Roegner A.F.,University of California at Davis |
Schirmer M.P.,University of the Republic of Uruguay |
Schirmer M.P.,Institute Higiene |
Puschner B.,University of California at Davis |
And 2 more authors.
Toxicon | Year: 2014
The freshwater cyanotoxins, microcystins (MCs), pose a global public health threat as potent hepatotoxins in cyanobacterial blooms; their persistence in drinking and recreational water has been associated with potential chronic effects in addition to acute intoxications. Rapid and accurate detection of the over 80 structural congeners is challenged by the rigorous and time consuming clean up required to overcome interference found in raw water samples. MALDI-MS has shown promise for rapid quantification of individual congeners in raw water samples, with very low operative cost, but so far limited sensitivity and lack of available and versatile internal standards (ISs) has limited its use. Two easily synthesized S-hydroxyethyl-Cys(7)-MC-LR and -RR ISs were used to generate linear standard curves in a reflectron MALDI instrument, reproducible across several orders of magnitude for MC-LR, -RR and -YR. Minimum quantification limits in direct water samples with no clean up or concentration step involved were consistently below 7 μg/L, with recoveries from spiked samples between 80 and 119%. This method improves sensitivity by 30 fold over previous reports of quantitative MALDI-TOF applications to MCs and provides a salient option for rapid throughput analysis for multiple MC congeners in untreated raw surface water blooms as a means to identify source public health threats and target intervention strategies within a watershed. As demonstrated by analysis of a set of samples from Uruguay, utilizing the reaction of different MC congeners with alternate sulfhydryl compounds, the m/z of the IS can be customized to avoid overlap with interfering compounds in local surface water samples. © 2013 Elsevier Ltd. All rights reserved.
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.
Gonzalez-Sapienza G.,Institute Higiene |
Rossotti M.A.,Institute Higiene |
Tabares-da Rosa S.,Institute Higiene
Frontiers in Immunology | Year: 2017
With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs) has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb)] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications. © 2017 Gonzalez-Sapienza, Rossotti and Tabares-da Rosa.
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.
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.
Herrera M.C.,Institute Higiene
Pharmacoeconomics - Spanish Research Articles | Year: 2012
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.