- research since 1985
- > 20 projects
- 10 different research locations
- 8 years ex-pat
- > 100 publications and achievements
Since the start of my career as a Biochemist in 1985 I have worked hands-on in more than 20 projects that have extensively tackled a wide variety of disciplines, including Organic Chemistry, Biophysics, Enzymology, Molecular Biology, Cell Biology, and Nanotechnology, among others. The biological systems investigated within these fields comprise the three main groups of biomacromolecules, namely, nucleic acids, proteins, and polysaccharides. This research has been performed in a total of 10 different research institutions, both private and public, mostly through fellowships, contracts, and grants that have been obtained in competitive calls where I was either main applicant or Principal Investigator. My research track includes stays in laboratories from Switzerland and the US for an accumulated period of more than 8 years. Despite constraints such as the frequent mobility, starting original and innovative research lines which were not the continuation of preexisting projects in the respective host institutions, being a small research group, and having limited research space, my work in all the subjects, systems, and centers mentioned above has been satisfactorily published in peer-reviewed journals indexed in PubMed , the main data base of reference in Biomedicine. This multidisciplinar background is reflected in the following scientific achievements.
Associate Research Professor
- Barcelona Institute for Global Health (ISGlobal)
- Head of the Nanomalaria Joint Unit
- Since May 2015
- Institute for Bioengineering of Catalonia (IBEC)
- Head of the Nanomalaria Joint Unit
- Since November 2006
Nanotechnology against malaria: origins (2010-2015)
There is a pressing need for the development of new therapeutic strategies against malaria because the currently available treatments will not guarantee its eradication; we use nanotechnological tools for the search of new antimalarial drugs and of valid strategies for their efficient administration.
Single-molecule approaches for the biodiscovery of new antimalarial drugs.
Using SMFS, we have probed the interaction between the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway essential for the viability of the malaria parasite, 1-deoxy-D-xylulose 5-phosphate synthase (DXS), and its two substrates, pyruvate and glyceraldehyde-3-phosphate (Figure 1). The DXS inhibitor fluoropyruvate has been detected in SMFS competition experiments at a concentration of 10 μM, improving by two orders of magnitude the sensitivity of conventional enzyme activity assays. This result highlights the potential of individual enzyme-substrate handling for the biodiscovery of new antimalarial and antibiotic compounds present in natural product extracts at concentrations well below the detection limits of current enzymatic assays.
Figure 1. High-sensitivity single-molecule nanosensor for the detection of DXS inhibitors.
(A) SMFS analysis of the effect of soluble fluoropyruvate on the binding between immobilized DXS and pyruvate. Temporal sequence from top to bottom: no soluble fluoropyruvate, addition of 1 μM fluoropyruvate, addition of 10 μM fluoropyruvate, removal of soluble fluoropyruvate. (B) Configuration showing the interaction between DXS bound to nanoscale sensor and pyruvate to mica surfaces. (C) A significant decrease in the binding of DXS to the pyruvate-functionalized surface indicates the presence of an inhibitor in solution. From Sisquella, X., de Pourcq, K., Alguacil, J., Robles, J., Sanz, F., Anselmetti, D., Imperial, S., and Fernàndez-Busquets, X. (2010) A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials. FASEB J. 24, 4203-4217.
Development of nanovectors for the targeted drug delivery of antimalarials.
Current administration methods of antimalarial drugs deliver the free compound in the blood stream, where it can be unspecifically taken up by all cells, and not only by Plasmodium-infected red blood cells (pRBCs). Nanosized carriers have been receiving special attention with the aim of minimizing the side effects of malaria therapy by increasing drug bioavailability and selectivity. Liposome encapsulation has been assayed for the delivery of compounds against murine malaria, but there is a lack of cellular studies on the performance of targeted liposomes in specific cell recognition and on the efficacy of cargo delivery, and very little data on liposome-driven antimalarial drug targeting to human-infecting parasites. We have used fluorescence microscopy to assess in vitro the efficacy of liposomal nanocarriers for the targeted delivery of their contents to pRBCs (Figure 2). 200-nm liposomes loaded with quantum dots were covalently functionalized with oriented, specific half-antibodies against P. falciparum late form-infected pRBCs. In less than 90 min, liposomes dock to pRBC plasma membranes and release their cargo to the cell. 100.0% of late form-containing pRBCs and 0.0% of non-infected RBCs in P. falciparum cultures are recognized and permeated by the content of targeted immunoliposomes. This immunoliposomal prototype has been shown to improve around tenfold the efficacy of commonly used antimalarial drugs.
Figure 2. Design of a nanovector for targeted antimalarial drug delivery. The cartoon on the left represents a liposomal nanovector functionalized with a targeting molecule (a half-antibody) and containing an antimalarial drug (pink spheres). The image on the right represents the outcome of having the nanovector in the presence of a mixture of infected and non-infected RBCs: a complete specificity of nanovector targeting and of antimalarial delivery. From Urbán, P., Estelrich, J., Cortés, A., and Fernàndez-Busquets, X. (2011) A nanovector with complete discrimination for targeted delivery to Plasmodium falciparum-infected versus non-infected red blood cells in vitro. J. Control. Release 151, 202-211.
5-year tenure track Ramón y Cajal position
- Research Center for Bioelectronics and Nanobioscience. Barcelona Science Park, Universitat de Barcelona, Spain.
- November 2001 - November 2006
- Study of single biomolecule interactions.
- Fine structure study of amyloid-beta peptide (Aβ) fibrillogenesis with atomic force microscopy.
- Study of proteoglycan mechanics by SMFS of allotypic cell adhesion glycans (Figure 3).
- Development of new nanobiosensors based on the measurement of interaction forces between individual enzyme-substrate pairs.
- Determination of the effect of sulfated polysaccharides on the fibrillogenetic process of Aβ (Figure 4).
- Identification of the sponge cell types involved in allogeneic recognition.
Figure 3. Single molecule force spectroscopy (SMFS) of cell adhesion proteoglycans. The self-adhesive properties of proteoglycan molecules were studied by SMFS through a process of molecular dissection that allowed us to track the self-binding site down to a glycan termed g200. This g200-g200 adhesion represents one of the first described carbohydrate-carbohydrate interactions that have an essential physiological function. (A) Detail of the interaction between two proteoglycan monomer subunits. (B) Scheme of a SMFS experiment to study the adhesive interactions between two proteoglycan molecules. (C) Typical SMFS approach-retract curve obtained in experiments performed with native proteoglycans, consistent with the cartoon representation from B. The numbers from 1 to 13 correspond to the breaking of the individual glycan interactions indicated in B. The 100-nm scale bar refers to parts B and C only. From Fernàndez-Busquets, X. (2007) The Sponge as a Model of Cellular Recognition. Sourcebook of Models for Biomedical Research, Conn, P.M., Ed., Humana Press Inc., Totowa, NJ, USA, 75-83.
Figure 4. Proposed model for Aβ1-42 smooth fibrils. (A) Wedged cylinder vs. protofilament stacking models. Hydrophobic boundaries are drawn as red lines. (B) Aβ1-42 sequence showing charged residues in the 1-17 tail, and cartoon depicting the possible role of sulfated polysaccharides in promoting the assembly of Aβ1-42 fibrils. From Bravo, R., Arimon, M., Valle-Delgado, J.J., García, R., Durany, N., Castel, S., Cruz, M., Ventura, S., and Fernàndez-Busquets, X. (2008) Sulfated polysaccharides promote the assembly of amyloid β1-42 peptide into stable fibrils of reduced cytotoxicity. J. Biol. Chem. 283, 32471-32483.
Postdoctoral position Plant Biotech Group
- Department of Biochemistry and Molecular Biology, School of Pharmacy, Universitat de Barcelona, Spain. Group of Drs. A. Boronat and A. Ferrer.
- May 1999 - November 2001
- Study of the biosynthetic routes of isoprenoid compounds in Arabidopsis thaliana.
- Establishment of the regulatory role of HMGR in plant sterol biosynthesis.
- Determination of the subcellular localization of HMGR in plant cells (Figure 5).
Figure 5. Subcellular localization of Arabidopsis thaliana 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) by immunofluorescence confocal microscopy. Previously unidentified vesicles loaded with HMGR are shown in green among autofluorescent chloroplasts (in red) after immunodetection with specific anti-HMGR antibodies. From Leivar, P., González, V.M., Castel, S., Trelease, R.N., López-Iglesias, C., Arró, M., Boronat, A., Campos, N., Ferrer, A., and Fernàndez-Busquets, X. (2005) Subcellular localization of Arabidopsis thaliana 3-hydroxy-3-methylglutarylcoenzyme A reductase. Plant Physiol. 137, 57-69.
Postdoctoral positions abroad
- Novartis AG-Friedrich Miescher Institut, Basel, Switzerland, and Marine Biological Laboratory, Woods Hole, USA. Group of Prof. M.M. Burger.
- April 1993 - April 1999
- Study of the molecular basis of cell adhesion using marine sponges as model animal.
- Use of rhodamine B isothiocyanate to detect proteoglycan core proteins in polyacrylamide gels.
- Probing of single biomolecules with atomic force microscopy.
- Hyaluronic acid-receptor binding demonstrated by synthetic adhesive proteoglycan peptide constructs and by cell receptors on the marine sponge Microciona prolifera.
- Cloning of the core proteins from the 2x104-kDa aggregation factor responsible for species-specific cell adhesion in M. prolifera.
- Demonstration of the accumulation in marine sponge grafts of the mRNA encoding the main proteins of the cell adhesion system.
- Demonstration of the enzymatic biosynthesis of N-linked glycan in M. prolifera.
- Elucidation of the supramolecular structure of a new family of circular proteoglycans (Figure 6).
Figure 6. High-resolution atomic force microscopy of cell adhesion proteoglycans. Unprecedented image definition allowed us to establish the molecular structure of the family of sponge proteoglycans termed aggregation factors, which play an essential role in species-specific cell recognition and adhesion. In combination with biochemical studies, it could be determined that the ring and the arms of the molecule are each formed by 20 subunits of two different proteins, MAFp3 and MAFp4, respectively. The resolution obtained was such that it permitted to identify folding domains in MAFp4 (B) and, most importantly, carbohydrate chains corresponding to the g200 glycan bound to MAFp3 (C). g200 is the self-adhesion site responsible for the interactions detected by single molecule force spectroscopy shown in Figure 3. From Jarchow, J., Fritz, J., Anselmetti, D., Calabro, A., Hascall, V.C., Gerosa, D., Burger, M.M., and Fernàndez-Busquets, X. (2000) Supramolecular structure of a new family of circular proteoglycans mediating cell adhesion in sponges. J. Struct. Biol. 132, 95-105
- Institute of Agroalimentary Research and Technology (IRTA), Cabrils, Spain. Group of Drs. P. Arús and R. Messeguer
- October 1992 - March 1993
- Genetic study of several Prunus species using Restriction Fragment Length Polymorphism (RFLP) and Random Amplified Polymorphic DNA (RAPD) techniques, with the objective of obtaining saturated genetic maps.
- Biochemical and biophysical study of the dynamics of protein-DNA interactions in the nucleosome.
- Elucidation of different mechanisms for in vitro formation of nucleosome core particles (Figure 7).
- Purification of the two strands of a DNA fragment by polyethylene glycol precipitation (Figure 8).
- Histones associated with single-stranded DNA do not preclude the formation of double-helical DNA.
- Mechanism of nucleosome dissociation produced by transcription elongation in a short chromatin template.
Figure 7. Supercoiling of circular DNA induced by histones transferred from oligonucleosome core particles complexed with excess core histones. M13 DNA was digested with topoisomerase I and treated with increasing amounts of oligonucleosomes containing excess core histones. From Aragay, A.M., Fernàndez-Busquets, X., and Daban, J.-R. (1991) Different mechanisms for in vitro formation of nucleosome core particles. Biochemistry 30, 5022- 5032.
Figure 8 Diagrammatic representation of the purification of the two strands of a DNA molecule. The method is based on the hybridization of the (-) strand of a thermally denatured DNA restriction fragment with the (+) strand of the same fragment inserted into the ssDNA form of a cloning vector. Polyethylene glycol differential precipitation is then used in a series of centrifugation steps to sequentially obtain both strands as represented in the scheme. From Fernàndez- Busquets, X. and Daban, J.-R. (1992) Purification of the two strands of a DNA fragment by polyethylene glycol precipitation. BioTechniques 13, 686-688.
- Zentrale Forschungslaboratorien, CIBA-GEIGY AG, Basel, Switzerland. Group of Drs. O. Ghisalba and G. Ramos.
- July - October 1985 & July - December 1986
- Study of enantioselective reactions in organic and aqueous media catalyzed by immobilized enzymes.
- Synthesis of both enantiomeric forms of 2-substituted 1,3-propanediol monoacetates starting from a common prochiral precursor, using enzymatic transformations in aqueous and in organic media (Figure 9).
Figure 9. Schematical representation of the approach followed for the synthesis of the enantiomers g and ent-g. Enzymatic hydrolysis of the diester f produces the monoester g, whereas the enzymatic acylation of the diol e should afford the enantiomeric monoacetate ent-g, provided that both processes take place on the same enantiotopic side of the substrates.From Ramos Tombo, G.M., Schär, H.P., Fernàndez-Busquets, X., and Ghisalba, O. (1987) Enantioselective reactions in aqueous and in organic media using carboxyl esterase fractions obtained from crude porcine pancreas lipase preparations. Biocatalysis in Organic Media, Laane, C., Tramper, J., and Lilly, M.D., Eds.,
- Fundamental Biology Institute, Universitat Autònoma de Barcelona, Spain. Group of Dr. F.-X. Avilés.
- December 1985 - July 1986
- Chromatographic separation and biochemical characterization of pig pancreas procarboxypeptidases and their fragments.