PCR Analysis of expR Gene Regulating Biosynthesis of Exopolysaccharides in Sinorhizobium meliloti

Exopolysaccharide (EPS) production by the rhizobacterium Sinorhizobium meliloti is essential for root nodule formation on its legume host (alfalfa), and for establishment of a nitrogen-fixing symbiosis between the two partners. Production of EPS II (galactoglucan) by certain S. meliloti strains results in a mucoid colony phenotype. Other strains that are unable to produce EPS II display a dry phenotype, due to the presence of an insertion element in the gene expR, a key regulator involved in many important cellular processes, including production of low-molecular-weight EPS II. We describe a series of three programmed undergraduate biochemistry laboratory classes teaching PCR and electrophoresis procedures to detect non-functional expR loci in S. meliloti.Fil: Sorroche, Fernando Guido. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Programmed Lab Experiments for Biochemical Investigation of Quorum-Sensing Signal Molecules in Rhizospheric Soil Bacteria

Biochemistry courses in the Department of Molecular Biology at the National University of Río Cuarto, Argentina, are designed for undergraduate students in biology, microbiology, chemistry, agronomy, and veterinary medicine. Microbiology students typically have previous coursework in general, analytical, and organic chemistry. Programmed sequences of lab experiments allow these students to investigate biochemical problems whose solution is feasible within the context of their knowledge and experience. We previously designed and reported a programmed lab experiment that familiarizes microbiology students with techniques for detection and characterization of quorum-sensing (QS) and quorum-quenching (QQ) signal molecules. Here, we describe a sequence of experiments designed to expand the understanding and capabilities of biochemistry students using techniques for extraction and identification of QS and QQ signal molecules from peanut rhizospheric soil bacteria, including culturing and manipulation of bacteria under sterile conditions. The program provides students with an opportunity to perform useful assays, draw conclusions from their results, and discuss possible extensions of the study.Fil: Nievas, Fiorela Lujan. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Bogino, Pablo Cesar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Effect of furans and a pyran on several quorum sensing factors in Ralstonia solanacearum

Ralstonia solanacearum race 2 is the causative agent of Bacterial Wilt in bananas, a disease also known as Moko disease. Once this bacterium becomes established within a plant, it causes the death of its host. Currently, no treatment is available for Bacterial Wilt, and the disease is able to destroy 100% of the crops in afflicted áreas.This study investigates a non-biocidal control method for R. solanacearum that attempts to nullify the pathogenic effects of R. solanacearum by interfering with the bacteria?s Quorum Sensing (QS) communication system. The effects of several molecules on R. solanacearum growth, biofilm formation and production of acylated homoserine lactone (AHL) autoinducers were analyzed. The results suggested that, of the molecules tested, 5,6 dihydro-2(H)- pyran-2-one most effectively inhibited growth, biofilm formation and AHL production. Furfural, 3-methyl-2(5H)- furanone and methyl 2-furoate also effectively inhibited growth. These molecules affected biofilm formation in a concentration-dependent manner. In addition, 3-methyl-2(5H)-furanone and methyl 2-furoate likewise inhibited AHL production. Therefore, all of these molecules could be useful for controlling R. solanacearum.Fil: Gallego Campos, Adriana. Academia Nacional de Ciencias de Buenos Aires. Centro de Estudios Filosóficos "Eugenio Pucciarelli"; Argentina. Universidad de Antioquia; Colombia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Rosero, Esteban. Universidad de Antioquia; ColombiaFil: Echeverri, Fernando. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentin

Roles of extracellular polysaccharides and biofilm formation in heavy metal resistance of rhizobia

Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs), in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti) produces two symbiosis-promoting EPSs: succinoglycan (or EPS I) and galactoglucan (or EPS II). Studies of the S. meliloti/alfalfa symbiosis model system have revealed numerous biological functions of EPSs, including host specificity, participation in early stages of host plant infection, signaling molecule during plant development, and (most importantly) protection from environmental stresses. We evaluated functions of EPSs in bacterial resistance to heavy metals and metalloids, which are known to affect various biological processes. Heavy metal resistance, biofilm production, and co-culture were tested in the context of previous studies by our group. A range of mercury (Hg II) and arsenic (As III) concentrations were applied to S. meliloti wild type strain and to mutant strains defective in EPS I and EPS II. The EPS production mutants were generally most sensitive to the metals. Our findings suggest that EPSs are necessary for the protection of bacteria from either Hg (II) or As (III) stress. Previous studies have described a pump in S. meliloti that causes efflux of arsenic from cells to surrounding culture medium, thereby protecting them from this type of chemical stress. The presence of heavy metals or metalloids in culture medium had no apparent effect on formation of biofilm, in contrast to previous reports that biofilm formation helps protect various microorganism species from adverse environmental conditions. In co-culture experiments, EPS-producing heavy metal resistant strains exerted a protective effect on AEPS-non-producing, heavy metal-sensitive strains; a phenomenon termed “rescuing” of the non-resistant strain.Fil: Nocelli, Natalia Estefanía. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Bogino, Pablo Cesar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Banchio, Erika. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Effects of Growth Regulators on Biomass and the Production of Secondary Metabolites in Peppermint (Mentha piperita) Micropropagated in Vitro

The effects of plant growth regulators on peppermint (Mentha piperita) cultured in vitro were studied for the purpose of maximizing growth and essential oil production in micropropagated plants. The basal medium was experimentally supplemented with the auxin 4-indol-3-ylbutyric acid (IBA) and the cytokinin 6-benzylaminopurine (BAP) individually and in combination. Supplementation with BAP alone resulted in the highest values for root length, root dry weight, shoot length, and numbers of nodes, leaves, and ramifications. Treatment with IBA alone or with IBA + BAP resulted in a ~50% increase in shoot fresh weight. The production of secondary metabolites was affected only by the addition of cytokinin, which resulted in a ~40% increase in the total yield of essential oils (EOs). Similar trends were observed for yields of the major EO components (menthone, menthol, pulegone, and menthofuran). Our findings demonstrate that the application of growth regulators increases EO production and biomass concomitantly in an herbaceous species rich in commercially valuable terpenes.Fil: Santoro, Valeria Maricel. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Nievas, Fiorela Lujan. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Zygadlo, Julio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Banchio, Erika. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The Role of Bacterial Biofilms and Surface Components in Plant-Bacterial Associations

The role of bacterial surface components in combination with bacterial functional signals in the process of biofilm formation has been increasingly studied in recent years. Plants support a diverse array of bacteria on or in their roots, transport vessels, stems, and leaves. These plant-associated bacteria have important effects on plant health and productivity. Biofilm formation on plants is associated with symbiotic and pathogenic responses, but how plants regulate such associations is unclear. Certain bacteria in biofilm matrices have been found to induce plant growth and to protect plants from phytopathogens (a process termed biocontrol), whereas others are involved in pathogenesis. In this review, we systematically describe the various components and mechanisms involved in bacterial biofilm formation and attachment to plant surfaces and the relationships of these mechanisms to bacterial activity and survival.Fil: Bogino, Pablo Cesar. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba; Argentina;Fil: Oliva, Maria de Las Mercedes. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba; Argentina;Fil: Sorroche, Fernando Guido. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba; Argentina;Fil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba; Argentina

Influence of drought stress and PGPR inoculation on essential oil yield and volatile organic compound emissions in Mentha piperita

Considering that inoculation with beneficial rhizobacteria promotes biomass and secondary metabolite biosynthesis and also the fact that drought stress can increase their production, we determined the effects when both of these situations were combined in EO yield. In addition, the levels of endogenous phytohormones and the gene expression of enzymes related to monoterpenes biosynthesis were measured. The experimental results showed that inoculation with PGPR in stressed plants produced the same effects on trichome density, the EO main components and total yield as in plants only inoculated or with moderate stress alone. In addition, the VOC emissions of inoculated stressed plants did not vary the release amount in relation to uninoculated stressed ones. The results observed have suggested a way of improving aromatic plant productivity, particularly that of peppermint, by managing drought stress through the inoculation of plants with PGPR. This inoculation under moderate drought stress is appropriate when the purpose of the crops is to obtain plants with a high secondary metabolites yield.Fil: Chiappero, Julieta. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Cappellari, Lorena del Rosario. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Palermo, Tamara Belen. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Banchio, Erika. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentin

Volatile organic compounds from rhizobacteria increase the biosynthesis of secondary metabolites and improve the antioxidant status in mentha Piperita L. Grown under salt stress

Salinity is a major abiotic stress factor that affects crops and has an adverse effect on plant growth. In recent years, there has been increasing evidence that microbial volatile organic compounds (mVOC) play a significant role in microorganism plant interactions. In the present study, we evaluated the impact of microbial volatile organic compounds (mVOC) emitted by Bacillus amyloliquefaciens GB03 on the biosynthesis of secondary metabolites and the antioxidant status in Mentha piperita L. grown under 0, 75 and 100 mM NaCl. Seedlings were exposed to mVOCs, avoiding physical contact with the bacteria, and an increase in NaCl levels produced a reduction in essential oil (EO) yield. Nevertheless, these undesirable effects were mitigated in seedlings treated with mVOCs, resulting in an approximately a six-fold increase with respect to plants not exposed to mVOCs, regardless of the severity of the salt stress. The main components of the EOs, menthone, menthol, and pulegone, showed the same tendency. Total phenolic compound (TPC) levels increased in salt-stressed plants but were higher in those exposed to mVOCs than in stressed plants without mVOC exposure. To evaluate the effect of mVOCs on the antioxidant status from salt-stressed plants, the membrane lipid peroxidation was analyzed. Peppermint seedlings cultivated under salt stress and treated with mVOC showed a reduction in malondialdehyde (MDA) levels, which is considered to be an indicator of lipid peroxidation and membrane damage, and had an increased antioxidant capacity in terms of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity in relation to plants cultivated under salt stress but not treated with mVOCs. These results are important as they demonstrate the potential of mVOCs to diminish the adverse effects of salt stress.Fil: Cappellari, Lorena del Rosario. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Chiappero, Julieta. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Palermo, Tamara Belen. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Banchio, Erika. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentin

Exopolysaccharide II Is Relevant for the Survival of Sinorhizobium meliloti under Water Deficiency and Salinity Stress

Sinorhizobium meliloti is a soil bacterium of great agricultural importance because of its ability to fix atmospheric nitrogen in symbiotic association with alfalfa (Medicago sativa) roots. We looked into the involvement of exopolysaccharides (EPS) in its survival when exposed to different environmental stressors, as well as in bacteria-bacteria and bacteria-substrate interactions. The strains used were wild-type Rm8530 and two strains that are defective in the biosynthesis of EPS II: wild-type Rm1021, which has a non-functional expR locus, and mutant Rm8530 expA. Under stress by water deficiency, Rm8530 remained viable and increased in number, whereas Rm1021 and Rm8530 expA did not. These differences could be due to Rm8530's ability to produce EPS II. Survival experiments under saline stress showed that viability was reduced for Rm1021 but not for Rm8530 or Rm8530 expA, which suggests the existence of some regulating mechanism dependent on a functional expR that is absent in Rm1021. The results of salinity-induced stress assays regarding biofilm-forming capacity (BFC) and autoaggregation indicated the protective role of EPS II. As a whole, our observations demonstrate that EPS play major roles in rhizobacterial survival.Fil: Primo, Emiliano David. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Bogino, Pablo Cesar. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Cossovich, Sacha. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Foresto, Emiliano. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Nievas, Fiorela Lujan. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentin

Effects of nutritional and environmental conditions on Sinorhizobium meliloti biofilm formation

Rhizobia are non-spore-forming soil bacteria that fix atmospheric nitrogen into ammonia in a symbiosis with legume roots. However, in the absence of a legume host, rhizobia manage to survive and hence must have evolved strategies to adapt to diverse environmental conditions. The capacity to respond to variations in nutrient availability enables the persistence of rhizobial species in soil, and consequently improves their ability to colonize and to survive in the host plant. Rhizobia, like many other soil bacteria, persist in nature most likely in sessile communities known as biofilms, which are most often composed of multiple microbial species. We have been employing in vitro assays to study environmental parameters that might influence biofilm formation in the Medicago symbiont Sinorhizobium meliloti. These parameters include carbon source, amount of nitrate, phosphate, calcium and magnesium as well as the effects of osmolarity and pH. The microtiter plate assay facilitates the detection of subtle differences in rhizobial biofilms in response to these parameters, thereby providing insight into how environmental stress or nutritional status influences rhizobial survival. Nutrients such as sucrose, phosphate and calcium enhance biofilm formation as their concentrations increase, whereas extreme temperatures and pH negatively affect biofilm formation.Fil: Rinaudi, Luciana Veronica. Universidad Nacional de Río Cuarto; ArgentinaFil: Fujishige, Nancy A.. University of California; Estados UnidosFil: Hirsch, Ann M.. University of California; Estados UnidosFil: Banchio, Erika. Universidad Nacional de Río Cuarto; ArgentinaFil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Giordano, Walter Fabian. Universidad Nacional de Río Cuarto; Argentin