Introduction:

Aitor Balmaseda and Pablo Revilla

Spanish Federation of Biotechnologist, Campus of Vegazana, s / n, 24071 León

From 10th to 12th of July of this year the Spanish Federation of Biotechnologists (https://febiotec.es/) organized the 13th edition of the Annual Biotechnology Congress (BAC).

This event, held in Madrid, allowed young students and professionals to share their results and knowledge with other students and senior professionals, part of the audience, being the setting of this network the purpose of BAC Madrid 2019. Biotechnologists from all over Spain could meet to see the last trends thank to lectures from academia researchers, but also business biotechnology. Participants could also share with the audience their results through oral communications and posters.

A sample of abstracts of those communications presented in BAC Madrid may be found bellow. We are looking forward to seeing you in the next edition of our Congress which will take place in Zaragoza in July 2020.

Lucas Dal Magro^1,2, Jakub F. Kornecki^1,4, Manuela P. Klein³, Rafael C. Rodrigues², Roberto Fernandez-Lafuente¹

¹Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Cantoblanco, ZC 28049, Madrid, Spain; ²Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil; ³Department of Nutrition, Federal University of Health Sciences of Porto Alegre (UFCSPA), ZC 90050-170, Porto Alegre, RS, Brazil; ⁴Orion High Technologies Calle Turín 15, Nave 3, 28983 Parla, Madrid

Correspondence: Jakub F. Kornecki (yakokornecki@hotmail.es)

Polygalacturonases (PGs) are enzymes that act over the pectic acid eliminating the α-1,4 bond between two non-esterified galacturonic acid residues. PGs from fungi present a high rate of activity and they perform best at slightly acidic pH values and an optimum temperature between 30°C and 50°C. The addition of PGs during juices production is focused on the degradation of pectin and other big molecules, reducing its viscosity and achieving a clear aspect for the juice, besides increasing the juice production and halving the required filtration time. PG from Aspergillus niger was immobilized using three different supports: glyoxyl, vinylsulfone and glutaraldehyde-activated amino support. The use of supports pre-activated with glutaraldehyde had the best results. PG immobilization was carried for 24h at pH 5, and at pH 5, 6.5 and 8 for 3h, and passed this time they were switched to pH 8 to complete the 24h. Another protocol used pH 8 adding 300 mM NaCl to prevent ionic exchange between the enzyme and the support. The immobilization under all conditions produced a significant increase in thermal stability during stress inactivation experiments at pHs from 4, up to 10. This permitted that at temperatures over 70°C or pH values that went over 7, the biocatalyst maintained significant levels of activity while the free enzyme was completely inactive. The immobilization conditions were key over enzyme activity, thermostability and operational stability, making us think that the different conditions applied, allowed PG to have different orientations while being immobilized. The interest on the performance of each biocatalyst depends on the parameter of most value (activity or stability) and the conditions used during the reaction. Optimal PG immobilized biocatalysts could be reused up to ten times without significant losses in enzyme activity and offered a very linear reaction courses.

Funding: This work was supported by grants and scholarships (L. Dal Magro) from Capes, CNPq (process 403505/2013-5) and FAPERGS (process 17/2551-0000939-8). We also gratefully recognize the economic support from the Comunidad Autónoma de Madrid (project Ref. IND2017/IND-7640) and the MICIU from Spanish Government, (project number CTQ2017-86170-R). The authors wish to thank Amazon group and LNF Latinoamericana for kindly supplying the enzymes used in this research.

Laia Bosch-Molist, Arnau Boix-Besora, Laura Cervera-Gràcia, Francesc Gòdia-Casablancas

Universitat Autònoma de Barcelona (UAB)

Correspondence: Laia Bosch-Molist (laiaboschm@gmail.com)

Virus-like particles (VLPs) are nanostructures that mimic the natural configuration of a virus [1]. They are based on the intrinsic ability of structural viral proteins to self-assemble into particles. Their capacity of generating a strong cellular and humoral immune response due to their repetitive subunits and not containing viral genetic material makes them good vaccine candidates [2]. HIV-1 VLPs are based on the polyprotein Gag which can form spherical structures when recombinantly expressed.

In this work, mammalian cell platforms are the selected systems for such complex and enveloped VLPs. This approach allows the incorporation of accurate post translational modifications into the VLP, which are important for vaccine efficacy. Production of recombinant Gag VLPs in HEK293 cultures can be achieved by transient gene expression (TGE) or stable gene expression (SGE) [3]. In TGE expression of the gene of interest is lost over time due to dilution in each cell division while SGE achieves a constitutive gene expression via direct integration of the gene of interest into the genome. CRISPR/Cas9 system introduces targeted double-stranded breaks (DSB) which may be repaired by homology-directed-repair (HDR) if a DNA template is used [4]. In here, we present an approach where HDR-mediated knock-in is used to generate an HIV-1 GagEGFP HEK293 stable cell line into the genomic safe harbour AAVS1.

References

[1] N. Kushnir, S. J. Streatfield, and V. Yusibov, “Virus-like particles as a highly efficient vaccine platform: Diversity of targets and production systems and advances in clinical development,” Vaccine, vol. 31, no. 1, pp. 58–83, 2012.

[2] W. A. Rodríguez-Limas, K. Sekar, and K. E. J. Tyo, “Virus-like particles: The future of microbial factories and cell-free systems as platforms for vaccine development,” Curr. Opin. Biotechnol., vol. 24, no. 6, pp. 1089–1093, 2013.

[3] L. Cervera, S. Gutiérrez-Granados, N. S. Berrow, M. M. Segura, and F. Gòdia, “Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement,” Biotechnol. Bioeng., vol. 112, no. 5, pp. 934–946, 2015.

[4] D. Paquet et al., “Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9,” Nature, vol. 533, no. 7601, 2016.

González-Gómez, R, Ruiz-López, E., Torres-Herrero, B., Schuhmacher, AJ

Aragon Health Research Institute (IIS Aragón)

Correspondence: González-Gómez, R. (ruthgo1994@gmail.com)

Despite the survival rate of cancer patients is higher than the last decades, new diagnosis tools are needed for the appropriate detection of tumours. ImmunoPET is a visual technique where antibodies are used to specifically detect a protein which is expressed in tumours, what allows to make an efficient diagnosis in a non-invasive manner [1].

The best candidate for this technique is a protein that is differentially expressed between normal and tumoral tissue. By bioinformatic analysis, ABCC3 showed to be a good target for immunoPET in Glioblastoma Multiforme (GBM), patient data revealed poor survival with high levels of ABCC3.

ABCC3 is a member of the subfamily of MRP (multi-drug resistance proteins) that belongs to ATP-binding cassettes (ABC) transporters, it may play a role in organic anions excretion in liver and intestine [2].

We aim to determine a correlation between ABCC3 expression and chemotherapy response. Our studies began with a cell line with high ABCC3 expression levels, we generate ABCC3 knock-out cells and they were more sensitive to cis-platin and temozolomide treatment compared with wild type cells. GBM cell lines were characterized at mRNA and protein levels of ABCC3, showing different expressing profiles.

ABCC3 overexpressing and knock-out cells of GBM cell lines were developed, demonstrating that the lack of ABCC3 in vitro causes sensitivity of the cells to chemotherapeutics, likewise high protein levels confer resistance to drugs.

O6-Benzylguanine (O6-BG) is a potent inhibitor of O6-methylguanine-DNA methyltransferase (MGMT). When inhibiting MGMT, cells are more sensitive to temozolomide administration [3]. Combination treatment between cis-platin or temozolomide with O6-BG did not synergize, however MGMT levels were altered when ABCC3 is knocked-out.

It would be interesting to further investigate ABCC3 and MGMT relation for treatment improvement. ABCC3 could be a favourable target for immunoPET to improve diagnosis and determine chemotherapy response in different tissues.

References

1. Bailly C, Cléry PF, Faivre-Chauvet A, Bourgeois M, Guérard F, Haddad F, Barbet J, Chérel M, Kraeber-Bodéré F, Carlier T, Bodet-Milin C. Immuno-PET for Clinical Theranostic Approaches. Internal Journal of Molecular Sciences. 2016 Dec 28;18(1):57.

2. Vasiliou, V., Vasiliou, K. and Nebert, DW. (2009). Human ATP-binding cassette (ABC) transporter family. Human Genomics 3(3): 281-290

3. Cai, W., Maldonado, NV., Cui, W., Harutyunyan, N., Ji, L., Sposto, R., Reynolds, CP. And Keshelava, N. (2010). Activity of irinotecan and temozolomide in the presence of O⁶-methylguanine-DNA methyltransferase inhibition in neuroblastoma pre-clinical models. British Journal of Cancer 103(9): 1369-1379.

Funding: FERO Foundation, ASPANOA, Carlos III Health Institute, Ramón y Cajal program.

Eduardo Ruiz-López, Ruth González-Gómez, Beatriz Torres-Herrero, Alberto J Schuhmacher

Aragon Health Research Institute (IIS ARAGON), Molecular Oncology Group, Zaragoza, Spain

Correspondence: Eduardo Ruiz-López (educalata21@gmail.com)

Glioblastoma (GBM) is the most prevalent and most aggressive brain tumor. Current techniques for GBM diagnosis -Magnetic Resonance Imaging- still being unaccurate and molecular imaging approaches are emmerging as non-invasive tools. Targeting of GBM with highly specific imaging probes allows non-invasive diagnosis, treatment stratification and monitoring of patients. Overexpressed GBM biomarkers have allowed the desing of monoclonal antibodies for molecular imaging. However, their large size impedes the penetration through the blood-brain barrier (BBB) and diffusion into the tumor. Smaller protein scaffold-based probes targeting GBM are being developed [target]: nanobodies [HGF], affibodies [PDGFRβ, EGFR, IGF-1R, VEGFR2], knottins [phosphatidyl inositide, MMP-2, integrins αvβ3, αvβ5], anticalins [HGF, VEGF-A], adnectins [VEGFR] and DARPins [HGF]. Their lower molecular weight enables better tumor uptake and faster clearance compared with monoclonal antibodies. Successful molecular imaging has been conducted on preclinical xenograft and orthotopic mice models.

We have bioinformatically identified molecular targets for the development of molecular imaging probes for GBM. We are developing nanobody-based targeting agents against these candidates following two strategies. First, we are actively immunizing dromedaries with a target-overexpression glioma cell line in order to clone the targeted-enriched repertoire of variable domain of camelid heavy chain. We will generate a nanobody-expressed bacteria library or nanobody nanothèque. Bacteria expressing high affinity nanobodies will be selected by E. coli display. Also, we are isolating nanobodies against GBM target from a gene library by phage display. Selected nanobody probes will be used for molecular imaging of xenograft and “avatar” mice models. For clinical development, we will fusion our nanobodies with molecular BBB shuttles (ApoE, FC5 nanobody) to improve brain access via transcytosis. Multitargeting nanobodies, dual labeling strategies and radionuclide theragnostics approaches are considered. In the future, genetic profile of patients may allow molecular personalized immunotargeting and same-day imaging of GBM.

Funding: Asociación Española contra el Cáncer (AECC), Programa Ramón y Cajal (RyC) y Fundación de Investigación Oncológica (FERO).

Carla Michelle Parada Sosa^1,2, Rosa del Carmen Rocha Gracia³, Magdalena Martínez Cañamero⁴, Antonio Cobo Molinos⁵ and Zaida Nelly Juárez¹

¹Facultad de Biotecnología. Decanato de Ciencias Biológicas, Universidad Popular Autónoma del Estado de Puebla, 21 Sur 1103, Colonia Santiago, 72410 Puebla, Puebla, México; ²Facultad de Ciencias Experimentales, Universidad de Jaén, 23071-Jaén, España; ³Centro de Investigaciones en Ciencias Microbiológicas, ICUAP, Benemérita Universidad Autónoma de Puebla. Complejo de Ciencias, edificio No. 103 J. Ciudad Universitaria, Colonia San Manuel, 72570 Puebla, México; ⁴Área de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaén, 23071-Jaén, España; ⁵Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, 18071-Granada, España

Correspondence: Carla Michelle Parada Sosa (carlamichelle.parada@upaep.edu.mx)

Antimicrobial resistance (AMR) spread is an alarming global health issue. Bacterial infections are not only linked to the hospital area, they are also an issue in the food area. A strategy against AMR is to strengthen the research on alternative antimicrobials [1].

Herbal extracts and their secondary metabolites are an option for treating infections or preserving food [2].

In this study, evaluations of antimicrobial activities of herbal extracts were carried out using in vitro antimicrobial susceptibility assays against Gram-positive and Gram-negative strains in order to propose the active samples as antimicrobials. Extracts of A. ludoviciana, Helianthus annuus var. Autumn Beauty y Lycopodium clavatum showed antibacterial properties against Gram-positive and Gram-negative strains. This property of herbal extracts could be an alternative for finding natural antimicrobials for future research and development of new products to treat hospital infections, food poisoning or as food preservatives.

References

1. Blair JMA, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ V. Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol. 2015;13(1):42–51.

2. Yang S, Low L, Yap PS, Yusoff K, Mai C, Lai K, et al. Plant-Derived Antimicrobials : Insights into Mitigation of Antimicrobial Resistance. Rec Nat Prod. 2018;4:1–22.

Javier Quero¹, Inés Mármol¹, Raquel Ibarz², Olga Martín-Belloso² and Mª Jesús Rodríguez-Yoldi¹

¹ Pharmacology and Physiology Department, Faculty of Veterinary, University of Zaragoza, Zaragoza, Spain; ² Food Technology Department, University of Lleida, Agrotecnio Center, Spain

Correspondence: Javier Quero (javierquero94@gmail.com)

Oxidative stress on intestinal mucosal barrier is closely related to the development of a wide range of chronic disorders such as Inflamatory Bowel Diseases and colorectal cancer (CRC). This exogenous oxidative stress can be triggered by multiple external factors such as diet, drugs, smoking or alcoholism, which increase reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the intestinal tissue [1,2].

Fruits and vegetables are well-known for their antioxidant potential given their composition in phenolic compounds and other bioactive compounds [3], which can be also obtained from forest and agro-industrial by-products.

We have analyzed the potential use of agroalimentary industry waste as protective agents for the mucosal barrier upon oxidative stress induction. In addition, influence of maltodextrin-encapsulation has been evaluated. The selected cellular intestinal barrier model were differentiated Caco-2 cells over 80% confluence, which were exposed to 10 mM H₂O₂ for 20 minutes.

Results suggest the potential role of agro-food by-products as protective agents for the intestinal barrier upon exogenous oxidative stress induction. Therefore, food waste might be of interest for the pharmaceutical industry on the management of oxidative stress-related diseases of the intestinal barrier, giving a second life to a great amount of waste generated by forest and agro-food industries.

References

[1] Reuter, S. et al. Oxidative stress, inflammation, and cancer: How are they linked? Free Radic Biol Med (2010) 49(11), 1603-1616.

[2] Perse, M. Oxidative stress in the pathogenesis of colorectal cancer: cause or consequence? Biomed Res Int (2013) 2013, 725710-725719.

[3] Tsao, R. Chemistry and biochemistry of dietary polyphenols Nutrients (2010) 2, 1231-1246.

Funding: Ministerio de Ciencia e Innovación (SAF2016-75441-R), Gobierno de Aragón/European Regional Development Fund (ERDF) (B16-R17) and Interreg Sudoe REDVALUE (SOE1/PI/E0123)

Nathalia Saraiva Rios^1,4, Davino M. Andrade Neto², José Cleiton Sousa dos Santos³,Pierre Basílio Almeida Fechine², Roberto Fernández-Lafuente⁴, Luciana Rocha Barros Gonçalves¹

¹Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Bloco 709, CEP 60455-760, Fortaleza, CE, Brazil; ²Departamento de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Av. Mister Hull s/n, Pici, 60455-760, Fortaleza,CECP12200, Brazil; ³Instituto de Engenharias e Desenvolvimento Sustentável, Universidade da Integração Internacional da Lusofonia Afro-Brasileira, 62785000 Acarape, CE, Brazil; ⁴Departamento de Biocatálisis. ICP-CSIC, Campus UAM-CSIC, Madrid, Spain

Correspondence: Nathalia Saraiva Rios (nathaliarios25@yahoo.com.br)

Lipases have been widely used in food industry, chemical and pharmaceutical processes, because their high activity and high chemo-, regio- and enantio-selectivity [1]. Therefore, the immobilization of enzymes has commonly used, since it enables the easy separation of the reaction medium and the reuse of the biocatalyst [2]. In this context, a novel NiZnFe₂O₄ superparamagnetic nanoparticle was used as support for immobilization of the lipase from Pseudomonas fluorescens (PFL). For this purpose, this nanoparticle was coated with silica by impregnation with tetraethoxysilane (TEOS) and further activated with p-benzoquinone (BQ) and divinylsulfone (DVS), to produce the covalent immobilized biocatalysts. BQ and DVS are bifunctional molecules that can react with different moieties of enzyme (amine, hydroxyl, thiol and among others), producing highly stable biocatalysts [3,4]. For the best of our knowledge, BQ has not been properly studied to stabilize enzymes via multipoint covalent attachment. Therefore, we use this activating agent to immobilize PFL comparing with DVS, which is a promising support activator. PFL also immobilized on TEOS coated NiZnFe₂O₄ nanoparticles without any activation to produce the adsorbed immobilized biocatalyst. BQ biocatalyst demonstrated the most active and stable preparation (Recovered activity: 89 % and t_1/2(60°C) over 1440 min), while the DVS covalent and adsorbed preparation presented a recovered activity of 82 and 55 % and half-lives of 225 and 150 min at 60°C, respectively. Therefore, this study shows a successful immobilization strategy, producing active and stable biocatalysts.

References

[1] D. Sharma, B. Sharma, A. K. Shukla, Biotechnological approach of microbial lipase: A review, Biotechnology. 10 (2011) 23–40.

[2] R. A. Sheldon, S. van Pelt, Enzyme immobilisation in biocatalysis: why, what and how., Chem. Soc. Rev. 42 (2013) 6223–6235.

[3] J.C.S. dos Santos, N. Rueda, O. Barbosa, J.F. Fernández-Sánchez, A.L. Medina-Castillo, T. Ramón-Márquez, M.C. Arias-Martos, M.C. Millán-Linares, J. Pedroche, M. del M. Yust, L.R.B. Gonçalves, R. Fernandez-Lafuente, Characterization of supports activated with divinyl sulfone as a tool to immobilize and stabilize enzymes via multipoint covalent attachment. Application to chymotrypsin, RSC Adv. 5 (2015) 20639–20649.

[4] N.S. Rios, D.M. Andrade Neto, J.C.S. dos Santos, P.B.A. Fechine, R. Fernández-lafuente, L.R.B. Gonçalves, Comparison of the immobilization of lipase from Pseudomonas fluorescens on divinylsulfone or p -benzoquinone activated support, Int. J. Biol. Macromol. 134 (2019) 936–945.

Funding: Nathalia S. Rios thanks to CNPq for a predoctoral fellowship (CNPq scholarship – Brazil). MICIU from Spanish Government, (project number CTQ2017-86170-R).

Miriam Esteban¹, Juan Pedro Navarro¹, Patricia Galán-Malo², Luis Mata², Mª Dolores Pérez¹, Lourdes Sánchez¹

¹ Departamento de Producción Animal y Ciencia de los Alimentos. Facultad de Veterinaria. Instituto Agroalimentario de Aragón (IA2) (Universidad de Zaragoza-CITA), Zaragoza, Spain; ² ZEULAB S.L., Zaragoza, Spain

Correspondence: Miriam Esteban (miriames93@gmail.com)

Clostridium tyrobutyricum is the major agent that causes the blowing defect problem in cheeses due to the germination of its dormant spores during the ripening [1]. As a result of the pressure exerted by the gases produced, many of the affected cheeses show cavities and cracks, which cause the product loss in most cases.

Our aim is to develop a fast method to detect the spores in milk instead of the traditional microbiological ones that are not specific and are time consuming. For this reason, we developed an approach based on previous digestion of milk with subtilisin for recovering the spores. For the real time PCR, we tested different methods for disruption of spores, which is a total challenge due to its resistant structure. The microwave treatment followed by a standard DNA purification has been found to be the most efficient disruption procedure. We were able to detect from 10¹ spores/mL to 10⁷ spores/mL, but we were not able to distinguish the quantity because of the low DNA recovery and also due to the effect of SASPs proteins on DNA packaging [2].

On the other hand, we C. tyrobutyricum spores detection by flow cytometry because it has the advantage of not disrupting the spores. The detection was done with a high affinity ligand as an alternative to antibodies produced in animals. The ligand was developed previously by Phage Display and its affinity for butyric spores analyzed by isothermal titration calorimetry (ITC). These results showed good binding of C. tyrobutyricum spores, but also for other clostridium species. Flow cytometry with specific ligands showed good results with a limit of detection of 10³ spores/mL and could be an alternative to the real time PCR to avoid the problem of DNA recovery from spores.

References

[1] Bassi, D., Puglisi, E., Cocconcelli, P.S., 2015. Understanding the bacterial communities of hard cheese with blowing defect. Food Microbiol. 52, 106–118. https://doi.org/10.1016/j.fm.2015.07.004

[2] Raju, D., Waters, M., Setlow, P., Sarker, M.R., 2006. Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat. BMC Microbiol. 13.

Funding: Aragon Government (Spain) and the European Social Fund under a DGA predoctoral grant and by the AGL2013-44130-R project financed by the Ministerio de Ciencia e Innovación of the Spanish Government.

Marta Pérez-Díaz¹, Elena G. Biosca², Belén Álvarez¹

¹ IMIDRA, A2 km 38.200, 28800 Alcalá de Henares, Madrid, Spain; ² University of Valencia, Avda. Dr. Moliner 50, 46100 Burjasot, Valencia, Spain

Correspondence: Belén Álvarez (mariabelen.alvarez@madrid.org)

Large-scale production of environmental microorganisms to exploit their biotechnological applications can be of great interest in agri-food industry. Activities of microorganisms with biotechnological use are the production of hydrolytic exoenzymes with diverse functions, the ability to supply essential nutrients to plants acting as biofertilizers, and the antagonistic effects they can exert against pathogenic microorganisms [1,2]. The beneficial consequences of their application can contribute to achieve a microbe-based sustainable agriculture [3].

A collection of soil and rhizosphere bacteria was isolated from cover crops of an olive grove in mainland Spain. All of them were identified and tested for their potential to produce exoenzymes related to the degradation and recycling of organic compounds, their ability to solubilize phosphates, fix atmospheric nitrogen, and capture iron, and their antagonism against relevant phytopathogenic fungi such as Verticillium dahliae, Phaeomoniella chlamydospora, Dactylonectria macrodidyma and Fusarium avenaceum.

Molecular identification of the isolates revealed a majority of Bacillus spp., and also Massilia spp., Brevibacterium spp., Pantoea spp., Pseudomonas spp., Enterococcus spp. and Chryseobacterium spp. as the main genera present. Production of exoenzymes as proteases, lipases, amylases, and DNases was positive for 96% of the isolates, whereas 92% was positive for phosphate solubilization, nitrogen fixation and/or iron uptake, and 41% displayed effective antagonism against, at least, one of the fungal pathogens tested. V. dahliae growth was inhibited by 33% of the isolates, one of them also by production of volatile organic compounds (VOCs). Inhibitory activity was detected against P. chlamydospora in 18% of the isolates and D. macrodidyma in 4%, without production of VOCs but, not against F. avenaceum. The high activity observed in soil and rhizosphere bacteria of the cover crops in the olive grove suggests a great potential to be produced in the biotechnological industry [4] for exoenzyme production, recycling of nutrients, reduction of agrochemicals, and/or biological control.

References

¹ Abada E, Al-Faifi Z, Osman M. 2017. Enzymes and nanoparticles produced by microorganisms and their applications in biotechnology. In: Fungal nanotechnology, fungal biology. Prasad R (Ed). Springer International Publishing. Doi: 10.1007/978-3-319-68424-6_7.

² Parnell JJ, Berka R, Young HA, Sturino JM, Kang Y, Barnhart DM, DiLeo MV. 2016. From the lab to the farm: an industrial perspective of plant beneficial microorganisms. Front. Plant Sci. 7:1110. Doi: 10.3389/fpls.2016.01110.

³ Umesha S, Singh PK, Singh RP. 2018. Microbial biotechnology and sustainable agriculture. In: Biotechnology for sustainable agriculture. Emerging approaches and strategies. pp. 185-205. Elsevier.

⁴ Prasad R, Gill SS, Tuteja N (Eds). 2018. New and future developments in microbial biotechnology and bioengineering. Crop improvement through microbial biotechnology. Elsevier. Doi: 10.1016/C2016-0-04330-9.

Funding: This work was funded by the AGRISOST S2013/ABI-2717 project, and BACPLANT-UVEG-Research Support

Sara Arana-Peña¹, Yuliya Lokha¹, Nathalia S. Rios^1,2, Carmen Méndez- Sánchez¹ and Roberto Fernández-Lafuente¹

¹Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica (ICP-CSIC), Campus UAM-CSIC, 28049 Madrid, Spain; ²Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, CEP 60455-760, Fortaleza, CE, Brazil

Correspondence: Sara Arana-Peña (s.arana@csic.es)

Lipases are among the most used enzymes due to their unique properties and may be found in all living beings. Lipase immobilization is mainly necessary if biocatalyst’s recovery and reuse is desired, as well as improve its properties. The lipases immobilization at low ionic strength on hydrophobic supports allows single step immobilization via interfacial activation, locking the lipase open form, and giving it a high stabilization [1]. It may be expected that immobilization conditions can have a significant impact on lipase conformation. In fact, the lipase from Thermomyces lanuginosus (TLL) has been described as sensible to the changes of the immobilization conditions [2]. Thus, in this work we intend to investigate if reversible immobilization of this enzyme on hydrophobic support under different conditions, such as pH and presence of some ions, may have an effect on their final stability and catalytic activity. Phosphate ions have proved to decrease the stability of many lipases [3], on the other hand, some cations (e.g., Ca2+) may increase enzyme stability, but mostly when immobilized on hydrophobic supports [4]. Octyl-agarose beads have been chosen as support, as they are widely used and agarose has some good properties. After performing the assays, we confirmed that immobilization conditions, using one lipase and the same support, can alter significantly the final catalytic activity and stability of TLL.

References

[1] J.M. Palomo, G. Muñoz, G. Fernández-Lorente, C. Mateo, R. Fernández-Lafuente, J. Guisan, Interfacial adsorption of lipases on very hydrophobic support (octadecyl-Sepabeads): Immobilization, hyperactivation and stabilization of the open form of lipases, J. Mol. Catal. B Enzym. 19 (2002) 279–286. doi:10.1016/S1381-1177(02)00178-9.

[2] E. Abreu Silveira, S. Moreno-Perez, A. Basso, S. Serban, R. Pestana Mamede, P.W. Tardioli, C. Sanchez Farinas, J. Rocha-Martin, G. Fernandez-Lorente, J.M. Guisan, Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium, BMC Biotechnol. 17 (2017) 88. doi:10.1186/s12896-017-0407-9.

[3] S. Arana-Peña, Y. Lokha, R. Fernández-Lafuente, Immobilization of Eversa Lipase on Octyl Agarose Beads and Preliminary Characterization of Stability and Activity Features, Catalysts. 8 (2018) 511. doi:10.3390/catal8110511.

[4] L. Fernandez-Lopez, R. Bartolome-Cabrero, M.D. Rodriguez, C.S. Dos Santos, N. Rueda, R. Fernandez-Lafuente, Stabilizing effects of cations on lipases depend on the immobilization protocol, RSC Adv. 5 (2015) 83868–83875. doi:10.1039/C5RA18344H.

Funding: MICIU from Spanish Government, (project number CTQ2017-86170-R)

El-Hocine Siar ^1,2, Sara Arana-Peña¹, Roberto Morellon-Sterling¹. Jakub F. Kornecki¹, Mohammed Nasreddine Zidoune², Oveimar Barbosa³, Roberto Fernandez-Lafuente¹

1. Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain; 2. Equipe TEPA, Laboratoire LNTA, INATAA, Université des Frères Mentouri Constantine 1, 25000 Constantine, Algeria; 3. Departamento de Química, Facultad de Ciencias, Universidad del Tolima, 730006299 Ibagué, Colombia

Correspondence: El-Hocine Siar (hocines1@hotmail.fr)

Enzymes are very useful tools in the modern biotechnology, because of their specificity and selectivity in the green chemistry. But some properties limit their use (inhibition, specificity and possibility of reuse). Immobilization and chemical modification of the free or immobilized enzyme are among the most proposed solutions to overcome these limitations. In this work, we studied the effects of amination using carbodiimide and ethylenediamine on the properties of immobilized/stabilized ficin on glyoxyl agarose and free ficin. The free ficin was first aminated then immobilized on glyoxyl agarose. Thus, the immobilized ficin on glyoxyl agarose was directly aminated. Ficin activity versus casein and BANA (benzoyl-arginine-p-nitroanilide) was determined and stability at different pH (5, 7 and 9) was checked. The activity of the aminated immobimized ficin was altered (versus casein and BANA), and while, activity versus casein slightly increased and versus BANA decreased. The heist effect was reported at pH 9, where activity vesus casein increased over 10% and activity versus BANA decreased more than 5 folds. Thus greatly altering the enzyme specificity. The amination improved the enzyme stability at pH 5 while stability was impoverished at pH 9. On the other hand, free aminated ficin, retained around 80% of activity versus BANA and 90% versus casein. After optimization of the immobilization protocol, the new biocatalyst was compared to the obtained using the non-aminated enzyme. Activity versus BANA decreases, but it increased versus casein. The aminated ficin biocatalyst was more stable than the non-aminated ficin biocatalyst mainly at pH 7. Amination of immobilized or free enzyme may be considered interesting for preparing improved biocatalyst of ficin, mainly in proteolytic applications. This may be also considered (mostly amination) as a possibility for further improves of enzyme immobilization.

Funding: We gratefully recognize the support from the MICIU from Spanish Government, (project number CTQ2017-86170-R) Mr. El-Hocine Siar thank Algerian Ministry of higher education and scientific research for their fellowships (Programme National Exceptionnel, Algeria P.N.E.

Tania García-Maceira¹, Ana B. Aragón Gómez¹, Verónica Luna-Guerrero¹, Gracia Montero-Peñalvo¹, Sara Gómez-Melero¹, Fé I. García-Maceira¹, José A González-Reyes², Elier Paz-Rojas¹

¹ Canvax Biotech, Córdoba, 14014, Spain ² Department of Cell Biology, Physiology and Immunology, University of Córdoba, 14014, Spain

Correspondence: Tania García-Maceira (t.garcia@canvaxbiotech.com)

The enzyme-linked immunosorbent assay (ELISA), is the most widely used and reliable clinical routine method for the detection of important protein markers in healthcare. Several methods have been developed to improve the ELISA sensitivity through immobilization of antibodies on the microtiter plates [1]. We have developed a highly sensitive ELISA strategy based on the preparation of acetylated chitosan surfaces in order to improve the antibodies orientation. For this purpose, we used capture antibodies fused to the chitin binding domain (ChBD) of chitinase A1 from Bacillus circulans WL-12 [2]. Chitin surfaces were obtained by mixing small quantities of chitosan and acetic anhydride in each well of a microtiter plate and allowing the mixture dry overnight [3]. Anti-c-myc 9E10 low affinity antibody fused to ChBD was cloned and expressed in CHO cells obtaining the anti-c-myc-ChBD antibody. We demonstrated that anti c-myc-ChBD binds specifically to the chitin surfaces in comparison with anti-c-myc 9E10 which did not bind to it. Chitin surface was used to develop a sandwich ELISA to detect the chimeric protein human c-myc-GST-IL8 cloned and expressed in Escherichia coli. The limit of detection and quantification of the high binding protein microtiter and chitin-treated plates were compared. The ELISA procedure developed on chitin surface were 6-fold more sensitive than the ELISA performed on standard surface with significant differences (p<0.0001). Acetylated chitosan surfaces allow the antibody orientation on the surface and may be an appropriated method to replace the standard surfaces given the stability in time and the low cost of its preparation.

References

1. Welch NG, Scoble JA, Muir BW, Pigram PJ. Orientation and characterization of immobilized antibodies for improved immunoassays, Biointerphases, 2017; 12(2): 02D301.

2. Hashimoto M, Ikegami T, Seino S, Ohuchi N, Fukada H, Sugiyama J, Shirakawa M, Watanabe T. Expression and characterization of the Chitin-Binding Domain of Chitinase A1 from Bacillus circulans WL-12. Journal of bacteriology. 2000; 182(11):3045-3054.

3. Bernard MP, Cao D, Myers RV, Moyle WR. Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan. Analytical Biochemistrry. 2004; 327:278-283

Funding: Reference: 76359 Project: 360229 Junta de Andalucía, España

Ximo García-Dominguez¹, Pablo Sopena-Novales², César D Vera-Donoso³, Juan V. Catret⁴, José S Vicente¹, Francisco Marco-Jiménez¹

¹ Institute for Animal Science and Technology, Universidad Politécnica de Valencia, 46022- Valencia, Spain; ² Department of Nuclear Medicine, Hospital Universitari i Politècnic La Fe, 46026- Valencia, Spain; ³ Department of Urology, Hospital Universitari i Politècnic La Fe, 46026- Valencia, Spain; ⁴ Oncovision, 46022 - Valencia, Spain

Correspondence: Ximo García-Dominguez (ximo.garciadominguez@gmail.com)

The idea of generating a functional kidney graft on demand would extend the option of kidney transplantation to more patients with end-stage kidney disease. Transplantation of embryonic kidneys (metanephroi) showed that if these intact primordia were transplanted into adult hosts, it can mature attracting a new vascular system from host, avoiding immune response and exhibiting functional properties [1]. Our purpose was to determine the excretory functionality of the developed metanephroi using an in vivo renal scintigraphy, as well as post-mortem examination and histological evaluation.

With this aim, using the rabbit model, 14 metanephroi were micro-dissected from 15-day-old foetuses and transplanted into the retroperitoneal fat of 3 non-immunosuppressed hosts, using a minimally invasive laparoscopic technique [2]. Three weeks after transplantation, cortical renal scintigraphy was performed using the Sentinella® 102 and 3mCi of technetium-99m DMSA 30 minutes before planar acquisition of the images. After that, recipients were euthanized and organoids were retrieved, evaluating morphologically and histologically. Host kidneys were used as control.

The renal scintigraphy analysis demonstrated the renal function in new kidneys developed from transplanted metanephroi. Renal activity was 4 times lower for new kidneys in comparison with host kidneys (10000 vs 41000 counts, respectively). Nevertheless, new kidneys were 21 times smaller than host kidneys (0.5±0.44 vs 11.2 ±0.18 g, respectively). Moreover, this renal activity was in concordance with histological results, as glomeruli developed from these new kidneys were mature and similar to those present in host kidneys (40±4 vs 41±2 cells/glomerulus, respectively). In addition, the hydronephrosis state of the new kidneys as consequence of the lack of connection between the new ureter and the host's bladder, prove the excretory function of the new kidneys. Therefore, this study could pave a way to an unlimited supply of kidney for transplantation that could put an end to the current long waiting lists.

Funding: This research was supported by ALCER TURIA (association for the fight against kidney diseases) and anonymous donation to a crowdfunding campaign (Precipita platform, FECYT).

Andrea Núñez-González^1,2, Estela Fernández-Alegre^1,2, Indira Álvarez-Fernández¹, Amer Salman¹, Juan Carlos Domínguez^1,3, Beatriz Martín-Fernández², Felipe Martínez-Pastor^1,4

¹Institute of Animal Health and Cattle Development (INDEGSAL), León, 24071, Spain; ²Bianor Biotech, León, 24071, Spain; ³Department of Medicine, Surgery and Veterinary Anatomy, Universidad de León, León, 24071, Spain, ⁴Department of Molecular Biology, Universidad de León, León, 24071, Spain

Correspondence: Andrea Núñez-González (annugo95@gmail.com)

Melatonin is a ubiquitous molecule that regulates numerous physiological functions and plays an important role in male reproduction, directly influencing testosterone levels and sperm quality [1]. The presence of sperm membrane melatonin receptors in several animal species supports the hypothesis of a direct action of this molecule on spermatozoa [2]. Sperm maturation, capacitation and survival may thus be affected by the presence of endogenous melatonin. This study aimed to assess the direct influence of melatonin on mitochondrial activity and reactive oxygen species (ROS) production of sperm from bull (Bos taurus taurus) and Iberian red deer (Cervus elaphus hispanicus). Sperm samples from five deer and five bulls, epididymal and ejaculated respectively, were treated with different concentrations of melatonin (1 μM, 10 nM, 100 pM, 1 pM) and then incubated (39 °C, 5% CO₂) for 4 h, under non-capacitating conditions (Hepes-buffered TALP) and capacitating conditions (Hepes-buffered TALP supplemented with 2 U/mL heparin). Flow cytometry analysis showed a significant increase (p<0.05) in the ratio of viable sperm cells with active mitochondria after melatonin treatment under capacitating conditions, for all tested concentrations. This effect was not seen when using non-capacitating conditions, except for 10 pM melatonin treatment on deer sperm, which could be a slightly capacitating environment in this species. Melatonin treatment under non-capacitating conditions lowered (p<0.05) the ratio of sperm cells with high mitochondrial superoxide production for all tested concentrations in both species. A similar effect was observed in deer sperm under capacitating conditions. Our results suggest that even the lowest concentrations of melatonin promote sperm survival during capacitation, presumably by stimulating signaling pathways through interaction with sperm membrane melatonin receptors. ROS function as second messengers for sperm capacitation [3], so the modulation of its levels could affect this process. The effect of melatonin on sperm cells may nevertheless vary considerably between species.

References

1. Li C, Zhou X. Melatonin and male reproduction. Clin Chim Acta. 2015;446:175–80.

2. Talpur H, Chandio I, Brohi R, Worku T, Rehman Z, Bhattarai D, et al. Research progress on the role of melatonin and its receptors in animal reproduction: A comprehensive review. Reprod Domest Anim. 2018;53(4):831–49.

3. de Lamirande E, O’Flaherty C. Sperm activation: Role of reactive oxygen species and kinases. Biochim Biophys Acta - Proteins Proteomics. 2008;1784(1):106–15.

Funding: This work was supported by MINECO (AGL2013-43328P and AGL2016-81890-REDT).

Eric Voltà-Durán ^1,2,3, Olivia Cano-Garrido ^1,4, Naroa Serna ^1,2,3, Hèctor López-Laguna ^1,2,3, Laura Sánchez-García ^1,2,3, Mireia Pesarrodona ^{1,2,3 ¥}, Ramón Mangues ^3,5, Antonio Villaverde ^1,2,3, Esther Vázquez ^1,2,3, Ugutz Unzueta ^3,5

¹ Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; ² Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; ³ CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), C/ Monforte de Lemos 3-5, 28029 Madrid, Spain; ⁴ Nanoligent SL, Edifici EUREKA, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; ⁵ Institut d'Investigacions Biomèdiques Sant Pau and Josep Carreras Research Institute, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; ^¥ Present address: Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain

Correspondence: Eric Voltà-Durán (eric.voltaduran@gmail.com)

The use of proteins as drug delivery systems is gaining interest in nanomedicine, especially when dealing with targeted therapies which require specific interaction and penetration into target cells [1]. By genetic fusion, proteins with multiple biological activities can be synthetized as single-chain polypeptides. This way, just a unique step of recombinant production is needed to obtain the final modular protein, in which each domain plays a determinant and distinct role [2]. In this context, the combination of both cationic peptides and polyhistidines into a protein structure has been proven as a universal self-assembling protein platform [3]. The paradigmatic example of this is the modular protein T22-GFP-H6, in which T22 (a potent ligand of the tumoral marker CXCR4) and H6 promote the formation of protein-only nanostructures around 11 nm (above renal filtration cut-off) which selectively bind and internalize CXCR4+ tumor cells in vitro and in vivo [4]. In here, we study the relevance of T22 and H6 positioning inside the modular structure by performing a morphological and functional characterization of alternatives in which T22 and H6 are rearranged in different locations. Interestingly, we prove that T22 must be placed at the amino terminus to allow CXCR4⁺ cell binding and specific internalization, while placing H6 at the carboxy terminus is crucial for promoting self-assembling. Moreover, the ability to oligomerize as regular protein nanoparticles increases cell penetrability in a cooperative way. Taken together, these data are of relevant interest for the design of smart nanoscale protein carriers for targeted drug delivery, as CXCR4 is a significant homing marker of many pathologies, such as many types of cancers and HIV infection, among others.

References

[1] Casanova I, Unzueta U, Arroyo-Solera I, Cespedes MV, Villaverde A, Mangues R, et al. Protein-driven nanomedicines in oncotherapy. Current opinion in pharmacology 2019;47:1-7.

[2] Sanchez-Garcia L, Martin L, Mangues R, Ferrer-Miralles N, Vazquez E, Villaverde A. Recombinant pharmaceuticals from microbial cells: a 2015 update. Microbial cell factories 2016;15:33.

[3] Rueda F, Cespedes MV, Conchillo-Sole O, Sanchez-Chardi A, Seras-Franzoso J, Cubarsi R, et al. Bottom-Up Instructive Quality Control in the Biofabrication of Smart Protein Materials. Advanced materials 2015;27:7816-22.

[4] Unzueta U, Ferrer-Miralles N, Cedano J, Zikung X, Pesarrodona M, Saccardo P, et al. Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles. Biomaterials 2012;33:8714-22.

Funding: We are indebted to Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE) to AV, AGAUR (2017SGR-229) to AV and 2017SGR-865 GRC to RM; CIBER-BBN (project NANOPROTHER) granted to AV and CIBER-BBN project 4NanoMets to RM; ISCIII (PI15/00272 co-founding FEDER) to EV and ISCIII (Co-founding FEDER) PIE15//00028 and PI18/00650 to RM, and to EU COST Action CA 17140. We are also indebted to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) that is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III

Eduardo Gomez Casales¹, Elena Fauste Alonso²; Marina Robas Mora³; Jesus presa⁵; Paola Otero Gómez²; María Isabel Panadero Antón²; Pedro Antonio Jiménez Gómez³; Agustín Probanza Lobo⁴; Carlos Bocos de Prada²

¹ Student. Faculty of Pharmacy, Universidad San Pablo CEU, Boadilla del Monte, Madrid, Spain; ²Biochemistry Area, Pharmaceutical and Health Sciences Department, Faculty of Pharmacy, Universidad San Pablo CEU, Boadilla del Monte, Madrid, Spain; ³Microbiology Area, Pharmaceutical and Health Sciences Department, Faculty of Pharmacy, Universidad San Pablo CEU, Boadilla del Monte, Madrid, Spain; ⁴Plant physiology Area, Pharmaceutical and Health Sciences Department, Faculty of Pharmacy, Universidad San Pablo CEU, Boadilla del Monte, Madrid, Spain; ⁵ Without ascription

Correspondence: Eduardo Gomez Casales (e.gomez53@usp.ceu.es)

Fructose consumption has increased considerably in the last decades associated with its incorporation in sugary drinks and processed foods [1]. This increase in consumption has been related to cardiovascular diseases, obesity and diabetes [2]. Recently, it has been shown that the gut microbiota is directly related to diet. This link affects both the functional activity and the composition of the gut microbiota [3]. Thus, the consumption of this sugar could affect the expression and profile of antibiotic resistance of said microbiota. This fact evidences the growing scientific interest in knowing those factors that may negatively affect the increase in antibiotic resistance, among which could potentially include the effect of this sweetener on the gut microbiota and its consequences.

The contribution of the present work consists of the biotechnological adaptation of techniques traditionally used for the evaluation of antibiograms of clinical isolates (phenotypic expression of population resistance) for the study of the behavior of the intestinal microbiota against antibiotics the coeno-antibiogram [4], insofar as it concerns the study of the phenotypic expression of the antibiotic resistance of the bacterial community as a whole (“coeno-“ from greek κοινός koinós 'common'). In this study, male Sprague-Dawley rats were used from mothers who received water during pregnancy (CONTROL) or mothers who consumed fructose in the drinking water (FRUCTOSA) who, after reaching adulthood, consumed fructose for 21 days (CF and FF), fructose with salt (components of the Western diet) (CX and FX) or water (CC and FC) together with a standard solid diet. The coeno-antibiogram (Epsilon test© and Vitek©) was studied in the feces of these animals. For the treatment of the data the Random Forest and Wilcoxon statistics were used.

The results of the gut microbial communities show an increase of Minimum Inhibitory Concentration (MIC) according to the following model: (CF) against piperacillin and amoxicillin; (FF) against cefuroxime and imipenem and (FX) against amoxicillin. Increases that, however, are not observed in the other groups (CC and CX).

References

1. Tappy, Luc y Lê, Kim-Anne Lê. “Health effects of fructose and fructose-containing caloric sweeteners: where do we stand 10 years after the initial whistle blowings?.” Current diabetes reports vol. 15, 2015

2. Riveros, María Jesús, Parada, Alejandra y Pettinelli, Paulina. "Consumo De Fructosa y Sus Implicaciones Para La Salud: Malabsorción De Fructosa e Hígado Graso no Alcohólico." Nutrición Hospitalaria 29.3, 2014

3. Astbury, Stuart et al., “High Fructose Intake During Pregnancy in Rats Influences the Maternal Microbiome and Gut Development in the Offspring.” Frontiers in genetics vol. 9, 2018

4. Robas Mora et al., “Effect of the Type of Vitis vinifera Cultivation in the Cenophenoresistome and Metabolic Profiling (CLPP) of Edaphic Bacterial Communities” Journal of Agricultural Science and Technology A 7 (8), 2017

Funding: Research support service (SAI-microbiología USPCEU)

Jorge Guío¹, Cristina Sarasa¹, Adrián Velázquez-Campoy^1,2,3, M. Teresa Bes¹, M. Luisa Peleato¹, María F. Fillat¹ and Emma Sevilla ¹

¹ Department of Biochemistry and Molecular and Cell Biology and Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza, Spain; ² Aragon Institute for Health Research (IIS Aragon), Zaragoza, Spain; ³ CIBER of Hepatic and Digestive Diseases (CIBERehd), Madrid, Spain; ARAID Fundation, Government of Aragon, Zaragoza, Spain

Correspondence: Jorge Guío (jguiomartinez@gmail.com)

FurA (Ferric Uptake Regulator) from the cyanobacteria Anabaena sp. PCC 7120 is a transcriptional regulator that controls not only iron homeostasis but also other important cellular processes such as oxidative stress response, nitrogen metabolism or cell morphology [1]. Furthermore FurA has been proposed to act as a redox sensor protein, due to its disulfide reductase activity and its ability to interact with heme [2]. 2-oxoglutarate (2-OG), a metabolite produced in the Krebs cycle, acts as a signal of carbon/nitrogen balance and, in cyanobacteria, it modulates DNA-binding activity of the key regulator for nitrogen metabolism NtcA [3]. As previous studies showed that FurA was able to control the transcription of ntcA and hetR genes [4] we wondered if this transcriptional regulator could also be sensing carbon/nitrogen balance via 2-OG. Isothermal Titration Calorimetry (ITC) assays proved that FurA was able to bind to 2-OG and Electrophoretic Mobility Shift Assays (EMSA) showed that FurA binding activity to the ntcA gene promoter region was modulated by this metabolite. In order to predict its binding site, a model of FurA tridimensional structure was built and docked with 2-OG, revealing that the potential binding site of this molecule was close to DNA binding domain and contained basic aminoacids. Multiple sequence alignments showed that these residues were present in Fur proteins from other cyanobacteria species, such as M. aeruginosa or N. spumigena, and docking simulations predicted that 2-OG was also able to bind to these structures. Taken together, these results suggest a putative role of Fur proteins as sensors of carbon/nitrogen balance in cyanobacteria.

References

1. González A, Angarica V, Sancho J, Fillat M. The FurA regulon in Anabaena sp. PCC 7120: in silico prediction and experimental validation of novel target genes. Nucleic Acids Research. 2014;42(8):4833-4846

2. Fillat M. The FUR (ferric uptake regulator) superfamily: Diversity and versatility of key transcriptional regulators. Archives of Biochemistry and Biophysics. 2014;546:41-52

3. Laurent S, Chen H, Bedu S, Ziarelli F, Peng L, Zhang C. Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proceedings of the National Academy of Sciences. 2005;102(28):9907-9912

4. González A, Valladares A, Peleato M, Fillat M. FurA influences heterocyst differentiation in Anabaena sp. PCC 7120. FEBS Letters. 2013;587(16):2682-2690

Funding: This work was supported by grants E35_17R from Gobierno de Aragón and BFU2016-77671-P/FEDER from MINECO

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13th Annual Biotechnology Congress (BAC Madrid 2019): abstract collection. BMC Biotechnol 19, 78 (2019). https://doi.org/10.1186/s12896-019-0569-8

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• Published: 04 December 2019

• DOI: https://doi.org/10.1186/s12896-019-0569-8

中文翻译：

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第十三届年度生物技术大会（BAC Madrid 2019）：摘要收藏：西班牙马德里。2019年7月10日至12日。

介绍：

Aitor Balmaseda和Pablo Revilla

西班牙生物技术专家联合会，贝加纳校区，电话：24071莱昂

今年7月10日至12日，西班牙生物技术专家联合会（https://febiotec.es/）组织了第13版的年度生物技术大会（BAC）。

该活动在马德里举行，使年轻的学生和专业人士可以与其他学生和高级专业人士（部分观众）分享他们的成果和知识，这是BAC Madrid 2019的目的。该网络的建立。来自西班牙各地的生物技术专家可以见面要了解最新趋势，不仅要感谢学术界研究人员的演讲，还要感谢商业生物技术。参与者还可以通过口头交流和海报与观众分享他们的成果。

可以在下面的BAC Madrid中找到这些通讯的摘要样本。我们期待在2020年7月在萨拉戈萨举行的下一届国会代表大会上见到您。

卢卡斯·达尔·马格罗^1,2，雅库布·F·科内基^1,4，曼努埃拉·克莱因³，拉斐尔·C·罗德里格斯²，罗伯托·费尔南德斯·拉富恩特¹

¹ ICP-CSIC生物催化系，坎塔布兰科UAM-CSIC校园，ZC 28049，西班牙马德里；²南里约热内卢联邦大学食品科学技术学院生物技术，生物过程和生物催化小组。BentoGonçalves，9500，PO Box 15090，ZC 91501-970，Porto Alegre，RS，Brazil; ³巴西阿雷格里港联邦卫生科学大学营养系（UFCSPA），巴西RS阿雷格里港ZC 90050-170；⁴ Orion High Technologies CalleTurín15，Nave 3，28983 Parla，Madrid

通讯： Jakub F. Kornecki（yakokornecki@hotmail.es）

聚半乳糖醛酸酶（PGs）是一种作用于果胶酸的酶，可消除两个未酯化的半乳糖醛酸残基之间的α-1,4键。来自真菌的PG具有很高的活性，它们在弱酸性pH值和30°C至50°C的最佳温度下表现最佳。在果汁生产过程中添加PG的重点在于果胶和其他大分子的降解，降低其粘度并为果汁提供清晰的外观，除了增加果汁产量并减少所需的过滤时间。黑曲霉的PG使用三种不同的载体固定乙二醛：乙醛基，乙烯基砜和戊二醛活化的氨基载体。使用戊二醛预活化的载体效果最佳。将PG固定在pH 5下进行24小时，然后在pH 5、6.5和8下进行3小时，然后将它们切换至pH 8以完成24小时。另一种方案是使用pH 8加入300 mM NaCl来防止酶和支持物之间的离子交换。在从4到10的pH值进行的应力灭活实验中，所有条件下的固定化都会显着提高热稳定性。这使得在温度超过70°C或pH值超过7的情况下，生物催化剂可以保持显着的活性水平，游离酶完全没有活性。固定条件是酶活性的关键，热稳定性和操作稳定性，使我们认为应用了不同的条件，使PG在固定时具有不同的方向。对每种生物催化剂性能的关注取决于最大价值的参数（活性或稳定性）和反应过程中使用的条件。最佳PG固定化生物催化剂可以重复使用多达十次，而不会显着降低酶的活性，并且提供了非常线性的反应过程。

资金：这项工作得到了来自Capes，CNPq（流程403505 / 2013-5）和FAPERGS（流程17 / 2551-0000939-8）的赠款和奖学金（L. Dal Magro）的支持。我们也衷心感谢马德里自治大学（项目编号IND2017 / IND-7640）和西班牙政府MICIU（项目编号CTQ2017-86170-R）的经济支持。作者要感谢Amazon集团和LNF Latinoamericana慷慨地提供了这项研究中使用的酶。

Laia Bosch-Molist，Arnau Boix-Besora，LauraCervera-Gràcia，Francesc Gdiadia-Casablancas

巴塞罗那自治大学（UAB）

通讯： Laia Bosch-Molist（laiaboschm@gmail.com）

病毒样颗粒（VLP）是模仿病毒自然结构的纳米结构[1]。它们基于结构病毒蛋白自组装成颗粒的内在能力。由于它们具有重复性亚基，并且不包含病毒遗传物质，因此它们能够产生强大的细胞和体液免疫反应，从而使其成为良好的候选疫苗[2]。HIV-1 VLP基于多蛋白Gag，重组表达时可形成球形结构。

在这项工作中，哺乳动物细胞平台是此类复杂且被包裹的VLP的选定系统。这种方法允许将准确的翻译后修饰结合到VLP中，这对疫苗效力非常重要。可以通过瞬时基因表达（TGE）或稳定基因表达（SGE）实现HEK293培养物中重组Gag VLP的生产[3]。在TGE中，由于每个细胞分裂中的稀释，目的基因的表达随着时间的流逝而丢失，而SGE通过将目的基因直接整合到基因组中来实现组成型基因表达。CRISPR / Cas9系统引入了靶向双链断裂（DSB），如果使用DNA模板，则可通过同源性定向修复（HDR）进行修复[4]。在这里，

参考文献

[1] N. Kushnir，SJ Streatfield和V. Yusibov，“病毒样颗粒作为高效疫苗平台：靶标和生产系统的多样性以及临床开发的进展”，《疫苗》，第1期。31号 1，第58-83页，2012年。

[2] WARodríguez-Limas，K。Sekar和KEJ Tyo，“病毒样颗粒：作为疫苗开发平台的微生物工厂和无细胞系统的未来，” Curr。in 生物技术。，卷 24号 6，第1089–1093页，2013年。

[3] L. Cervera，S。Gutiérrez-Granados，NS Berrow，MM Segura和F.Gòdia，“通过培养基交换和重复瞬时转染来扩展基因表达，以提高重组蛋白的产量，” Biotechnol。生恩 ，卷 112号 5，第934–946页，2015年。

[4] D. Paquet等。，“使用CRISPR / Cas9有效引入特定的纯合和杂合突变”，《自然》，第 533号 7601，2016年。

González-Gómez，R，Ruiz-López，E.，Torres-Herrero，B.，Schuhmacher，AJ

阿拉贡健康研究所（IISAragón）

通讯： González-Gómez，R.（ruthgo1994@gmail.com）

尽管癌症患者的存活率高于过去的几十年，但仍需要新的诊断工具来适当地检测肿瘤。ImmunoPET是一种视觉技术，其中使用抗体特异性检测在肿瘤中表达的蛋白质，从而可以以非侵入性方式进行有效诊断[1]。

该技术的最佳候选者是在正常组织和肿瘤组织之间差异表达的蛋白质。通过生物信息学分析，ABCC3被证明是多形性胶质母细胞瘤（GBM）中免疫PET的良好靶标，患者数据显示，高水平的ABCC3可使存活率降低。

ABCC3是MRP（多药抗性蛋白）亚家族的成员，属于ATP结合盒（ABC）转运蛋白，它可能在肝脏和肠道中的有机阴离子排泄中起作用[2]。

我们旨在确定ABCC3表达与化疗反应之间的相关性。我们的研究始于具有高ABCC3表达水平的细胞系，我们产生了ABCC3敲除细胞，与野生型细胞相比，它们对顺铂和替莫唑胺的治疗更为敏感。GBM细胞系以ABCC3的mRNA和蛋白水平表征，显示出不同的表达特征。

已开发出GBM细胞系的ABCC3过表达和敲除细胞，表明体外缺乏ABCC3会导致细胞对化学治疗药物敏感，同样，高蛋白水平也会赋予药物抗药性。

O6-苄基鸟嘌呤（O6-BG）是O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的有效抑制剂。当抑制MGMT时，细胞对替莫唑胺给药更加敏感[3]。顺铂或替莫唑胺与O6-BG的联合治疗没有协同作用，但是当敲除ABCC3时，MGMT水平发生了改变。

进一步研究ABCC3和MGMT的关系以改善治疗将是有趣的。ABCC3可能是免疫PET改善诊断并确定不同组织化学反应的理想靶标。

参考文献

1. Bailly C，CléryPF，Faivre-Chauvet A，Bourgeois M，GuérardF，Haddad F，Barbet J，ChérelM，Kraeber-BodéréF，Carlier T，Bodet-Milin C.用于临床治疗方法的Immuno-PET。内部分子科学学报。2016年12月28日; 18（1）：57。

2. V. Vasiliou，K。Vasiliou和DW Nebert。（2009）。人ATP结合盒（ABC）转运蛋白家族。人类基因组学3（3）：281-290

3. Cai，W.，内华达州的Maldonado，Cui，W.，北卡罗来纳州Harutyunyan，Ji，L.，R。Sposto，Reynolds，CP。和Keshelava，N.（2010）。在成神经细胞瘤临床前模型中，在存在O ^6-甲基鸟嘌呤-DNA甲基转移酶抑制的情况下，伊立替康和替莫唑胺的活性。英国癌症杂志103（9）：1369-1379。

资金来源：FERO基金会，ASPANOA，卡洛斯三世健康研究所，Ramóny Cajal计划。

爱德华多·鲁伊斯·洛佩斯（EduardoRuiz-López），露丝·冈萨雷斯（RuthGonzález-Gómez），比阿特丽斯·托雷斯（Beatriz Torres-Herrero），阿尔贝托·J·舒马赫（Alberto J Schuhmacher）

西班牙萨拉戈萨分子肿瘤小组Aragon健康研究所（IIS ARAGON）

通讯： EduardoRuiz-López（educalata21@gmail.com）

胶质母细胞瘤（GBM）是最普遍和最具侵略性的脑肿瘤。GBM诊断的当前技术-磁共振成像-仍不准确，分子成像方法正在成为非侵入性工具。使用高特异性成像探针靶向GBM可以进行非侵入性诊断，治疗分层和监测患者。高表达的GBM生物标志物已允许设计单克隆抗体进行分子成像。但是，它们的大尺寸阻碍了通过血脑屏障（BBB）的渗透和扩散到肿瘤中。正在开发针对GBM的基于蛋白质支架的较小探针[目标]：纳米抗体[ HGF ]，亲和力[ PDGFRβ，EGFR，IGF-1R，VEGFR2 ]，结蛋白[磷脂酰肌苷，MMP-2，整联蛋白αvβ3，αvβ5，anticalins [ HGF，VEGF-A ]，adnectins [ VEGFR ]和DARPins [ HGF ]。与单克隆抗体相比，它们的较低分子量可实现更好的肿瘤吸收和更快的清除率。已经在临床前异种移植和原位小鼠模型上进行了成功的分子成像。

我们已经通过生物信息学确定了分子靶标，用于GBM分子成像探针的开发。我们正在按照两种策略开发针对这些候选物的基于纳米抗体的靶向剂。首先，我们正在积极地用靶标过表达的神经胶质瘤细胞系对单峰骆驼进行免疫，以克隆骆驼科重链可变域的靶标富集库。我们将生成表达纳米抗体的细菌文库或纳米抗体nanothèque。表达高亲和力纳米抗体的细菌将通过大肠杆菌进行选择显示。此外，我们正在通过噬菌体展示从基因库中分离出针对GBM靶标的纳米抗体。选定的纳米抗体探针将用于异种移植和“化身”小鼠模型的分子成像。为了进行临床开发，我们将纳米抗体与BBB分子穿梭分子（ApoE，FC5纳米抗体）融合在一起，以通过转胞吞作用改善大脑的通行性。考虑了多靶点纳米抗体，双重标记策略和放射性核素治疗学方法。将来，患者的遗传特征可能会允许分子个性化免疫靶向和GBM的当日成像。

资金来源：西班牙社会发展协会（AECC），拉蒙·卡哈尔计划（RyC）和Oncológica研究基金（FERO）。

卡拉米歇尔帕拉达Sosa的^1,2，罗莎卡门罗沙Gracia的³，莱纳·马丁内斯卡尼亚梅罗⁴，安东尼奥科博诺斯⁵和再打莉雷斯¹

¹ Facultad deBiotecnología。Decanato de CienciasBiológicas，普埃布拉大学人民大学，1103年1月21日，圣地亚哥科洛尼亚，72410普埃布拉，墨西哥普埃布拉；²哈恩大学，法国科学研究院，23071-哈恩，西班牙；³ ICUAP微生物研究中心，普埃布拉自治省贝内梅里塔大学。Cencias Complejo de Ciencias，第103 J. Ciudad Universitaria建筑，科洛尼亚圣曼努埃尔，墨西哥72570普埃布拉；⁴德拉萨洛德·德·西恩西亚斯·德拉·萨洛德分院微生物研究所，哈恩大学，法国科学研究院，23071-Jaén，西班牙；⁵格拉纳达大学西恩西亚斯分校微生物系，18071-格拉纳达，西班牙

通讯：卡拉·米歇尔·帕拉达·索萨（carlamichelle.parada@upaep.edu.mx）

抗菌素耐药性（AMR）传播是一个令人震惊的全球健康问题。细菌感染不仅与医院地区有关，而且在食品领域也是一个问题。对抗AMR的策略是加强对替代抗生素的研究[1]。

草药提取物及其次生代谢产物是治疗感染或保存食物的一种选择[2]。

在这项研究中，使用体外针对革兰氏阳性和革兰氏阴性菌株的药敏试验对草药提取物的抗菌活性进行了评估，以提出活性样品作为抗菌剂。提取物A. ludoviciana，向日葵变种。Autumn Beauty和Lycopodium clavatum对革兰氏阳性和革兰氏阴性菌株均表现出抗菌性能。草药提取物的这一特性可以替代天然抗菌剂，以用于将来研发用于治疗医院感染，食物中毒或用作食品防腐剂的新产品。

参考文献

1. Blair JMA，Webber MA，Baylay AJ，Ogbolu DO，Piddock LJV。抗生素耐药性的分子机制。Nat Rev Microbiol。2015; 13（1）：42–51。

2. Yang S，Low L，Yap PS，Yusoff K，Mai C，Lai K等。植物衍生的抗菌剂：减轻抗菌素耐药性的见解。Rec Nat Prod。2018; 4：1-22。

哈维尔·奎罗（Javier Quero）¹，伊内斯·马尔莫（InésMármol）¹，拉奎尔·伊巴尔兹（Raquel Ibarz）²，奥尔加·马丁·贝洛索（OlgaMartín-Belloso）²和穆·耶苏斯·罗德里格斯（MYJesúsRodríguez-Yoldi）¹

¹西班牙萨拉戈萨萨拉戈萨大学兽医学院药理和生理学系；²莱里达大学食品技术系，西班牙Agrotecnio中心

通讯： Javier Quero（javierquero94@gmail.com）

肠粘膜屏障上的氧化应激与多种慢性疾病（如炎症性肠病和结直肠癌（CRC））的发展密切相关。这种外源性氧化应激可以由多种外部因素触发，例如饮食，药物，吸烟或酒精中毒，这些因素会增加肠道组织中的活性氧（ROS）和活性氮（RNS）[1,2]。

水果和蔬菜的抗氧化剂潜力众所周知，因为它们在酚类化合物和其他生物活性化合物中的组成[3]，也可以从森林和农业工业副产品中获得。

我们已经分析了农业食品工业废料在氧化应激诱导下作为粘膜屏障保护剂的潜在用途。另外，已经评价了麦芽糖糊精包囊的影响。选择的细胞肠屏障模型是融合的80％融合的Caco-2细胞，将其暴露于10 mM H ₂ O ₂中20分钟。

结果表明，农业食品副产物在外源性氧化应激诱导下作为肠屏障保护剂的潜在作用。因此，食物残渣可能对制药业的肠屏障氧化应激相关疾病的治疗很感兴趣，从而使森林和农业食品业产生的大量废物获得第二生命。

参考文献

[1] Reuter，S。等。氧化应激，炎症和癌症：它们如何联系？Free Radic Biol Med（2010）49（11），1603-1616。

[2] Perse，M.大肠癌发病机理中的氧化应激：原因还是后果？生物医学研究杂志（2013）2013，725710-725719。

[3] Tsao，R.膳食多酚营养素的化学和生物化学（2010）2，1231-1246。

资金来源：部长级公民创新（SAF2016-75441-R），戈比诺·德拉贡/欧洲区域发展基金（ERDF）（B16-R17）和Interreg Sudoe REDVALUE（SOE1 / PI / E0123）

Nathalia萨赖瓦Rios的^1,4，达维诺M.安德拉德内托²，何克莱顿Sousa的多斯桑托斯³，皮尔巴西尔奥·阿尔梅达Fechine ²，罗伯特·费尔南德斯Lafuente的⁴，卢西亚娜罗沙巴罗斯贡萨尔维斯¹

¹巴西联邦联邦塞纳大学，德比西校区Engenharia昆米卡分校，CEP 60455-760，CE，福尔萨萨，巴西；²锡米西亚联邦中央大学，西米西亚联邦财政部，塞阿里亚联邦大学，大道2。赫尔先生（Spicen），皮奇（Pipic），60455-760，福塔莱萨（Fortaleza），CECP12200，巴西; ³巴西国际大学联盟非洲可持续发展研究所，可持续发展研究所，62785000巴西巴西阿卡拉佩；⁴生物降解部门。ICP-CSIC，校园UAM-CSIC，西班牙马德里

通讯： Nathalia Saraiva Rios（nathaliarios25@yahoo.com.br）

脂肪酶因其高活性和高化学选择性，区域选择性和对映体选择性而被广泛用于食品工业，化学和制药工艺[1]。因此，酶的固定化是常用的，因为它使反应介质的分离和生物催化剂的再利用变得容易[2]。在这种情况下，一种新型的NiZnFe ₂ O ₄超顺磁性纳米颗粒被用作固定荧光假单胞菌（PFL）中脂肪酶的载体。为此目的，通过用四乙氧基硅烷（TEOS）浸渍将该纳米颗粒涂覆二氧化硅，并进一步用p活化。-苯醌（BQ）和二乙烯基砜（DVS），以生产共价固定的生物催化剂。BQ和DVS是双功能分子，可以与酶的不同部分（胺，羟基，硫醇等）反应，生成高度稳定的生物催化剂[3,4]。就我们所知，尚未对BQ进行适当的研究以通过多点共价连接稳定酶。因此，与有希望的支持活化剂DVS相比，我们使用该活化剂固定PFL。PFL也固定在TEOS包覆的NiZnFe ₂ O ₄纳米颗粒上，没有任何活化作用，从而产生吸附的固定化生物催化剂。BQ生物催化剂显示出最活跃，最稳定的制备方法（回收率：89％，t _1/2（在1440分钟内达到60°C），而DVS共价和吸附的制剂在60°C下的回收率分别为82％和55％，半衰期分别为225和150分钟。因此，本研究显示了成功的固定化策略，可产生活性和稳定的生物催化剂。

参考文献

[1] D. Sharma，B。Sharma，AK Shukla，微生物脂肪酶的生物技术方法：综述，生物技术。10（2011）23-40。

[2] RA Sheldon，S。van Pelt，生物催化中的酶固定化：为什么，什么和如何。Soc。修订版42（2013）6223-6235。

[3] JCS dos Santos，N。Rueda，O。Barbosa，JFFernández-Sánchez，AL Medina-Castillo，T。Ramón-Márquez，MC Arias-Martos，MCMillán-Linares，J。Pedroche，M。del M. Yust，LRBGonçalves，R。Fernandez-Lafuente，用二乙烯基砜活化的支持物的表征，该工具通过多点共价连接固定和稳定酶。胰凝乳蛋白酶的应用，RSC Adv。5（2015）20639-20649。

[4] NS Rios，DM Andrade Neto，JCS dos Santos，PBA Fechine，R。Fernández-lafuente，LRBGonçalves，将荧光假单胞菌中的脂肪酶固定在二乙烯基砜或对苯二酚活化载体上的比较，Int。J.Biol。大分子 134（2019）936–945。

资金来源：Nathalia S. Rios感谢CNPq的博士前研究金（CNPq奖学金–巴西）。西班牙政府的MICIU（项目号CTQ2017-86170-R）。

仪埃斯特¹，娟佩德罗纳瓦罗¹，帕特里夏加兰圣马洛²，路易斯马塔²，Mª多洛雷斯佩雷斯¹，路德桑切斯¹

^1个阿雅德Producción动物ý西恩西亚洛斯ALIMENTOS。Facultad de Veterinaria。西班牙萨拉戈萨市阿拉贡农业研究所（IA2）（萨拉戈萨-CITA大学）；² ZEULAB SL，西班牙萨拉戈萨

通讯： Miriam Esteban（miriames93@gmail.com）

酪丁酸梭状芽胞杆菌是导致奶酪起泡缺陷问题的主要因素，这是由于其熟化过程中休眠孢子的发芽所致[1]。由于产生的气体施加的压力，许多受影响的奶酪会出现空腔和裂纹，这在大多数情况下会导致产品损失。

我们的目标是开发一种快速的方法来检测牛奶中的孢子，而不是非特异性且费时的传统微生物。因此，我们开发了一种基于以前用枯草杆菌蛋白酶消化牛奶的方法来回收孢子的方法。对于实时PCR，我们测试了破坏孢子的不同方法，由于其抗性结构，这是一个挑战。已经发现，微波处理后进行标准的DNA纯化是最有效的破坏程序。我们能够检测到10 ¹孢子/ mL至10 ⁷孢子/ mL，但由于DNA回收率低以及SASPs蛋白对DNA包装的影响，我们无法区分数量[2]。

另一方面，我们通过流式细胞仪检测酪丁酸梭菌的孢子，因为它具有不破坏孢子的优点。使用高亲和力的配体作为动物中产生的抗体的替代品进行检测。该配体先前由Phage Display开发，并通过等温滴定热法（ITC）分析了其对丁酸孢子的亲和力。这些结果表明酪丁酸梭菌孢子具有良好的结合力，但其他梭菌也具有良好的结合力。使用特定配体的流式细胞仪显示了良好的结果，检测极限为10 ³孢子/ mL，可以代替实时PCR以避免从孢子中回收DNA的问题。

参考文献

[1] Bassi，D.，E。Puglisi，E.，Cocconcelli，PS，2015年。了解带有吹气缺陷的硬质奶酪的细菌群落。食品微生物。52，106–118。https://doi.org/10.1016/j.fm.2015.07.004

[2] Raju，D.，Waters，M.，Setlow，P.，Sarker，MR，2006。研究小型，酸溶性孢子蛋白（SASP）在产气荚膜梭菌孢子对热的抵抗力中的作用。BMC微生物。13

资金来源：阿拉贡政府（西班牙）和欧洲社会基金，获得了DGA博士前资助，并由西班牙政府的部长Ciencia eInnovación资助了AGL2013-44130-R项目。

玛尔塔佩雷斯迪亚兹¹，埃琳娜G. Biosca ²，贝伦阿尔瓦雷斯¹

¹ IMIDRA，A2公里38.200，28800Alcaláde Henares，马德里，西班牙；²瓦伦西亚大学，阿夫达分校。Moliner 50，46100 Burjasot，巴伦西亚，西班牙

通讯： BelénÁlvarez（mariabelen.alvarez@madrid.org）

大规模生产环境微生物以利用其生物技术应用在农业食品工业中可能引起巨大兴趣。具有生物技术用途的微生物的活动是产生具有多种功能的水解外酶，向作为生物肥料的植物提供必需营养素的能力，以及它们对病原微生物的拮抗作用[1,2]。其应用的有益结果可以有助于实现基于微生物的可持续农业[3]。

从西班牙大陆橄榄树林的农作物中分离出土壤和根际细菌的集合。鉴定并测试了它们的全部潜力，可产生与有机化合物的降解和循环利用相关的外切酶，它们溶解磷酸盐，固定大气中的氮和捕获铁的能力，以及它们对相关植物病原真菌（如大黄萎病菌，Phaeomoniella）的拮抗作用衣原体，拟密线虫和镰刀菌。

分离物的分子鉴定揭示了大部分的芽孢杆菌属，还有Massilia属，短杆菌属，泛菌属，假单胞菌属，肠球菌属。和Chryseobacterium spp。作为目前的主要属。蛋白酶，脂肪酶，淀粉酶和DNase等外酶的产生在96％的分离物中呈阳性，而92％的磷酸盐溶解，固氮和/或铁吸收呈阳性，而41％显示出对至少一种有效的拮抗作用。被测试的真菌病原体。大丽花33％的分离物抑制了细菌的生长，其中之一还通过挥发性有机化合物（VOC）的产生而受到抑制。针对检测到的抑制活性P. chlamydospora在分离株和18％D. macrodidyma在4％，不产生VOC的，但不是针对F. avenaceum。在橄榄林中，农作物的根际土壤和根际细菌中观察到的高活性表明，在生物技术产业中[4]可以产生大量的外源酶，养分循环利用，减少农用化学品和/或生物防治潜力。

参考文献

¹ Abada E，Al-Faifi Z，Osman M.，2017年。微生物产生的酶和纳米颗粒及其在生物技术中的应用。在：真菌纳米技术，真菌生物学。普拉萨德（Ed）。施普林格国际出版社。Doi：10.1007 / 978-3-319-68424-6_7。

² Parnell JJ，Berka R，Young HA，Sturino JM，Kang Y，Barnhart DM，DiLeo MV。2016。从实验室到农场：植物有益微生物的工业视角。面前。植物科学 7：1110。Doi：10.3389 / fpls.2016.01110。

³ Umesha S，辛格PK，辛格RP。2018.微生物生物技术与可持续农业。在：生物技术促进可持续农业。新兴的方法和策略。第185-205页。爱思唯尔。

⁴ Prasad R，Gill SS，Tuteja N（编辑）。2018年。微生物生物技术和生物工程学的新的和未来的发展。通过微生物生物技术改善作物。爱思唯尔。Doi：10.1016 / C2016-0-04330-9。

资金：这项工作由AGRISOST S2013 / ABI-2717项目和BACPLANT-UVEG-研究支持提供资金

萨拉阿拉纳-尼亚¹，尤丽娅Lokha ¹，Nathalia S. Rios的^1,2，卡门Méndez-桑切斯¹和Roberto费尔南德斯- Lafuente的¹

¹西班牙加泰罗尼亚石油化学研究所（ICP-CSIC）生物催化部门，UAM-CSIC园区，西班牙马德里28049；²巴西联邦联邦大学塞纳分校的EngenhariaQuímica部门，CEP 60455-760，CE，福塔雷萨，巴西

通讯： SaraArana-Peña（s.arana@csic.es）

脂肪酶由于其独特的特性而成为最常用的酶之一，并且可以在所有生物中发现。如果需要生物催化剂的回收和再利用，以及改善其性能，固定化脂肪酶是必不可少的。以低离子强度将脂肪酶固定在疏水性支持物上，可以通过界面活化作用一步固定，锁定脂肪酶开放形式，并使其具有较高的稳定性[1]。可以预期的是，固定化条件会对脂肪酶的构象产生重大影响。实际上，来自嗜热单胞菌的脂肪酶（TLL）被描述为对固定条件的改变很敏感[2]。因此，在这项工作中，我们打算研究在不同条件下（例如pH和某些离子的存在）将该酶可逆地固定在疏水性载体上是否会对它们的最终稳定性和催化活性产生影响。磷酸根离子已被证明会降低许多脂肪酶的稳定性[3]，另一方面，某些阳离子（例如Ca2 +）可能会增加酶的稳定性，但大多数时候是固定在疏水性支持物上的[4]。已选择辛基-琼脂糖珠作为载体，因为它们被广泛使用并且琼脂糖具有一些良好的性能。进行测定后，我们确认使用一种脂肪酶和相同的支持物的固定条件可以显着改变TLL的最终催化活性和稳定性。

参考文献

[1] JM Palomo，G。Muñoz，G。Fernández-Lorente，C。Mateo，R。Fernández-Lafuente，J。Guisan，脂酶在非常疏水的载体上的界面吸附（十八烷基-Sepabeads）：固定化，超活化和稳定化脂肪酶的开放形式，J。Mol。卡塔尔。B酶。19（2002）279–286。doi：10.1016 / S1381-1177（02）00178-9。

[2] E. Abreu Silveira，S。Moreno-Perez，A。Basso，S。Serban，R。Pestana Mamede，PW Tardioli，C。Sanchez Farinas，J。Rocha-Martin，G。Fernandez-Lorente，JM Guisan，通过生物催化剂工程对油在完全无水的培养基BMC Biotechnol中进行乙醇裂解的生物催化剂工程来调节嗜热单胞菌脂肪酶的区域选择性。17（2017）88. doi：10.1186 / s12896-017-0407-9。

[3] S.Arana-Peña，Y。Lokha，R。Fernández-Lafuente，将Eversa脂肪酶固定在辛基琼脂糖微珠上和稳定性和活性特征的初步表征，催化剂。8（2018）511. doi：10.3390 / catal8110511。

[4] L. Fernandez-Lopez，R。Bartolome-Cabrero，MD Rodriguez，CS Dos Santos，N。Rueda，R。Fernandez-Lafuente，阳离子对脂肪酶的稳定作用取决于固定化方案，RSC Adv。5（2015）83868–83875。doi：10.1039 / C5RA18344H。

资金：西班牙政府的MICIU（项目号CTQ2017-86170-R）

El-Hocine Siar ^1,2，萨拉·阿拉纳-佩尼亚¹，罗伯托·莫雷伦-斯特林¹。Jakub F. Kornecki ¹，穆罕默德·纳斯雷丁·齐多恩²，奥韦马尔·巴尔博萨³，罗伯托·费尔南德斯·拉富恩特¹

1.西班牙天主教大学CSIC研究所生物催化部门，西班牙马德里28049；2. Equipe TEPA，Inventa LNTA实验室，门托里大学弗朗斯·门托里·康斯坦丁1号，阿尔及利亚25000年康斯坦丁；3.托利马大学，西恩西亚斯大学，昆米卡分校，730006299哥伦比亚伊巴圭

通讯： El-Hocine Siar（hocines1@hotmail.fr）

酶由于在绿色化学中的特异性和选择性，在现代生物技术中是非常有用的工具。但是某些属性限制了它们的使用（抑制，特异性和重复使用的可能性）。游离或固定化酶的固定化和化学修饰是克服这些限制的最提出的解决方案之一。在这项工作中，我们研究了使用碳二亚胺和乙二胺的胺化作用对乙醛酸琼脂糖和游离丝蛋白固定化/稳定化丝蛋白的性能的影响。首先将游离的丝氨酸胺化，然后固定在乙醛酸琼脂糖上。因此，直接固定在乙醛酸琼脂糖上的固定蛋白。丝蛋白活性与酪蛋白和BANA（苯甲酰精氨酸-p测定（硝基硝基苯胺）并检查在不同pH（5、7和9）下的稳定性。胺化的免疫蛋白的活性发生了改变（酪蛋白和BANA相对），而酪蛋白的活性略有增加，而酪蛋白的活性则下降。据报道在pH 9时发生了heist效应，其中酪蛋白酶含量增加了10％以上，而与BANA相比活性降低了5倍以上。因此大大改变了酶的特异性。胺化提高了pH值为5时酶的稳定性，而pH值为9时稳定性却很差。另一方面，游离胺化的丝状蛋白相对于BANA保留了约80％的活性，相对于酪蛋白保留了90％的活性。优化固定方案后，将新的生物催化剂与使用非胺化酶获得的生物催化剂进行比较。相对于BANA的活性降低了，但是相对于酪蛋白却增加了。胺化的非生物素蛋白生物催化剂主要在pH值为7时比非胺化的生物素催化剂更稳定。固定化或游离酶的胺化对于制备改良的生物素的生物催化剂（主要是在蛋白水解应用中）被认为是令人感兴趣的。也可以将其（主要是胺化）视为进一步改善酶固定化的可能性。

资金来源：我们非常感谢西班牙政府MICIU的支持（项目号CTQ2017-86170-R）。El-Hocine Siar先生感谢阿尔及利亚高等教育和科学研究部的研究金（阿尔及利亚PNE国家计划特别委员会）

塔尼亚加西亚-马塞拉¹，安娜B.阿拉贡戈麦斯¹，婆婆纳露娜-格雷罗¹，Gracia的蒙特罗-Peñalvo ¹，萨拉戈麦斯- Melero ¹，铁I.加西亚-马塞拉¹，何甲冈萨雷斯-雷耶斯²，Elier帕兹-Rojas的^1个

¹ Canvax Biotech，西班牙科尔多瓦14014 ^{2 2}科尔多瓦大学细胞生物学，生理学和免疫学系，14014

通讯： TaniaGarcía-Maceira（t.garcia@canvaxbiotech.com）

酶联免疫吸附测定（ELISA）是检测医疗保健中重要蛋白质标志物的最广泛使用和最可靠的临床常规方法。已经开发了几种通过将抗体固定在微量滴定板上来提高ELISA灵敏度的方法[1]。我们已经基于乙酰化壳聚糖表面的制备开发了一种高度灵敏的ELISA策略，以改善抗体的方向。为此，我们使用了捕获抗体，该抗体与来自芽孢杆菌的几丁质酶A1的几丁质结合域（ChBD）融合WL-12 [2]。几丁质表面是通过在微量滴定板的每个孔中混合少量的壳聚糖和乙酸酐并使混合物干燥过夜而获得的[3]。克隆与ChBD融合的抗c-myc 9E10低亲和力抗体，并在CHO细胞中表达以获得抗c-myc-ChBD抗体。我们证明，与未结合的抗c-myc 9E10相比，抗c-myc-ChBD特异性结合了几丁质表面。几丁质表面用于开发夹心ELISA，以检测在大肠杆菌中克隆和表达的嵌合蛋白人c-myc-GST-IL8。比较了高结合蛋白微量滴定板和几丁质处理板的检出限和定量限。在几丁质表面上开发的ELISA程序比在标准表面上进行的ELISA灵敏度高6倍，差异有统计学意义（p <0.0001）。乙酰化的壳聚糖表面允许抗体在表面上定向，并且鉴于时间的稳定性和其制备的低成本，其可能是替代标准表面的合适方法。

参考文献

1. Welch NG，Scoble JA，Muir BW，Pigram PJ。固定化抗体的定位和表征，以改善免疫测定，生物间期，2017年; 12（2）：02D301。

2.桥本M，池上T，Seino S，大内N，深田H，杉山J，白川M，渡边T。来自圆形芽孢杆菌WL-12的几丁质酶A1的几丁质结合结构域的表达和表征。细菌学杂志。2000; 182（11）：3045-3054。

3.伯纳德议员，曹D，迈尔斯RV，莫伊尔WR。几丁质结合域标记的蛋白与乙酰化壳聚糖包被的表面紧密结合。分析生物化学。2004; 327：278-283

资金来源：参考：76359项目：360229西班牙安塔卢西亚（Junta deAndalucía）

细末加西亚-格斯¹，巴勃罗Sopena-德诺瓦莱斯²塞萨尔d维拉-多诺索³，涓V. Catret ⁴，何维森特¹，旧金山马科-门尼斯¹

¹西班牙瓦伦西亚政治大学动物科学与技术研究所，46022-西班牙瓦伦西亚；²西班牙政治大学拉菲医院核医学系，46026-西班牙巴伦西亚；³西班牙政治大学拉菲医院泌尿外科，46026-西班牙巴伦西亚；⁴ Oncovision，46022-西班牙巴伦西亚

通讯： XimoGarcía-Dominguez（ximo.garciadominguez@gmail.com）

按需生成功能性肾移植物的想法将把肾脏移植的选择范围扩展到更多患有终末期肾脏疾病的患者。胚胎肾脏（metanephroi）的移植表明，如果将这些完整的原基移植到成年宿主中，它可以成熟并吸引新的宿主血管系统，从而避免免疫反应并表现出功能特性[1]。我们的目的是使用体内肾脏闪烁显像技术以及验尸检查和组织学评估来确定已发展的后肾的排泄功能。

为此，在兔模型中，使用微创腹腔镜技术从15日龄的胎儿中显微解剖出14个肾上腺，并移植到3个非免疫抑制宿主的腹膜后脂肪中[2]。移植后三周，在平面采集图像前30分钟，使用102和3mCi的99 99m DMSA进行皮质肾闪烁显像。之后，对接受者实施安乐死并取回类器官，进行形态学和组织学评估。宿主肾脏用作对照。

肾脏闪烁显像分析表明，由移植的后肾移植出的新肾脏具有肾脏功能。与宿主肾脏相比，新肾脏的肾脏活性低4倍（分别为10000 vs 41000）。尽管如此，新肾脏比宿主肾脏小21倍（分别为0.5±0.44和11.2±0.18 g）。此外，这种肾脏活动与组织学结果一致，因为从这些新肾脏发育而来的肾小球已经成熟并且与宿主肾中存在的肾小球相似（分别为40±4 vs. 41±2个细胞/肾小球）。另外，由于新输尿管与宿主膀胱之间缺乏连接而导致的新肾脏的肾积水状态证明了新肾脏的排泄功能。因此，

资金：这项研究得到了ALCER TURIA（抗击肾脏疾病协会）的支持，以及对众筹活动（Precipita平台，FECYT）的匿名捐赠。

安德烈努涅斯冈萨雷斯^1,2，埃斯特拉费尔南德斯港^1,2，迪拉阿尔瓦雷斯-费尔南德斯¹，阿梅尔萨勒曼¹，卡洛斯多明格斯^1,3，比阿特丽马丁-费尔南德斯²，菲利普马丁内斯-牧师^1,4-

¹西班牙莱昂动物卫生和牛群发展研究所（INDEGSAL），24071；² Bianor Biotech，莱昂，24071，西班牙；³内科，外科及兽医解剖学，大学莱昂，莱昂，24071，西班牙，系⁴分子生物学，大学莱昂，莱昂，24071，西班牙系

通讯： AndreaNúñez-González（annugo95@gmail.com）

褪黑素是一种无处不在的分子，调节多种生理功能，并在男性生殖中起重要作用，直接影响睾丸激素水平和精子质量[1]。在几种动物中，精子膜褪黑激素受体的存在支持了这种分子对精子的直接作用的假说[2]。内源性褪黑激素的存在可能会影响精子的成熟，获能和存活。这项研究旨在评估褪黑激素对公牛（Bos taurus taurus）和伊比利亚马鹿（Cervus elaphus hispanicus）精子线粒体活性和活性氧（ROS）产生的直接影响）。用不同浓度的褪黑激素（1μM，10 nM，100 pM，1 pM）处理5只鹿和5只公牛的精子样本，分别附睾和射精，然后孵育（39°C，5％CO ₂）在非容性条件下（Hepes缓冲的TALP）和容性条件下（Hepes缓冲的TALP补充2 U / mL肝素）4 h。流式细胞仪分析显示，在所有条件下，在褪黑素处理后，在充能条件下，具有活性线粒体的活精子细胞比例显着增加（p <0.05）。当使用非容性条件时，除了对鹿精子进行10 pM褪黑素处理（在该物种中可能具有轻微的容性环境）外，未观察到这种效果。在两个物种中所有测试浓度下，在非容性条件下进行褪黑素处理均会降低（p <0.05）线粒体超氧化物生成量较高的精子细胞比例。在电容器条件下，在鹿精子中观察到类似的效果。我们的结果表明，即使是最低浓度的褪黑素，也可能通过与精子膜褪黑素受体相互作用来刺激信号传导途径，从而在获能期间提高了精子的存活率。ROS作为精子获能的第二信使[3]，因此其水平的调节可能会影响该过程。然而，褪黑素对精子细胞的作用在物种之间可能有很大的不同。

参考文献

1.李丙，周X。褪黑激素和雄性生殖。Clin Chim Acta。2015; 446：175–80。

2. Talpur H，Chandio I，Brohi R，Worku T，Rehman Z，Bhattarai D等。褪黑素及其受体在动物繁殖中的作用研究进展：综述。复制Domest动画。2018; 53（4）：831–49。

3. de Lamirande E，O'FlahertyC。精子活化：活性氧和激酶的作用。Biochim Biophys Acta-蛋白质组学。2008; 1784（1）：106-15。

资金：这项工作得到了MINECO（AGL2013-43328P和AGL2016-81890-REDT）的支持。

埃里克·沃尔塔·杜兰^1,2,3，奥利维亚·卡诺·加里多^1,4，纳洛阿·塞纳^1,2,3，埃克托·洛佩斯·拉古纳^1,2,3，劳拉·桑切斯·加尔西亚^1,2,3，米雷亚·佩萨罗多纳^{1， 2,3¥}，拉蒙Mangues ^3,5-，安东尼奥比利亚韦德^1,2,3，埃丝特克斯^1,2,3，Ugutz Unzueta ^3,5-

¹西班牙巴塞罗那自治大学University ofAutònomade Biotecnologia i de Biomedicina生物医学研究所，西班牙巴塞罗那08193；²巴塞罗那自治大学微生物学系，西班牙贝拉泰拉，08193，西班牙；³ CIBER deBioingeniería，生物材料与纳米医学（CIBER-BBN），C / Monforte de Lemos 3-5，西班牙马德里28029；⁴ Nanoligent SL，Edifici EUREKA，巴塞罗那自治大学，贝拉特拉，08193，西班牙；⁵西班牙圣克鲁医院圣保罗生物医学研究所和约瑟夫·卡雷拉斯研究所，西班牙08041；^¥ 现在的地址：西班牙巴塞罗那科学技术研究所生物医学研究所（IRB Barcelona），西班牙巴塞罗那08028

通讯： EricVoltà-Durán（eric.voltaduran@gmail.com）

蛋白质作为药物传递系统的使用在纳米医学中引起了人们的兴趣，尤其是在处理需要特异性相互作用并渗透到靶细胞中的靶向治疗时[1]。通过遗传融合，可以将具有多种生物活性的蛋白质合成为单链多肽。这样，仅需重组生产的独特步骤即可获得最终的模块蛋白，其中每个结构域都起决定性和独特的作用[2]。在这种情况下，阳离子肽和聚组氨酸两者结合成蛋白质结构已被证明是通用的自组装蛋白质平台[3]。典型的例子是模块蛋白T22-GFP-H6，体外和体内[4]。在这里，我们通过对T22和H6在不同位置重新排列的替代方案进行形态和功能表征，研究T22和H6在模块结构内部定位的相关性。有趣的是，我们证明T22必须放置在氨基末端以允许CXCR4 ⁺细胞结合和特异性内在化，同时将H6置于羧基末端对于促进自组装至关重要。此外，作为常规蛋白质纳米颗粒的低聚能力以协同方式增加了细胞的渗透性。综上所述，这些数据对于设计用于靶向药物递送的智能纳米级蛋白质载体具有重要意义，因为CXCR4是许多病理（例如许多类型的癌症和HIV感染）的重要归巢标记。

参考文献

[1] Casanova I，Unzueta U，Arroyo-Solera I，Cespedes MV，Villaverde A，Mangues R等。肿瘤治疗中蛋白质驱动的纳米药物。药理学最新观点2019; 47：1-7。

[2] Sanchez-Garcia L，Martin L，Mangues R，Ferrer-Miralles N，Vazquez E，VillaverdeA。微生物细胞的重组药物：2015年更新。微生物细胞工厂2016; 15：33。

[3] Rueda F，Cespedes MV，Conchillo-Sole O，Sanchez-Chardi A，Seras-Franzoso J，Cubarsi R等。智能蛋白质材料生物制造中的自下而上的指导性质量控制。先进材料2015; 27：7816-22。

[4] Unzueta U，Ferrar-Miralles N，Cedano J，Zikung X，Pesarrodona M，Saccardo P等。非淀粉样肽标签可用于仅蛋白质纳米颗粒的可调节自组装。生物材料2012; 33：8714-22。

资金：我们欠Agencia Estatal deInvestigación（AEI）和Fondo Europeo de Desarrollo Regional（FEDER）（授予BIO2016-76063-R，AEI / FEDER，UE），AV，AGAUR（2017SGR-229），AV和2017SGR -865 GRC为RM; 向AV授予CIBER-BBN（NANOPROTHER项目），向RM授予CIBER-BBN项目4NanoMets；EV的ISCIII（PI15 / 00272与FEDER共同创建）和PIE15 // 00028的ISCIII（与RM共同创建）以及RM的PI18 / 00650，以及EU COST Action CA17140。我们还欠了生物工程网络研究中心的责任，生物材料和纳米医学（CIBER-BBN），由VI-VI国家R＆D＆I计划2008-2011，Iniciativa Ingenio 2010，Consolider计划，CIBER Actions资助，并由Salud Carlos III研究所资助

爱德华多戈麦斯卡萨莱斯¹，埃琳娜福斯特阿隆索² ; 玛丽娜罗巴斯莫拉³ ; 耶稣^5节; 保拉奥特罗戈麦斯² ; 玛利亚伊莎贝尔Panadero安东² ; 佩德罗安东尼奥吉门尼斯戈麦斯³ ; 阿古斯丁Probanza Lobo ⁴ ; 卡洛斯·博科斯·德·普拉达²

^1名学生。西班牙马德里圣巴勃罗大学CEU药学院 ²西班牙马德里圣巴勃罗大学CEU药学院药物与健康科学系，生物化学领域；³西班牙马德里圣巴勃罗大学CEU药学院药物与健康科学系微生物学领域；⁴西班牙马德里圣巴勃罗大学CEU药学院药物与健康科学系植物生理学系；⁵不带归属

通讯： Eduardo Gomez Casales（e.gomez53@usp.ceu.es）

在过去的几十年中，果糖的消费量与含糖饮料和加工食品的摄入量有关[1]。消费的增加与心血管疾病，肥胖症和糖尿病有关[2]。最近，已经表明肠道菌群与饮食直接相关。这种联系会影响肠道菌群的功能活性和组成[3]。因此，该糖的消耗可影响所述微生物群的抗生素抗性的表达和分布。这一事实证明了对了解可能会对抗生素抗性增加产生负面影响的因素的兴趣日益浓厚，其中可能包括这种甜味剂对肠道菌群的影响及其后果。

目前的工作包括对传统上用于评估临床分离株的抗菌谱（群体抗性的表型表达）的技术进行生物技术改造，以研究肠道菌群对抗生素的行为-异位抗菌谱[4]，就整个细菌群落的抗生素抗性的表型表达的研究而言（来自希腊κοινόςkoinós“ common”的“ coeno-”）。在这项研究中，雄性SD大鼠从怀孕谁（期间接受水的母亲常用CONTRO L）或母亲谁在饮用水消耗果糖（FRUCTOSA）在成年后食用果糖21天（CF和FF），果糖加盐（西方饮食的成分）（CX和FX）或水（CC和FC）以及标准固体饮食。在这些动物的粪便中研究了古柯抗原图（Epsilon test©和Vitek©）。为了处理数据，使用了Random Forest和Wilcoxon统计数据。

肠道微生物群落的结果显示，根据以下模型，最低抑菌浓度（MIC）有所提高：（CF）对抗哌拉西林和阿莫西林；（FF）对抗头孢呋辛和亚胺培南，（FX）对抗阿莫西林。但是，其他组（CC和CX）中未观察到这种增加。

参考文献

1. Tappy，Luc yLê，Kim-AnneLê。“果糖和含果糖的热量甜味剂对健康的影响：第一次吹口哨后10年，我们站在哪里？” 当前糖尿病报告卷。2015年15月15日

2. Riveros，MaríaJesús，Parada，Alejandra y Pettinelli和Paulina。“ Consumo De Fructosa y Sus Implicaciones Para La Salud：MalabsorciónDe Fructosa eHígadoGraso noAlcohólico。” NUTRICION Hospitalaria 29.3 2014

3. Astbury，Stuart等人， “大鼠妊娠期间高果糖摄入量会影响后代的母体微生物组和肠道发育。” 遗传学前沿 2018年9月9日

4. Robas Mora等人，“葡萄栽培类型在浅层细菌群落的先天性雌激素组和代谢谱（CLPP）中的作用”农业科学学报A 7（8）， 2017年

资金：研究支持服务（SAI-microbiologíaUSPCEU）

豪尔赫·吉奥¹，克里斯蒂娜·萨拉萨¹，阿德里安·贝拉斯克斯-坎皮伊^1,2,3，特里萨·贝斯¹，路易莎·佩莱亚托¹，玛丽亚·菲亚特¹和埃玛·塞维利亚¹

¹萨拉戈萨大学生物化学与分子和细胞生物学系以及复杂系统生物计算与物理研究所，西班牙萨拉戈萨Pedro Cerbuna 12，50009；²西班牙萨拉戈萨阿拉贡卫生研究所（IIS Aragon）；³西班牙马德里，CIBER肝和消化系统疾病（CIBERehd）；ARAID基金，西班牙萨拉戈萨省阿拉贡市政府

通讯： JorgeGuío（jguiomartinez@gmail.com）

蓝细菌鱼腥藻的FurA（铁摄取调节剂）。PCC 7120是一种转录调节因子，它不仅控制铁体内平衡，而且还控制其他重要的细胞过程，例如氧化应激反应，氮代谢或细胞形态[1]。此外，由于FurA的二硫键还原酶活性及其与血红素相互作用的能力，已被提议用作氧化还原传感器蛋白[2]。2-克戊二酸（2-OG）是克雷布斯循环中产生的代谢产物，可作为碳/氮平衡的信号，在蓝细菌中，它可调节氮代谢NtcA关键调节剂的DNA结合活性[3]。如先前的研究表明，FurA能够控制ntcA和hetR的转录基因[4]，我们想知道这种转录调节因子是否也可以通过2-OG感应碳/氮平衡。等温滴定量热法（ITC）分析证明FurA能够结合2-OG，电泳迁移率移动分析（EMSA）表明FurA与ntcA基因启动子区域的结合活性受到该代谢产物的调节。为了预测其结合位点，建立了FurA三维结构模型并与2-OG对接，表明该分子的潜在结合位点接近DNA结合域并包含碱性氨基酸。多序列比对表明，这些残基在蛋白质的毛皮存在来自其他蓝藻物种，如铜绿微囊藻或N. spumigena以及对接模拟预测2-OG也能够与这些结构结合。综上所述，这些结果表明Fur蛋白在蓝细菌中作为碳/氮平衡传感器的假定作用。

参考文献

1.GonzálezA，Angarica V，Sancho J，FillatM。Anabaena sp。的FurA regulon 。PCC 7120：新型靶基因的计算机预测和实验验证。核酸研究。2014; 42（8）：4833-4846

2. Fillat M. FUR（铁摄取调节剂）超家族：关键转录调节剂的多样性和多功能性。生物化学和生物物理学档案。2014; 546：41-52

3. Laurent S，Chen H，Bedu S，Ziarelli F，Peng L，ZhangC。2-氧代戊二酸酯的不可代谢类似物在阻遏条件下诱导鱼腥藻属异囊分化。PCC7120。美国国家科学院院刊。2005; 102（28）：9907-9912

4.GonzálezA，Valladares A，Peleato M，FillatM。FurA影响Anabaena sp。的异囊分化。PCC7120。FEBS字母。2013; 587（16）：2682-2690

资金：这项工作得到了Gobierno deAragón的E35_17R和MINECO的BFU2016-77671-P / FEDER的支持

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第十三届年度生物技术大会（BAC Madrid 2019）：摘要集。BMC生物技术 19， 78（2019）。https://doi.org/10.1186/s12896-019-0569-8

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• 发表时间：2019年12月04日

• DOI ：https ：//doi.org/10.1186/s12896-019-0569-8