Search

24 p.-10 fig.-3 tab., D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma., This research was funded by Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO), grant number UGP-19-063, and co-supported by PRECIPITA crowdfunding platform from Spanish Foundation for Science and Technology (FECYT) and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to M.S., Miguel Servet Program from Instituto de Salud Carlos III (CP19/00095) to C.d.J.R. and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to J.M.S. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).

BACKGROUND The α1,3-galactosyltransferase gene-knockout (GalT KO) mice are able to produce natural anti-αGal antibodies apparently without any specific immunization. GalT KO mice are commonly used as a model immunological system for studying anti-αGal responses to Gal-positive xenografts in human. In this study, we compared the specificity of mouse and human αGal antibodies to realize the adequacy of the murine model. METHODS Using hapten-specific affinity chromatography antibodies against Galα1-3Galβ1-4GlcNAcβ epitope were isolated from both human and GalT KO mice blood sera. Specificity of isolated antibodies was determined using a printed glycan array (PGA) containing 400 mammalian glycans and 200 bacterial polysaccharides. RESULTS The quantity of isolated specific anti-Galα antibodies corresponds to a content of

19 p.-8 fig.-1 tab., D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair., This research was funded by Spanish Instituto de Salud Carlos III, grant number PI01202025, and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to MS and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to JMS. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

Antibody-dependent enhancement (ADE) of infection occurs when nonneutralizing antibodies facilitate the infectivity of pathogens. In humans, antibodies involved in ADE of infections may include those naturally produced against α-galactosyl (αGal) epitopes. Here, we investigate whether the removal of circulating anti-αGal antibodies using a soluble αGal glycopolymer confers protection against Gram-negative bacterial infections. We demonstrate that the intra-corporeal removal of anti-αGal antibodies in an α1,3-galactosyltransferase knockout mouse model is associated with protection against mortality from Gram-negative sepsis after cecal ligation and puncture. Additionally, inhibition of anti-αGal antibodies in vitro from human serum increases the bactericidal effect against E. coli O86:B7 and multi-drug resistant Pseudomonas aeruginosa and Klebsiella pneumoniae. Our results show that protective immunity against Gram-negative bacterial pathogens can be elicited and infectious diseases can be prevented by removing anti-αGal antibodies. The intracorporeal removal of non-neutralizing anti-carbohydrate antibodies with glycopolymers may represent a new approach to providing protection against infectious diseases.

44 p.-6 fig.-2 tab.-5 fig.supl., Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application of these biopolymers contemplates its utilization as supports for protein immobilization. Here, the PHA-binding domain of the Pseudomonas putida KT2440 PhaF phasin (BioF polypeptide) was investigated as affinity tag for in vitro functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF- β-galactosidase, containing the choline-binding module C-LytA and the β-galactosidase enzyme, respectively). Protein-biopolyester interaction was strong and stable in a wide range of pH and temperature, and the bound protein was highly protected from self-degradation, while binding strength could be modulated upon particle coating with amphiphilic compounds. Finally, a mini-bioreactor with immobilized BioF- β-galactosidase displayed a very stable enzymatic activity after several continuous activity+washing cycles. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials., Importance: Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The BioF tag wide substrate range presumably allows protein immobilization in vitro to virtually all natural PHAs as well as blends, copolymers or artificial, chemically-modified derivatives with novel physical-chemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading., This work was supported by Spanish Ministry of Economy and Competitiveness (grants BFU2010-17824, BIO2013-44878-R, BIO2013-47684-R and BIO2016-79323-R) and Community of Madrid (Spain) (NanoBIOSOMA S20 1473 3/M lT-2807)

12 p.-6 fig.-2 tab., The presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof-of-concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase-like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40-fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45-90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters., This work was supported by Spanish Ministry of Economy and Competitiveness (grants BIO2013-47684-R and BIO2016-79323-R),IVACE (Institut Valenciá de Competitivitat Empresarial, SPAIN) and FEDER (Fondo Europeo de Desarrollo Regional, Europe).

We present and describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Neisseria meningitidis. The tagging cassette is designed for carboxyl-terminal tagging of proteins and it contains only two repeats of IgG-binding units. P64k protein from N. meningitidis was chosen to fuse at these new affinity tags. This protein is well recognized in immunoassays by serum from human convalescent meningococcal disease and it is highly immunogenic in animals. To continue the characterization of this meningococcal antigen, we designed and constructed two vectors for use in TAP purification method. We also carried-out preliminary test to check the correct expression of the protein fused in these vectors.

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. This work was supported by grants BFU2010–17824, CTQ2008-04492-E and CTQ2010-18570 (Spanish Ministerio de Economia y Competitividad) and FPA-2010-055 (Valentian Government, Spain).