Rna-seq: a glanee at technologies and methodologies / RNA-Seq: un vistazo sobre las tecnologías y metodologías

RNA Sequencing (RNA-Seq) is a newly born tool that has revolutionized the post-genomic era. The data produced by RNA-Seq, sequencing technologies and use of bioinformatics are exploding rapidly. In recent years, RNA-Seq has been the method of choice for profiling dynamic transcriptome taking advantage of high throughput sequencing technologies. RNA-Seq studies have shown the transcriptome magnitude, notion and complexity. From 2008, as its introduction year, the relevant reports on RNA-Seq have been multiplied by more than 2822 times just in 6 years. RNA-Seq also contributes a more accurate gene expression and transcript isoform estimation than other methods. Furthermore, some of the potential applications for RNA-Seq cannot be conducted by other methods and as yet are unique to RNA-Seq. As RNA-Seq approaches increase in speed and decrease in cost, more distinct researches are applied and become more common and accurate. RNA-Seq is a cross and interdisciplinary method that interconnects biology to other scientific topics. This article describes RNA-Seq approach, technologies, methodologies, implementation, and methods done so far in characterizing and profiling transcriptomes.

En los últimos años, la técnica RNA-Seq ha tenido un desarrollo acelerado y se ha convertido en el método de elección para el estudio y la caracterización de los transcriptomas dinámicos, aprovechando las tecnologías de secuenciación de alto rendimiento. Estudios aplicando RNA-Seq han mostrado la magnitud, noción y complejidad del transcripotma. A partir de 2008, año de introducción de la técnica, los estudios con RNA-Seq se han multiplicados por más de 2822 veces sólo en 6 años. Al compararse con otros métodos, los estudios empleando RNA-Seq contribuyen a una estimación más precisa de la expresión génica y de las isoformas de los transcriptos. Además, algunas de las aplicaciones potenciales de RNA-Seq no se pueden llevar a cabo con otros métodos. El uso de RNA-Seq aumenta la velocidad de obtención de información y disminuye los costos, logrando con su uso, que investigaciones diversas se vuelvan más frecuentes y precisas. RNA-Seq es un método interdisciplinario que interconecta la biología a otros temas científicos. En este artículo se describe el planteamiento de la tecnología RNA-Seq, metodologías y los métodos realizados en la caracterización de transcriptomas.

Development on neurodegenerative disorder modeling with induced pluripotent stem cells / 中国药理学通报

Remarkable advances in cellular reprogramming have made it possible to investigate relevant cell populations derived from induced pluripotent stem cells ( iPSCs ) of patients. Be-cause many diseases have its specific genetic information, using the cells to convert into iPSCs can build up a set of genetic pro-file of diseases. The iPSCs which contain the genetic contribution of the donor can be expanded and differentiated into cells of the affected lineages to show aberrant phenotypes in culture. To date, over fifty such disease models have been reported, and while the field is young and hurdles remain, we can foresee the huge potential of it in drug screening. Recent studies using iP-SCs to model various neurogenetic disorders are summarized. Compared to the traditional methods, we analyze the future de-velopment of iPSC based disease models and its past application on high-throughput screening ( HTS) and high-content screening ( HCS) .

Review article: High-throughput screening: the hits and leads of drug discovery- an overview.

The mechanism-based approach which corresponds to the target-based approach screens for compounds with a specific mode of action. The highly effective nature of high-throughput screening (HTS) for identification of highly target specific compounds is attributed to its precise focus on single mechanism. This logical development of receptor technology is closely connected with the changes in strategy of chemical synthesis. The vast number of compounds produced by combinatorial chemistry and the possibility of testing many compounds, including natural products, in a short period of time by HTS attracted attention of many workers. Various detection techniques like fluorescence resonance energy transfer (FRET), Homogeneous time resolved fluorescence (HTRF), etc are available, and the screening of more than 100,000 samples per day is possible. With the introduction of robotics, automation and miniaturization techniques, it became feasible to screen 50,000 compounds a day with complex work-stations. High-throughput screening methods are also used to characterize metabolic and pharmacokinetic data about new drugs. With the use of Cassette dosing techniques even the pharmacokinetic data can be assessed for large number of drug candidates, though not free of drawbacks, yet an effective technique to further increase the drug discovery and development rate. The objective of this article is to give an overview to the High-Throughput screening methodologies used in industries as well as in academic research programmes.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Functional assay and high throughput screening for G-protein coupled receptors / 中国药理学通报

G-protein coupled receptor (GPCR) family is the large st group of therapeutic targets. High throughput screening (HTS) plays critical ro les in early stage of drug discovery. Based on signaling pathway stimulated by l igand binding on the GPCRs, many functional assay technologies have been develop ed for HTS. These technologies include Fluorometric microvolume assay technology (FMAT), Fluorescence polarization (FP), Competitive enzyme-linked immunosorben t assay (ELISA), Scintillation proximity assay (SPA), Melanophore assay, Reporte r gene assay and Calcium assay. The principles and applic- ations of these technologies were summarized in this review. We particularly emphasize those techniques of nonradioactive, su bstrate and cofactor free assays, which will be the major approaches for HTS, su ch as reporter gene and calcium assays.

HTS model for Poly(ADP-ribose) polymerase-1 inhibitors / 中国药理学通报

Aim To introduce a high throughput screen model for PARP-1 inhibitors. Methods Setting up an assay for PARP-1 activity relies on the conversion of NAD~+into a highly fluorescent compound. The inhibitory effects of 9 280 samples(including pure organic compounds, extracts from plants and extracts from microorganism)were screened by the high throughout assay. Results 148 compounds had inhibitory effects over 70%. Ultimately, three inhibitors were identified as PARP-1 inhibitor with high activity.Conclusion The high throughput screening was a highly sensitive, inexpensive, and operationally simple assay method in identifying PARP-1 inhibitors.

HTS model for protein tyrosine kinase inhibitors / 中国药理学通报

Aim To introduce a high throughput screen model for PTK inhibitors. Methods With tyrosine containing polypeptides as substrate, in reaction buffer PTK extracts catalyzed the phosphorylation of tyrosine residues. ELISA method was used to measure the activity of protein tyrosine kinase. Resutls The IC_ 50 of genistein was 110 ?mol?L -1. From more than 7000 samples, 16 were found to possess some degree of PTK inhibiting activity. Conclusion This HTS model for screening PTK inhibitors was stable and sensitive.