Abstract:

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

Author affiliation: Prieto, Daniel. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Aparicio, Gonzalo. Universidad de la República; Uruguay

Author affiliation: Morande, Pablo Elías. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Zolessi, Flavio. Universidad de la República; Uruguay. Instituto Pasteur de Montevideo; Uruguay

Abstract:

Chagas disease is a major unsolved health issue in Latin America and an emerging threat worldwide. New drugs are urgently needed for chemotherapy as those available (benznidazole and nifurtimox) have variable efficacy and elevated toxicity. Efforts are actually oriented to improve tools and technologies (e.g. transgenic parasites, flow cytometry or image-based systems) for the screening of large numbers of candidate compounds for their activity against Trypanosoma cruzi (T. cruzi). Methods that test drug efficacy and selectivity in the same assay are suitable to accelerate the process of drug discovery. Here, we developed a GFP expressing T. cruzi from a moderate virulence stock and confirmed that the transgenic parasite retained the biological characteristics of the parental strain. With this tool, we established a flow cytometer-based method to simultaneously test drug activity against intracellular amastigotes and toxicity to the host cell. This one-step procedure allows determining the selectivity index of the tested compound in a sensitive and accurate manner even with low infection rates. This method can provide additional information on the interactions between drug, parasites and host cell and could be adapted to other trypanosomatids and protozoa with intracellular multiplication.

Author affiliation: Miranda, Cristian Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Médica; Argentina

Author affiliation: Solana, Maria Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Médica; Argentina

Author affiliation: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina

Author affiliation: Lammel, Estela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Médica; Argentina

Author affiliation: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; Argentina

Author affiliation: Alba Soto, Catalina Dirney. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones En Microbiología y Parasitología Médica; Argentina

Abstract:

Chagas disease is a major unsolved health issue in Latin America and an emerging threat worldwide. New drugs are urgently needed for chemotherapy as those available (benznidazole and nifurtimox) have variable efficacy and elevated toxicity. Efforts are actually oriented to improve tools and technologies (e.g. transgenic parasites, flow cytometry or image-based systems) for the screening of large numbers of candidate compounds for their activity against Trypanosoma cruzi (T. cruzi). Methods that test drug efficacy and selectivity in the same assay are suitable to accelerate the process of drug discovery. Here, we developed a GFP expressing T. cruzi from a moderate virulence stock and confirmed that the transgenic parasite retained the biological characteristics of the parental strain. With this tool, we established a flow cytometer-based method to simultaneously test drug activity against intracellular amastigotes and toxicity to the host cell. This one-step procedure allows determining the selectivity index of the tested compound in a sensitive and accurate manner even with low infection rates. This method can provide additional information on the interactions between drug, parasites and host cell and could be adapted to other trypanosomatids and protozoa with intracellular multiplication.

Author affiliation: Miranda, Cristian Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

Author affiliation: Solana, Maria Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

Author affiliation: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Lammel, Estela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

Author affiliation: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Author affiliation: Alba Soto, Catalina Dirney. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina

Abstract:

In this study, we describe a flow cytometry (FC) system for detecting antibodies to factor VIII (FVIII) and compare its results with those of enzyme-linked immunosorbent assay (ELISA) that detects both inhibitory (I-Ab) and non-inhibitory (NI-Ab) antibodies and the Nijmegen modification of the Bethesda method, detecting I-Ab. FC was set up in our laboratory. Recombinant FVIII (rFVIII) was coupled to microspheres (FVIII-m) and reacted with different plasma dilutions. Microspheres without rFVIII were used as control (control-m). Captured anti-FVIII antibodies were detected using anti-human IgG. Plasma samples from the following patients with severe haemophilia A (SHA) patients were evaluated: 17 P (patients without I-Ab, <0.5BUmL-1); 13 PI (patients with I-Ab, 1.1-8200BUmL-1). Of these 13, two PI were referred during immune tolerance induction (ITI), and plasmas from 12 healthy donors (HD) were evaluated. Semiquantitative results were given as an index (the highest mean fluorescence intensity ratio between FVIII-m and control-m multiplied by the inverse of the corresponding plasma dilution). Both plasma and serum were suitable for the test. FC agreed with the Bethesda method (r=0.8; P=0.0001). FC and ELISA had 80% of coincidence. Four of 17 patients (23.5%) had NI-Ab by FC, and two of them developed high levels of I-Ab later on. This test provides a useful alternative for measuring FVIII antibodies supplementing Bethesda assay. FC is fast and easy to perform. No more than 200μL of plasma or serum is required especially making it useful for paediatric patients. © 2010 Blackwell Publishing Ltd.

Author affiliation: Irigoyen, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Primiani, L.. Fundación Argentina de Hemofilia; Argentina

Author affiliation: Felippo, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Candela, M.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Perez Bianco, R.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: De Bracco, M. M. DE E.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Galassi, Nora Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Abstract:

The tribe Leucocoryneae is taxonomically andcytogenetically complex, mainly due to its extraordinarymorphological and karyological variation. Robertsoniantranslocations had long been recognized as a central factorcontributing to karyotype diversity within the Leucocoryneae,but so far no major tendency prevailing on theobserved complexity of karyotype formula among specieshas been identified. The assessment of nuclear DNAcontents by flow cytometry using propidium iodide in 23species, representing all genera within the tribe, showed amonoploid genome size variation of 1Cx = 9.07?30.46 pgdenoting a threefolds fluctuation. A highly significant linearassociation between the average DNA content per chromosomearm (2C/FN) and the monoploid genome size (1Cx)is reported for the first time and identified as a novel indicatorof a trend governing karyotype diversity within Leucocoryneae.This trend shows that a reduction in DNA contentper chromosome arm is influencing and has shaped karyotypeevolution of different monophyletic groups within thetribe despite the complex karyotype diversity and apparentlycontrasting patterns of genome sizes.

Author affiliation: Sassone, Agostina Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; Argentina

Author affiliation: López, Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; Argentina

Author affiliation: Hojsgaard, Diego Hernan. Goethe Universitat Frankfurt; Alemania

Author affiliation: Giussani, Liliana Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; Argentina

Abstract:

Introduction: Immune humoral neutropenia (Np) could be the consequence of anti-polymorphonuclear neutrophil (PMN) antibodies, circulating immune complexes (CIC) and/or antibodies against myeloid precursors. Granulocyte immunofluorescence test (GIFT) and a leukoagglutination technique (LAGT) assays are recommended for its diagnosis. Methods: Fifty adult patients with secondary Np were screened for anti-PMN. GIFT by flow cytometry from viable PMN and LAGT were employed. In addition, CIC levels, low expression of CD16b (CD16 blow), PMN phenotype and sera tumor necrosis factor- alpha (TNF-α) were also evaluated. Results: Direct IgG-PMN binding (dir-GIFT) was positive in 16% of the patients. Antibodies against autologous PMN were detected in 32% of the samples by indirect (ind)-GIFT and demonstrated in 70% of the sera by both ind-GIFT and/ or LAGT. Predominance of human neutrophil alloantigen (HNA)-1b and HNA-2 expression was confirmed. CD16b low was detected in 16% of the patient's PMN and TNF-α in 68% of sera patients. Conclusion:Our results suggest that diagnosis of immune Np in the laboratory may be improved by focusing on patient's PMN together with the assessment of cellular markers. © 2009 Blackwell Publishing Ltd.

Author affiliation: Riera Cervantes, Norma Edith. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Rosso Saltó, M.. Asociación Española Primera de Socorros Mutuos Montevideo; Uruguay

Author affiliation: Galán, V.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Canalejo, K.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Khoury, M.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Aixalá, M.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Kantor, G. L.. Hospital Durand; Argentina

Author affiliation: Vermeulen, Elba Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Bengló, R.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: de Elizalde, Maria Marta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina

Abstract:

Bleeding and thrombosis in myeloproliferative disorders (MPD) are common events, sometimes both are present in the same patient during the course of the disease. Platelet activation in patients with MPD is often suggested. The present study analyses the presence of circulating activated platelets, using simultaneously flow cytometry and aggregometric studies in MPD. We studied 28 patients: 13 with polycythaemia vera, seven with essential thrombocythaemia, and eight chronic myeloid leukaemia. We performed functional tests, aggregation and adenosine triphosphate (ATP) release and flow cytometric assays (mepacrine staining and platelet activation markers CD62, CD63 and fibrinogen binding (B-FG)). Twenty-one MPD samples (75%) had reduced aggregation and ATP release. Acquired δ-SPD was detected in 11 of 28 MPD patients (39%), and we found no association between reduced mepacrine labelling and abnormal ATP release. High levels of activation markers were obtained: CD62 in 19 of 28 patients (68%), CD63 in 13 of 28 patients (46%) and B-FG in 19 of 28 patients (68%). The most prevalent abnormality was a reduced aggregation and ATP release. The lack of association between ATP release and mepacrine labelling suggests that other mechanisms, besides the deficit of intraplatelet ATP/adenosine diphosphate, might occur. High levels of activation markers were also observed. We conclude that both tests are complementary and necessary to understand the functional status of platelets in MPD.

Author affiliation: Bermejo, Emilse. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Alberto, Maria Fabiana. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Meschengieser, Susana S.. Academia Nacional de Medicina de Buenos Aires; Argentina

Author affiliation: Lazzari, María Ángela. Academia Nacional de Medicina de Buenos Aires; Argentina

Abstract:

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.

Author affiliation: Porro, Valentina. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Pagotto, Romina María del Luján. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Harreguy, María Belén. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Ramírez, Sofía. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Crispo, Martina. Instituto Pasteur de Montevideo; Uruguay

Author affiliation: Santamaría, Clarisa Guillermina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

Author affiliation: Luque, Enrique Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

Author affiliation: Rodriguez, Horacio Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

Author affiliation: Bollati Fogolín, Mariela. Instituto Pasteur de Montevideo; Uruguay

Abstract:

Cytogenetic characterization and determination of DNA content by flow cytometry of five species of MecardoniaRuiz et Pavon, 1798 (Gratiolae, Plantaginaceae) was performed. This is the first study of nuclear DNA content carried out in the genus. Mitotic analysis revealed a base chromosome number x = 11 for all entities and different ploidy levels, ranging from diploid (2n = 2x = 22) to hexaploid (2n = 6x = 66). The results include the first report of the chromosome numbers for M. flagellaris(Chamisso & Schlechtendal, 1827) (2n = 22), M. grandiflora(Bentham) Pennell, 1946 (2n = 22), M. kamogawaeGreppi & Hagiwara, 2011 (2n = 66), and Mecardoniasp. (2n = 44). The three ploidy levels here reported suggest that polyploidy is common in Mecardoniaand appear to be an important factor in the evolution of this genus. The 2C- and 1Cx-values were also estimated in all the species. The 2C-values ranged from 1.91 to 5.29 pg. The 1Cx-values ranged from 0.88 to 1.03 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level. The significance of the results is discussed in relation to taxonomy of the genus.

Author affiliation: Sosa, María de las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

Author affiliation: Angulo, Maria Betiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentina

Author affiliation: Greppi, Julián Alejandro. Instituto Nacional de Tecnología Agropecuaria; Argentina

Author affiliation: Bugallo, Verónica. Instituto Nacional de Tecnología Agropecuaria; Argentina

Abstract:

Chrysolaena flexuosa is a South American species of potential ornamental value (basic chromosome number of x=10). Diploid (n=10) and tetraploid (n=20) cytotypes have been reported for its distribution area, although one hexaploid (n=30-32ca.) cytotype has been reported for its most southern distribution. To investigate if ploidy and latitude are positively related in Ch. flexuosa natural populations and if sexual polyploidization could have had a role in the origin of the polyploid cytotypes, we determined chromosome numbers, DNA content, and pollen viability and size, and analyzed microsporogenesis in samples of seven Argentinian accessions. Two of the northeastern accessions were diploid and one was tetraploid, whereas the four southeastern accessions were hexaploid. Ploidy levels determined both by chromosome countings and flow cytometry coincided, although monoploid genome size significantly decreased with increasing ploidy. In all accessions, variability was observed for pollen viability and size, as well as for large (presumably 2n) pollen production. This variability was underlined by abnormal cytological events in meiosis and at the tetrad stage (lagging chromosomes, parallel spindles, triads). The results would indicate that there is, apparently, a positive relation between ploidy and latitude, and suggest a likely role of sexual polyploidization in the origin, establishment and expansion of Ch. flexuosa populations.

Número de cromosomas, anomalías meióticas y formación de polen 2n en accesiones de la especie silvestre Chrysolaena flexuosa (Vernonieae, Compositae) de su rango de distribución en Argentina. Chrysolaena flexuosa es una especie sudamericana de potencial valor ornamental. Para el área principal de su distribución fueron reportados citotipos diploides (n=10) y tetraploides (n=20) mientras que para el área más austral sólo existe un registro correspondiente a un citotipo hexaploide (n=30-32ca.). Para investigar, en poblaciones naturales de Ch. flexuosa, una relación positiva entre la ploidía y la latitud y la posible participación de la poliploidización sexual en el origen de los citotipos poliploides, se determinó, en siete introducciones argentinas, el número cromosómico, contenido de ADN y tamaño y viabilidad de polen y se analizó la microesporogénesis. Las introducciones del noreste resultaron diploides y tetraploides mientras que las introducciones del sudeste resultaron hexaploides. El nivel de ploidía determinado a partir de conteos cromosómicos coincidió con lo obtenido mediante citometría de flujo; el tamaño genómico se redujo significativamente con la ploidía. Todas las introducciones presentaron variabilidad en viabilidad y tamaño de polen así como producción de granos grandes (presumiblemente polen 2n). Esta variabilidad fue acompañada por eventos citológicos anormales en meiosis y en el estadio de tétrada. Los resultados evidencian una potencial relación entre la ploidía y la latitud y la posibilidad de poliploidización sexual en el origen, establecimiento y expansión de las poblaciones de Ch. flexuosa.

Author affiliation: Echeverría, María Lis. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina

Author affiliation: Camadro, Elsa Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina