Exploring drug-receptor interaction kinetics: Lessons from a sigma-1 receptor transmembrane biosensor

An important field of study in pharmacology comprises the investigation of drug-target interaction kinetics. Thus, assessing both the lifetime of a drug on its receptor (i.e., drug-target residence time; Copeland, 2016) and the magnitude of drug-mediated receptor activation (i.e., drug efficacy) across the time are critical to understand in vivo pharmacological activity of small-molecule drugs. Of note, while classical in vitro methods view drug-receptor interaction in terms of equilibrium affinity, the residence time model considers the dynamics of receptor conformational rearrangements, which affect drug association and dissociation. Although, classical binding experiments can also address kinetics questions, they are tedious and very time consuming. Accordingly, monitoring drug-receptor interaction dynamics by means of receptor biosensors has become fundamental for understanding how drugs trigger receptor activity over the time. Precisely, in the last years, a number of Fluorescence Resonance Energy Transfer (FRET)-based assays have been developed to accurately display drug-receptor interaction in real time (Lohse et al., 2012). Indeed, one of the most outstanding methods consists of assessing intramolecular conformational rearrangements upon receptor challenge by monitoring intramolecular FRET changes (Vilardaga et al., 2009). Thus, a FRET-based receptor biosensor is built by fusing both donor and acceptor fluorophores to the receptor sequence (Vilardaga et al., 2009). Importantly, a general consensus has prompted to basically attach these molecules (i.e., cyan and yellow fluorescent proteins, CFP and YFP, respectively) intracellularly, this is, in the cytosolic side of the receptor's structure (Figure 1A). Accordingly, when the receptor is activated and a conformational rearrangement occurs the distance and/or orientation of the fluorophores within the receptor biosensor changes and it is possible to monitor FRET changes in real time, thus permitting to finely characterize receptor's activation. Needless to say, although precision is higher than that obtained in classical binding assays, the present biosensors cannot discern between receptors expressed at the cell surface or intracellularly, thus much effort is needed in order to exactly elucidate ligand-receptor kinetics constants

Development of a novel 1 receptor biosensor based on its heterodimerization with binding immunoglobulin protein in living cells

The σ1 receptor (S1R) is a ligand-regulated non-opioid intracellular receptor involved in several pathological conditions. The development of S1R-based drugs as therapeutic agents is a challenge due to the lack of simple functional assays to identify and classify S1R ligands. We have developed a novel nanoluciferase binary technology (NanoBiT) assay based on the ability of S1R to heteromerize with the binding immunoglobulin protein (BiP) in living cells. The S1R-BiP heterodimerization biosensor allows for rapid and accurate identification of S1R ligands by monitoring the dynamics of association-dissociation of S1R and BiP. Acute treatment of cells with the S1R agonist PRE-084 produced rapid and transient dissociation of the S1R-BiP heterodimer, which was blocked by haloperidol. The effect of PRE-084 was enhanced by calcium depletion, leading to a higher reduction in heterodimerization even in the presence of haloperidol. Prolonged incubation of cells with S1R antagonists (haloperidol, NE-100, BD-1047, and PD-144418) increased the formation of S1R-BiP heteromers, while agonists (PRE-084, 4-IBP, and pentazocine) did not alter heterodimerization under the same experimental conditions. The newly developed S1R-BiP biosensor is a simple and effective tool for exploring S1R pharmacology in an easy cellular setting. This biosensor is suitable for high-throughput applications and a valuable resource in the researcher's toolkit

Identification of the begomovirus resistance genes Ty‑2 and Ty‑3 in tomato genotypes

O  objetivo  deste  trabalho foi  identificar  a  presença  dos genes  Ty‑2 e Ty‑3, de resistência a begomovírus, em acessos de tomateiro do Banco de Germoplasma de Hortaliças da Universidade Federal de Viçosa. Os oligonucleotídeos TO302 F/R e FLUW25 F/R foram utilizados em reações de PCR, para verificar a presença de marcadores relacionados aos genes Ty‑2 e Ty‑3, respectivamente. Observou-se a presença do gene Ty‑2, em heterozigose na subamostra BGH-6881 (Solanum peruvianum), e do gene Ty‑3, em homozigose nas subamostras BGH-6878, BGH-6897 (S. lycopersicum)  e  em heterozigose  na subamostra BGH-6881. A identificação dos genes de resistência, com reações de PCR, representa um avanço para os programas de melhoramento de tomateiro no Brasil.The objective of this work was to identify the presence of the genes begomovirus resistence Ty‑2 and Ty‑3 in tomato accessions of the Banco de Germoplasma de Hortaliças of the Universidade Federal de Viçosa. The oligonucleotides TO302 F/R and FLUW25 F/R were used in PCR analysis to verify the presence of markers associated to the genes Ty‑2 and Ty‑3, respectively. The Ty‑2 gene was observed in heterozygosis in the accession BGH-6881 (Solanum peruvianum), and the Ty‑3 gene in homozygosis in the accessions BGH-6878, BGH-6897 (S. lycopersicum), and in heterozygosis in acession BGH-6881. The identification of resistance genes, using PCR analysis, represents an advance for tomato breeding programs in Brazil

Exploring drug-receptor interaction kinetics: Lessons from a sigma-1 receptor transmembrane biosensor

An important field of study in pharmacology comprises the investigation of drug-target interaction kinetics. Thus, assessing both the lifetime of a drug on its receptor (i.e., drug-target residence time; Copeland, 2016) and the magnitude of drug-mediated receptor activation (i.e., drug efficacy) across the time are critical to understand in vivo pharmacological activity of small-molecule drugs. Of note, while classical in vitro methods view drug-receptor interaction in terms of equilibrium affinity, the residence time model considers the dynamics of receptor conformational rearrangements, which affect drug association and dissociation. Although, classical binding experiments can also address kinetics questions, they are tedious and very time consuming. Accordingly, monitoring drug-receptor interaction dynamics by means of receptor biosensors has become fundamental for understanding how drugs trigger receptor activity over the time. Precisely, in the last years, a number of Fluorescence Resonance Energy Transfer (FRET)-based assays have been developed to accurately display drug-receptor interaction in real time (Lohse et al., 2012). Indeed, one of the most outstanding methods consists of assessing intramolecular conformational rearrangements upon receptor challenge by monitoring intramolecular FRET changes (Vilardaga et al., 2009). Thus, a FRET-based receptor biosensor is built by fusing both donor and acceptor fluorophores to the receptor sequence (Vilardaga et al., 2009). Importantly, a general consensus has prompted to basically attach these molecules (i.e., cyan and yellow fluorescent proteins, CFP and YFP, respectively) intracellularly, this is, in the cytosolic side of the receptor's structure (Figure 1A). Accordingly, when the receptor is activated and a conformational rearrangement occurs the distance and/or orientation of the fluorophores within the receptor biosensor changes and it is possible to monitor FRET changes in real time, thus permitting to finely characterize receptor's activation. Needless to say, although precision is higher than that obtained in classical binding assays, the present biosensors cannot discern between receptors expressed at the cell surface or intracellularly, thus much effort is needed in order to exactly elucidate ligand-receptor kinetics constants

Exploring drug-receptor interaction kinetics: Lessons from a sigma-1 receptor transmembrane biosensor

An important field of study in pharmacology comprises the investigation of drug-target interaction kinetics. Thus, assessing both the lifetime of a drug on its receptor (i.e., drug-target residence time; Copeland, 2016) and the magnitude of drug-mediated receptor activation (i.e., drug efficacy) across the time are critical to understand in vivo pharmacological activity of small-molecule drugs. Of note, while classical in vitro methods view drug-receptor interaction in terms of equilibrium affinity, the residence time model considers the dynamics of receptor conformational rearrangements, which affect drug association and dissociation. Although, classical binding experiments can also address kinetics questions, they are tedious and very time consuming. Accordingly, monitoring drug-receptor interaction dynamics by means of receptor biosensors has become fundamental for understanding how drugs trigger receptor activity over the time. Precisely, in the last years, a number of Fluorescence Resonance Energy Transfer (FRET)-based assays have been developed to accurately display drug-receptor interaction in real time (Lohse et al., 2012). Indeed, one of the most outstanding methods consists of assessing intramolecular conformational rearrangements upon receptor challenge by monitoring intramolecular FRET changes (Vilardaga et al., 2009). Thus, a FRET-based receptor biosensor is built by fusing both donor and acceptor fluorophores to the receptor sequence (Vilardaga et al., 2009). Importantly, a general consensus has prompted to basically attach these molecules (i.e., cyan and yellow fluorescent proteins, CFP and YFP, respectively) intracellularly, this is, in the cytosolic side of the receptor's structure (Figure 1A). Accordingly, when the receptor is activated and a conformational rearrangement occurs the distance and/or orientation of the fluorophores within the receptor biosensor changes and it is possible to monitor FRET changes in real time, thus permitting to finely characterize receptor's activation. Needless to say, although precision is higher than that obtained in classical binding assays, the present biosensors cannot discern between receptors expressed at the cell surface or intracellularly, thus much effort is needed in order to exactly elucidate ligand-receptor kinetics constants

Identificación y análisis de factores de éxito en la gerencia de proyectos con equipos virtuales en el sector de Tecnologías de la Información (TI)

The research work proposes the identification and analysis of success factors of the management of projects that include virtual teams in the sector of Information Technology (IT). In order to contribute in the probability of success of project management with virtual teams in the IT sector, through identify the most common success factors in the environment and thus be able to implement them in the different projects from the beginning of these.El trabajo de investigación propone la identificación y análisis de factores de éxito de la gerencia de proyectos que incluyen equipos virtuales en el sector de Tecnologías de la Información (TI). Con el fin de contribuir en la probabilidad de éxito de la gerencia de proyectos con equipos virtuales en el sector TI, a través de identificar los factores de éxito más comunes en el medio y así poder implementarlos en los diferentes proyectos desde un principio de estos.MaestríaMagíster en Desarrollo y Gerencia Integral de Proyecto

Identificação dos genes Ty-2 e Ty-3 de resistência a begomovírusem genótipos de tomateiro

O objetivo deste trabalho foi identificar a presença dos genes Ty-2 e Ty-3, de resistência a begomovírus, em acessos de tomateiro do Banco de Germoplasma de Hortaliças da Universidade Federal de Viçosa. Os oligonucleotídeos TO302 F/R e FLUW25 F/R foram utilizados em reações de PCR, para verificar a presença de marcadores relacionados aos genes Ty-2 e Ty-3, respectivamente. Observou-se a presença do gene Ty-2, em heterozigose na subamostra BGH-6881 (Solanum peruvianum), e do gene Ty-3, em homozigose nas subamostras BGH-6878, BGH-6897 (S. lycopersicum) e em heterozigose na subamostra BGH-6881. A identificação dos genes de resistência, com reações de PCR, representa um avanço para os programas de melhoramento de tomateiro no Brasil.The objective of this work was to identify the presence of the genes begomovirus resistence Ty-2 and Ty-3 in tomato accessions of the Banco de Germoplasma de Hortaliças of the Universidade Federal de Viçosa. The oligonucleotides TO302 F/R and FLUW25 F/R were used in PCR analysis to verify the presence of markers associated to the genes Ty-2 and Ty-3, respectively. The Ty-2 gene was observed in heterozygosis in the accession BGH-6881 (Solanum peruvianum), and the Ty-3 gene in homozygosis in the accessions BGH-6878, BGH-6897 (S. lycopersicum), and in heterozygosis in acession BGH-6881. The identification of resistance genes, using PCR analysis, represents an advance for tomato breeding programs in Brazil

Response of tomato (Solanum L. section Lycopersicon Mill.) germplasm to begomovirus inoculation under controlled and field conditions

Ninety-five tomato accessions belonging to the Vegetable Crops Germplasm Bank of the Federal University of Viçosa (BGH-UFV) were evaluated in a protected environment (biolistic inoculation under greenhouse conditions) and in the field (natural infection) for resistance to bipartite begomoviruses present in Brazil. Plants were assessed by the visual evaluation of symptoms, and viral infection was confirmed by non-radioactive molecular hybridization. Univariate statistical analyses and correlations were made between the traits. High and significant correlations were found between the percentages of visual symptoms and of viral replication, with values of 0.67 in the protected environment and 0.60 in the field. We concluded that under the protected environment, the accessions BGH-2144, BGH-2150, BGH-6878 and BGH-6881 displayed resistance to infection. Under field conditions with natural infection, the best results were obtained for the accessions BGH-2080 and BGH-6881. Only the BGH-6881 (Solanum peruvianum L.) accession excelled in both conditions; this resistance was attributed to the presence of the Ty-2 and Ty-3 resistance gene alleles in heterozygosity. In general, all of the accessions selected in both experimental conditions can serve as sources for the development of cultivars tolerant or resistant to the bipartite begomoviruses present in Brazil