High-throughput screening in two dimensions: binding intensity and off-rate on a peptide microarray.

We report a high-throughput two-dimensional microarray-based screen, incorporating both target binding intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation are measured for labeled tumor necrosis factor alpha (TNF-alpha) bound to a peptide microarray. The time-resolved dissociation is fitted to a one-component exponential decay model, from which relative dissociation rates are determined for all peptides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR).

O Homem Que Calculava em Libras: Tradução, Contação de História e Matemática / The Man Who Counted in Libras: Translation, Storytelling and Mathematics

RESUMO: Esta pesquisa teve por objetivo compreender os desafios e as possibilidades de tradução e contação em Língua Brasileira de Sinais (Libras) das histórias presentes no livro O homem que Calculava, do escritor modernista brasileiro Malba Tahan. Tratase de uma investigação qualitativa que se debruçou sobre o processo de tradução audiovisual das histórias presentes no livro, as adaptações feitas em suas narrativas, os recursos de preparação para a produção e edição dos vídeos, a implementação de recursos gráficos e visuais e, por fim, sua divulgação como produto da pesquisa. As análises indicam o potencial didático que emerge na interação entre os envolvidos no processo de tradução das histórias: professores, intérpretes e discentes (surdo e ouvinte), possibilitando formas de expressão artísticas que exploram a visualidade inerente da língua de sinais aliada a recursos gráficos, tomando as narrativas como elemento central para o desenvolvimento criativo ancorado no ensino da Matemática por meio da leitura e da interpretação de histórias.

ABSTRACT: This research aimed to understand the challenges and possibilities of translation and storytelling in the Brazilian Sign Language (known by the acronym LIBRAS) of the stories present in the book O homem que calculava [The man who counted] by the Brazilian modernist writer Malba Tahan. This is a qualitative investigation that focused on the audiovisual translation process of the stories present in the book, on the adaptations made in its narratives, the preparation resources for the production and editing of the videos, the implementation of graphic and visual resources and, finally, its dissemination as a research product. The analyzes indicate the didactic potential that emerges in the interaction between those involved in the process of translating the stories: teachers, interpreters and students (deaf and hearing), enabling forms of artistic expression that explore the inherent visuality of sign language combined with graphic resources, taking the narratives as a central element for the creative development anchored in the teaching of Mathematics through the reading and interpretation of stories.

Rapid detection of a dengue virus RNA sequence with single molecule sensitivity using tandem toehold-mediated displacement reactions.

A DNA tetrahedron having a single-strand DNA edge was used to detect a dengue virus RNA sequence. Novel tandem toehold-mediated displacement reactions (tTMDR) were developed to amplify the fluorescence signal from the DNA tetrahedron. Using an excess of the DNA tetrahedron each target RNA was recycled about 103 times during the tTMDR process. This amplification process was used for the sensitive detection of dengue virus RNA in this study. As few as 6 copies of RNA per sample could be detected using a photon count technique with single molecule sensitivity.

Study of a two species microbial community by an inferential comparative genomic analysis tool: Spatial Analytical Microbial Imaging.

Most molecular fingerprinting techniques, including denaturing gradient gel electrophoresis (DGGE) [1], comparative genomic hybridization (CGH) [2], real-time polymerase chain reaction (RT-PCR) [3], destroy community structure and/or cellular integrity, therefore lost the info. of the spatial locus and the in situ genomic copy number of the cells. An alternative technique, fluorescence in situ hybridization (FISH) doesn't require sample disintegration but needs to develop specific markers and doesn't provide info. related to genomic copy number. Here, a microbial analysis tool, Spatial Analytical Microbial Imaging (SAMI), is described. An application was performed with a mixture of Synechocystis sp. PCC 6803 and E. coli K-12 MG1655. The intrinsic property of their genome, reflected by the average fluorescence intensity (AFI), distinguished them in 3D. And their growth rates were inferred by comparing the total genomic fluorescence binding area (GFA) with that of the pure culture standards. A 93% of accuracy in differentiating the species was achieved. •SAMI does not require sample disintegration and preserves the community spatial structure.•It measures the 3D locus of cells within the mixture and may differentiate them according to the property of their genome.•It allows assessment of the growth rate of the cells within the mixture by comparing their genomic copy number with that of the pure culture standards.

Microsecond time scale rotation measurements of single F1-ATPase molecules.

A novel method for detecting F(1)-ATPase rotation in a manner sufficiently sensitive to achieve acquisition rates with a time resolution of 2.5 micros (equivalent to 400,000 fps) is reported. This is sufficient for resolving the rate at which the gamma-subunit travels from one dwell state to another (transition time). Rotation is detected via a gold nanorod attached to the rotating gamma-subunit of an immobilized F(1)-ATPase. Variations in scattered light intensity allow precise measurement of changes in the angular position of the rod below the diffraction limit of light. Using this approach, the transition time of Escherichia coli F(1)-ATPase gamma-subunit rotation was determined to be 7.62 +/- 0.15 (standard deviation) rad/ms. The average rate-limiting dwell time between rotation events observed at the saturating substrate concentration was 8.03 ms, comparable to the observed Mg(2+)-ATPase k(cat) of 130 s(-)(1) (7.7 ms). Histograms of scattered light intensity from ATP-dependent nanorod rotation as a function of polarization angle allowed the determination of the nanorod orientation with respect to the axis of rotation and plane of polarization. This information allowed the drag coefficient to be determined, which implied that the instantaneous torque generated by F(1) was 63.3 +/- 2.9 pN nm. The high temporal resolution of rotation allowed the measurement of the instantaneous torque of F(1), resulting in direct implications for its rotational mechanism.

Holliday junction dynamics and branch migration: single-molecule analysis.

The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg2+ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.

DNA-binding interactions and conformational fluctuations of Tc3 transposase DNA binding domain examined with single molecule fluorescence spectroscopy.

The fluorescent dye tetramethylrhodamine (TMR) was conjugated to a synthetic peptide containing the sequence-specific DNA binding domain of Tc3 transposase. Steady-state and single molecule fluorescence spectroscopy was used to investigate protein conformational fluctuations and the thermodynamics of binding interactions. Evidence is presented to show that the TMR-Tc3 conjugate exists in at least two conformational states. The most stable conformation is one in which the TMR fluorescence is quenched. Upon binding to DNA, the total fluorescence from TMR-Tc3 increases by three- to fourfold. Single molecule measurements of TMR-Tc3 bound to DNA shows that this complex also fluctuates between a fluorescent and quenched form. The fluorescent form of the conjugate is stabilized when bound to DNA, and this accounts for part of the increase in total fluorescence. In addition, the inherent photodynamics of the dye itself is also altered (e.g., fluorescent lifetime or triplet yield) in such a way that the total fluorescence from the conjugate bound to DNA is enhanced relative to the unbound form.