Montevideo, Uruguay
Montevideo, Uruguay

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Giglio J.,Catedra de Radioquimica | Fernandez S.,Catedra de Radioquimica | Pietzsch H.-J.,Helmholtz Center Dresden | Dematteis S.,Catedra de Inmunologia | And 3 more authors.
Nuclear Medicine and Biology | Year: 2012

The evaluation of oxygenation status of solid tumors is an important field of radiopharmaceutical research. With the aim to develop new potential 99mTc-radiopharmaceuticals for imaging hypoxia, we have synthesized two novel isocyanide derivatives of metronidazole, which has demonstrated high affinity for hypoxic tumors in vitro and in vivo. Methods: Metronidazole derivatives 4-isocyano-N-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]butanamide (M1) and 1-(4-isocyanobutanoyl)-4-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]piperazine (M2) were synthesized, and labeling was performed through preparation of their corresponding 99mTc-(4+1) complexes, 99mTc-NS3M1 and 99mTc-NS3M2. The structure of the technetium complexes was corroborated by preparation and characterization of the corresponding rhenium complexes. We have studied the main physicochemical properties (stability, lipophilicity and plasma protein binding). Biological behavior in HCT-15 cells both in oxia and in hypoxia was assessed. Biodistribution in normal mice and in animals bearing induced 3LL Lewis murine lung carcinoma was also studied. Results: Metronidazole derivatives were successfully synthesized. Labeling with high radiochemical purity was achieved for both ligands. 99mTc complexes were stable in labeling milieu and human plasma. However, presence of the piperazine linker in M2 resulted in higher lipophilicity and protein binding. Although cell uptake in hypoxic conditions was observed for both radiotracers, 99mTc-NS3M2 biodistribution was considered unsuitable for a potential radiopharmaceutical due to high liver uptake and poor blood clearance. However, 99mTc-NS3M1 demonstrated a very favorable in vivo profile both in normal mice and in mice bearing induced tumors. Conclusion: Selective uptake and retention in tumor together with favorable tumor/muscle ratio make 99mTc-NS3M1 a promising candidate for further evaluation as potential hypoxia imaging agent in tumors. © 2012 Elsevier Inc..


Kim H.-J.,University of California at Davis | McCoy M.,University of California at Davis | Gee S.J.,University of California at Davis | Gonzalez-Sapienza G.G.,Institute Higiene | Hammock B.D.,University of California at Davis
Analytical Chemistry | Year: 2011

Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination. © 2010 American Chemical Society.


Brower C.S.,California Institute of Technology | Veiga L.,California Institute of Technology | Veiga L.,Institute Higiene | Jones R.H.,California Institute of Technology | And 2 more authors.
Journal of Biological Chemistry | Year: 2010

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional PAte1/Dfa promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3′-terminal exon, a 217-residue protein termed DfaA. Other Dfa mRNAs also contain this exon. DfaA is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse DfaA that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed DfaA. Furthermore, DfaA was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that DfaA acts as a repressor of TATA-box transcriptional promoters. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Rossotti M.,Institute Higiene | Tabares S.,Institute Higiene | Alfaya L.,Institute Higiene | Leizagoyen C.,Parque Lecoq | And 2 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2015

Background Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. Methods Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. Results The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. Conclusions This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. General significance This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. © 2015 Elsevier B.V.


Alonso E.D.,Instituto Nacional Of Endocrinologia Inen | Jo A.S.,Institute Higiene | Galan Y.,Instituto Nacional Of Oncologia Y Radiobiologia Inor
Revista Cubana de Salud Publica | Year: 2011

Objectives To identify differences in the integral burden (combined mortality and morbidity) associated to lung cancer between sexes and among provinces, and to describe the evolution in 1990, 1995, 2000 and 2002. Methods The Potential Years of Life Lost due to mortality were calculated on the basis of estimated Life Expectancy for quinquennial groups of age. The Potential Years of Life Lost were calculated per death as an average. The Potential Years of Life Lost due to morbidity were estimated on the basis of severity, incidence and average duration. Results The rate of potential years of life lost due to premature mortality increased for both sexes in the 1990-2002 period; it was 6.07 to 7.45 per 1 000 inhabitants in males and 2.52 to 4.21 per 1 000 inhabitants in females. The provinces with the highest rates for males in 1990 and 2002 were Ciudad de La Habana, Matanzas, La Habana and Isla de la Juventus whereas the highest rates for females were found in Pinar del Río, Villa Clara, Ciudad de La Habana, Isla de la Juventud and Ciego de Avila provinces in the same years. There was found an increase in the rate of Potential Years of Life Lost due to morbidity for both sexes from 1990 to 2002; it was 0.42 to 0.52 and 0.19 to 0.28 per 1 000 inhabitants in males and females respectively. The rate of Disability Adjusted Years of Life also showed unfavourable evolution in both sexes. The highest figures were seen in La Habana, Ciudad de La Habana and Villa Clara for both sexes. The males were more affected in terms of mortality and morbidity. Conclusions The impact of lung cancer in healthy years of life lost had unfavourable evolution in Cuba in the selected years of the 1990-2002 period.


Bieczynski F.,CONICET | De Anna J.S.,CONICET | Pirez M.,Institute Higiene | Brena B.M.,Institute Higiene | And 2 more authors.
Aquatic Toxicology | Year: 2014

We studied Abcc mediated-transport in middle and posterior intestine of the rainbow trout, Oncorhynchus mykiss. Luminal and serosal transport were evaluated in everted and non-everted intestinal sacs, respectively, incubated with 1-chloro-2,4-dinitrobenzene (CDNB; 200μM). CDNB enters the cells and is conjugated with glutathione via glutathione S-transferase (GST) to form 2,4-dinitrophenyl-S-glutathione (DNP-SG), a known Abcc substrate. DNP-SG concentration in the bath was recorded every 10min, in order to calculate the mass-specific transport rate. For evaluating the possible involvement of Abcc proteins in microcystin-LR (MCLR) transport, 1.135μM MCLR was added to the bath or inside the sacs, in everted or non-everted preparations, respectively. Both luminal and serosal DNP-SG efflux were significantly inhibited by MCLR. A concentration-response curve obtained using strips from middle intestine yielded an IC50 value of 1.33μM MCLR. The Abcc inhibitor, MK571 produced concentration-dependent inhibition of DNP-SG similar to that produced by MCLR. Since competition of MCLR and CDNB as GST substrates could bias the DNP-SG transport results, we evaluated the effects of MCLR on calcein efflux, which does not depend on GST activity. We applied the non-fluorescent, cell-permeant compound calcein-AM (0.25μM) to middle intestinal strips and recorded the efflux of its hydrolysis product, the fluorescent Abcc substrate calcein. 2.27μM MCLR and 3μM MK571 inhibited calcein efflux (17.39 and 20.2%, respectively). Finally, MCLR interaction with Abcc transporters was evaluated by measuring its toxic intracellular effects. Middle intestinal segments were incubated in saline solution with 1.135μM MCLR (MC1), 2.27μM MCLR (MC2), 3μM MK571 (MK) or 1.135μM MCLR+3μM MK571 (MC1/MK). After 1h, GSH concentration, protein phosphatase 1 and 2A (PP1, PP2A) and GST activities were measured in each segment. MC1did not produce significant effect while MC1/MK and MC2 significantly inhibited PP1and PP2A in similar proportions (34-49%). MK alone significantly increased PP2A activity (40%) with no effect in any other variable. GST activity and GSH concentration were not affected by any treatment. Concentration-response curves for MCLR (1.135 to 13.62μM) alone or plus 3 or 6μM MK571 were obtained using PP1 activity as response variable. The IC50 values were 1.0, 0.52, and 0.37μM, respectively. Our results suggest that O. mykiss enterocytes are capable of eliminating MCLR by GST-mediated conjugation and luminal excretion through an Abcc-like apical transporter. This mechanism would prevent toxic effects and reduce the toxin uptake into the blood, which is likely mediated by basolateral Abccs. © 2014 Elsevier B.V.


Objective: A literature review on the pharmacoeconomic evaluation for the National Health Systems of Cuba was performed, in order to assess the economic and social value to improve efficiency. Methods: A literature review was carried out to search for international and national information to support the incorporation of pharmacoeconomics in the health system of Cuba. Results: Thus, the required levels of pharmacoeconomics efficiency justifying the use of drugs in the health budgets were considered. For this reason, pharmacoeconomics applied to pharmacotherapy is considered to be an additional element contributing to improve rational prescription, which leads to the efficient use of health resources. Conclusions: As shown by the literature review, pharmacoeconomics had a very special place in the topic of drug efficiency. Using pharmacoeconomic techniques we can define the research lines based not only on effectiveness but also on efficiency, and improve the efficiency within the National Health System of Cuba. Adis © 2012 Springer International Publishing AG.


Seuc A.H.,Institute Higiene | Dominguez E.,Instituto Nacional Of Endocrinologia
Cadernos de Saude Publica | Year: 2010

The objective of this study was to estimate the evolution of the burden of disease in Cuba for 20 major causes at five year intervals from 1990 to 2005, in terms of mortality and years of life lost due to premature death (YLL), using national mortality registries. Six summary measures were computed for each of the 20 major causes of death which characterized the evolution of the disease burden over the period studied. The 20 causes were then grouped according to their behaviour in these summary measures; hierarchical cluster analysis was used to support this grouping process. We compute YLL results with and without age-weighting and time discounting (3%). The 20 major causes were grouped into 12 subgroups, each with a particular pattern. The burden of disease in Cuba during the period 1990-2005 has a peculiar pattern that does not reproduce the one characteristic of other low- and middle-income countries. The approach used in this study supports a better description of mortality and YLL trends for major causes, for identifying possible explanations, and for supporting public health policy making. It seems convenient to reproduce this analysis using shorter time intervals, e.g. annually.


Lassabe G.,Institute Higiene | Rossotti M.,Institute Higiene | Gonzalez-Techera A.,Institute Higiene | Gonzalez-Sapienza G.,Institute Higiene
Analytical Chemistry | Year: 2014

Small compounds cannot bind simultaneously to two antibodies, and thus, their immunodetection is limited to competitive formats in which the analyte is indirectly quantitated by measuring the unoccupied antibody binding sites using a competing reporter. This limitation can be circumvented by using phage-borne peptides selected for their ability to specifically react with the analyte-antibody immunocomplex, which allows the detection of these small molecules in a noncompetitive format (PHAIA) with increased sensitivity and a positive readout. In an effort to find substitutes for the phage particles in PHAIA, we explore the use of the B subunit of the Shiga-like toxin of Escherichia coli, also known as verotoxin (VTX), as a scaffold for multivalent display of anti-immunocomplex peptides. Using the herbicides molinate and clomazone as model compounds, we built peptide-VTX recombinant chimeras that were produced in the periplasmic space of E. coli as soluble pentamers, as confirmed by multiangle light scattering analysis. These multivalent constructs, which we termed nanopeptamers, were conjugated to a tracer enzyme and used to detect the herbicide-antibody complex in an ELISA format. The VTX-nanopeptamer assays performed with over a 10-fold increased sensitivity and excellent recovery from spiked surface and mineral water samples. The carbon black-labeled peptide-VTX nanopeptamers showed great potential for the development of a lateral-flow test for small molecules with a visual positive readout that allowed the detection of up to 2.5 ng/mL of clomazone. © 2014 American Chemical Society.


Arevalo F.J.,National University of Rio Cuarto | Gonzalez-Techera A.,Institute Higiene | Zon M.A.,National University of Rio Cuarto | Gonzalez-Sapienza G.,Institute Higiene | Fernandez H.,National University of Rio Cuarto
Biosensors and Bioelectronics | Year: 2012

Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC 50 of 0.15ngmL -1 (linear range from 4.4×10 -3 to 10ngmL -1). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9×1000nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors. © 2011 Elsevier B.V.

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