancing activity by heparin. The analysis of the PCPE-1 interaction network based on Gene Ontology terms suggests that, besides its role in collagen deposition, PCPE-1 might be involved in tumour growth, neurodegenerative diseases and angiogenesis. In vitro assays have indeed shown that the CUB1CUB2 (where CUB is complement protein subcomponents C1r/C1s, urchin embryonic growth factor and BMP-1) fragment of PCPE-1 inhibits angiogenesis.

Transglutaminase-2: a new endostatin partner in the extracellular matrix of endothelial cells.

Endostatin, a C-terminal fragment of collagen XVIII, binds to TG-2 (transglutaminase-2) in a cation-dependent manner. Recombinant human endostatin binds to TG-2 with an affinity in the nanomolar range (Kd=6.8 nM). Enzymatic assays indicated that, in contrast with other extracellular matrix proteins, endostatin is not a glutaminyl substrate of TG-2 and is not cross-linked to itself by the enzyme. Two arginine residues of endostatin, Arg27 and Arg139, are crucial for its binding to TG-2. They are also involved in the binding to heparin [Sasaki, Larsson, Kreuger, Salmivirta, Claesson-Welsh, Lindahl, Hohenester and Timpl (1999) EMBO J. 18, 6240-6248], and to alpha5beta1 and alphavbeta3 integrins [Faye, Moreau, Chautard, Jetne, Fukai, Ruggiero, Humphries, Olsen and Ricard-Blum (2009) J. Biol. Chem. 284, 22029-22040], suggesting that endostatin is not able to interact simultaneously with TG-2 and heparan sulfate, or with TG-2 and integrins. Inhibition experiments support the hypothesis that the GTP-binding site of TG-2 is a potential binding site for endostatin. Endostatin and TG-2 are co-localized in the extracellular matrix secreted by endothelial cells under hypoxia, which stimulates angiogenesis. This interaction, occurring in a cellular context, might participate in the concerted regulation of angiogenesis and tumorigenesis by the two proteins.

Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications.

Fibrillar collagens are the more abundant extracellular proteins. They form a metazoan-specific family, and are highly conserved from sponge to human. Their structural and physiological properties have been successfully used in the food, cosmetic, and pharmaceutical industries. On the other hand, the increase of jellyfish has led us to consider this marine animal as a natural product for food and medicine. Here, we have tested different Mediterranean jellyfish species in order to investigate the economic potential of their collagens. We have studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using Rhizostoma pulmo oral arms and the pepsin extraction method (2-10 mg collagen/g of wet tissue). Although a significant yield was obtained with Cotylorhiza tuberculata (0.45 mg/g), R. pulmo was used for further experiments, this jellyfish being considered as harmless to humans and being an abundant source of material. Then, we compared the biological properties of R. pulmo collagen with mammalian fibrillar collagens in cell cytotoxicity assays and cell adhesion. There was no statistical difference in cytotoxicity (p > 0.05) between R. pulmo collagen and rat type I collagen. However, since heparin inhibits cell adhesion to jellyfish-native collagen by 55%, the main difference is that heparan sulfate proteoglycans could be preferentially involved in fibroblast and osteoblast adhesion to jellyfish collagens. Our data confirm the broad harmlessness of jellyfish collagens, and their biological effect on human cells that are similar to that of mammalian type I collagen. Given the bioavailability of jellyfish collagen and its biological properties, this marine material is thus a good candidate for replacing bovine or human collagens in selected biomedical applications.

Versatile lysine dendrigrafts and polyethylene glycol hydrogels with inherent biological properties: in vitro cell behavior modulation and in vivo biocompatibility.

Poly(ethylene glycol) (PEG) hydrogels have been extensively used as scaffolds for tissue engineering applications, owing to their biocompatibility, chemical versatility, and tunable mechanical properties. However, their bio-inert properties require them to be associated with additional functional moieties to interact with cells. To circumvent this need, we propose here to reticulate PEG molecules with poly(L-lysine) dendrigrafts (DGL) to provide intrinsic cell functionalities to PEG-based hydrogels. The physico-chemical characteristics of the resulting hydrogels were studied in regard of the concentration of each component. With increasing amounts of DGL, the cross-linking time and swelling ratio could be decreased, conversely to mechanical properties, which could be tailored from 7.7 ± 0.7 to 90 ± 28.8 kPa. Furthermore, fibroblasts adhesion, viability, and morphology on hydrogels were then assessed. While cell adhesion significantly increased with the concentration of DGL, cell viability was dependant of the ratio of DGL and PEG. Cell morphology and proliferation; however, appeared mainly related to the overall hydrogel rigidity. To allow cell infiltration and cell growth in 3D, the hydrogels were rendered porous. The biocompatibility of resulting hydrogels of different compositions and porosities was evaluated by 3 week subcutaneous implantations in mice. Hydrogels allowed an extensive cellular infiltration with a mild foreign body reaction, histological evidence of hydrogel degradation, and neovascularization.

Interaction of the coiled-coil domain with glycosaminoglycans protects angiopoietin-like 4 from proteolysis and regulates its antiangiogenic activity.

Angiopoietin-like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled-coil domain (CCD) and fibrinogen-like domain (FLD) fragments. The full-length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM-bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure-function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full-length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule-like formation. In endothelial cells, ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor alpha1-PDX and abolished by the mutation of the (161)RRKR(164) cleavage site without modification of the ECM binding and release. These data suggest that the full-length form, which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.

The Utilization of Linear Polylysine Coupled with Mechanic Forces to Extract Microbial DNA from Different Matrices.

Molecular approaches are powerful tools that are used for medical or environmental diagnoses. However, the main limitations of such a tools are that they extract low levels of DNA and they do not remove the inhibitors of polymerase chain reaction (PCR). Although the use of polycation to complex and purify DNA has been described in the literature, elution often requires a high ionic strength or pH levels not compatible with molecular analyses. In this paper, we described a new process that is based on the complexation of DNA with linear polylysine, followed by capturing the complex by a cation exchange resin. The originality of the process consisted of using mechanic force to elute DNA from the complex. The extraction method showed several advantages when compared to existing methods, such as being compatible with pH levels that range from 5 to 11, as well as high levels of DNA recovery and elimination of PCR inhibitors from complex samples. This method was successfully applied to different types of samples, such as environmental samples, beverage samples, and medical samples. Furthermore, it was proven to be a good solution for removing PCR inhibitors and assuring good DNA recovery yield.

Photochemistry on the Space Station-Antibody Resistance to Space Conditions after Exposure Outside the International Space Station.

Antibody-based analytical instruments are under development to detect signatures of life on planetary bodies. Antibodies are molecular recognition reagents able to detect their target at sub-nanomolar concentrations, with high affinity and specificity. Studying antibody binding performances under space conditions is mandatory to convince space agencies of the adequacy of this promising tool for planetary exploration. To complement previous ground-based experiments on antibody resistance to simulated irradiation, we evaluate in this paper the effects of antibody exposure to real space conditions during the EXPOSE-R2 mission outside the International Space Station. The absorbed dose of ionizing radiation recorded during the 588 days of this mission (220 mGy) corresponded to the absorbed dose expected during a mission to Mars. Moreover, samples faced, at the same time as irradiation, thermal cycles, launch constraints, and long-term storage. A model biochip was used in this study with antibodies in freeze-dried form and under two formats: free or covalently grafted to a solid surface. We found that antibody-binding performances were not significantly affected by cosmic radiation, and more than 40% of the exposed antibody, independent of its format, was still functional during all this experiment. We conclude that antibody-based instruments are well suited for in situ analysis on planetary bodies.

Photochemistry on the Space Station-Aptamer Resistance to Space Conditions: Particles Exposure from Irradiation Facilities and Real Exposure Outside the International Space Station.

Some microarray-based instruments that use bioaffinity receptors such as antibodies or aptamers are under development to detect signatures of past or present life on planetary bodies. Studying the resistance of such instruments against space constraints and cosmic rays in particular is a prerequisite. We used several ground-based facilities to study the resistance of aptamers to various types of particles (protons, electrons, neutrons, and carbon ions) at different energies and fluences. We also tested the resistance of aptamers during the EXPOSE-R2 mission outside the International Space Station (ISS). The accumulated dose measured after the 588 days of this mission (220 mGy) corresponds to the accumulated dose that can be expected during a mission to Mars. We found that the recognition ability of fluorescently labeled aptamers was not significantly affected during short-term exposure experiments taking into account only one type of radiation at a time. However, we demonstrated that the same fluorescent dye was significantly affected by temperature variations (-21°C to +58°C) and storage throughout the entirety of the ISS experiment (60% of signal loss). This induced a large variability of aptamer signal in our analysis. However, we found that >50% of aptamers were still functional after the whole EXPOSE-R2 mission. We conclude that aptamer-based instruments are well suited for in situ analysis on planetary bodies, but the detection step requires additional investigations.

A gold standard method for the evaluation of antibody-based materials functionality: Approach to forced degradation studies.

The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)-based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab-based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality. Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti-HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti-HRP antibody (anti-HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within-assay precision less than 7.1%, and 3) the inter-day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 1012 to 2.98 × 1012 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab-surfaces to storage, pH and aqueous-organic solvent mixture stresses.

Biosynthetic support based on dendritic poly(L-lysine) improves human skin fibroblasts attachment.

Poly(L-lysine) (PLL) dendrigrafts (DGLs) are arborescent biosynthetic polymers of regular and controlled structures. They have specific properties such as biocompatibility and non-immunogenicity, and their surface density of NH2 functions can be easily modified and therefore appears as a powerful tool for the functionalization of hydrophobic polymers used in the context of tissue engineering. In this study, we evaluated several criteria of human skin fibroblasts when cultured with DGL of generations 2, 3 and 4, with linear PLL polymer as reference. In aqueous phase, DGLs and PLL displayed a similar cytotoxicity towards fibroblasts. Plastic culture plates grafted with DGLs were further characterized as homogeneous surfaces by atomic force microscopy and surface characterization by amino density estimation by colorimetric assay. Proliferation of fibroblasts was increased when cultured onto PLL and DGLs monolayers when compared with crude plates. Cellular adhesion was increased by 20% on DGLs in comparison to PLL. Integrin α5 subunit protein expression level was increased after 48 h of culture on DGLs, in comparison to control or PLL-coated surfaces. The presence of DGLs did not lead to overexpression or activation of matrix metalloproteinases 2 and 9. Finally, fibroblasts adhesion was increased by 40% on poly-(lactic-co-glycolic acid) matrices functionalized with DGLs when compared to PLL. Overall, these features make DGL promising candidates for the surface engineering of biomaterials in tissue engineering.

Grafting of poly-L-lysine dendrigrafts onto polypropylene surface using plasma activation for ATP immobilization - Nanomaterial for potential applications in biotechnology.

The present work describes a new environmental friendly strategy for the development of surfaces with high amine density via the grafting of native or modified poly-L-lysine dendrigraft (DGL G3) onto plasma activated polypropylene (PP), polystyrene (PS), polyimide, and polytetrafluoroethylene (PTFE) surface. Modified DGL G3 was prepared by replacement of few peripheral amines by various functionalities. Grafting efficiency was determined by wettability measurements, IRTF, XPS, AFM, and by colorimetry using optimized Coomassie Brilliant Blue method tailored for surface analysis. It was shown that a 4-7nm DGL G3 monolayer with 4×10(14)aminecm(-)(2) was covalently grafted onto various surfaces. Immobilization of adenosine triphosphate on the DGL-g-PP material from dilute solution was studied by bioluminescence and proved the ability of the material to interact with polyanionic biological compounds: 1 ATP complex with 5 amine groups. So, this material has a potential use in diagnostic and more widely for biotechnology due to its high capacity for biomolecule immobilization.

From protected trialkoxysilyl-peptide building blocks to bioorganic-silica hybrid materials.

A straightforward method for the preparation of hybrid bioorganic-inorganic materials is reported. Common strategies to synthesize such promising materials require special surface modifications of silica followed by grafting of the organic moiety via chemoselective ligation. In this context, we set up a general and bottom-up strategy relying on modified peptides functionalized with a trialkoxysilane group. Used in mixtures with TEOS and a surfactant as the structure directing agent, these hybrid building blocks allow one step direct synthesis of bioorganic-inorganic hybrid materials. Two examples were chosen to demonstrate our general approach. (1) An antifouling surface was prepared by dip coating of a sol containing an antibacterial silylated peptide. (2) Organized mesoporous silica displaying a peptide catalyst in the pores was prepared in one step and tested.

In vivo evidence for a bridging role of a collagen V subtype at the epidermis-dermis interface.

Collagen V is the defective product in most cases of classical Ehlers-Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V)(2)α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V)(3) minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V)(3) homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V)(3)-containing fibrils leads to ultrastructural modifications at the epidermis-dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal-dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.

Antibody-based surfaces: rapid characterization using two complementary colorimetric assays.

Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A(2)HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.

The first draft of the endostatin interaction network.

Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds to heparin/heparan sulfate and to a number of proteins, but its molecular mechanisms of action are not fully elucidated. We have used surface plasmon resonance (SPR) arrays to identify new partners of endostatin, and to give further insights on its molecular mechanism of action. New partners of endostatin include glycosaminoglycans (chondroitin and dermatan sulfate), matricellular proteins (thrombospondin-1 and SPARC), collagens (I, IV, and VI), the amyloid peptide Abeta-(1-42), and transglutaminase-2. The biological functions of the endostatin network involve a number of extracellular proteins containing epidermal growth factor and epidermal growth factor-like domains, and able to bind calcium. Depending on the trigger event, and on the availability of its members in a given tissue at a given time, the endostatin network might be involved either in the control of angiogenesis, and tumor growth, or in neurogenesis and neurodegenerative diseases.

Molecular interplay between endostatin, integrins, and heparan sulfate.

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

[Collagens associated with basement membranes and their matricryptins]. / Les collagènes associés aux membranes basales et leurs matricryptines.

The superfamily of collagens is comprised of 27 members (reviewed by Myllyharju & Kivirikko, 2004; Ricard-Blum & Ruggiero, 2005; Ricard-Blum et al., 2005), which are classified into several subgroups according to their structural features and supramolecular assemblies. Fibrillar collagens and FACITS (Fibril-Associated Collagens with Interrupted Triple helix) are described in the paper by Ruggiero et al. in this issue. Our paper reports recent advances on collagens associated to basement membranes. It focuses on the multiplexin family (including collagens XV and XVIII) and on membrane collagens present in skin, namely collagens XIII and XVII. The mechanisms leading to the shedding of their ectodomain from cell membrane and the biological roles of their shedded domains are discussed. The last part of the paper is devoted to several fragments of basement membrane collagens, called matricryptins or matrikins, and to their biological activities.