pinecone/core-2020-05-10-deduplication huggingface dataset:
The Whereabouts of 2D Gels in Quantitative Proteomics.

International audienceTwo-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using 2D gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of 2D gels is performed
Establishment of the DNA repair-defective mutants in DT40 cells.

The chicken B cell line DT40 has been widely used as a model system for reverse genetics studies in higher eukaryotes, because of its advantages including efficient gene targeting and ease of chromosome manipulation. Although the genetic approach using the RNA interference technique has become the standard method particularly in human cells, DT40 still remains a powerful tool to investigate the regulation and function of genes and proteins in a vertebrate system, because of feasibility of easy, rapid, and clear genetic experiments. The use of DT40 cells for DNA repair research has several advantages. In addition to canonical assays for DNA repair, such as measurement of the sensitivities toward DNA damage reagents, it is possible to measure homologous recombination and translesion synthesis activities using activation-induced deaminase (AID)-induced diversification of the immunoglobulin locus. In this chapter, we would describe a detailed protocol for gene disruption experiments in DT40 cells
Animal Models of Helicobacter-Induced Disease: Methods to Successfully Infect the Mouse [chapter]

Animal models of microbial diseases in humans are an essential component for determining fulfillment of Koch’s postulates and determining how the organism causes disease, host response(s), disease prevention, and treatment. In the case of Helicobacter pylori, establishing an animal model to fulfill Koch’s postulates initially proved so challenging that out of frustration a human volunteer undertook an experiment to become infected with H. pylori and to monitor disease progression in order to determine if it did cause gastritis. For the discovery of the organism and his fulfillment of Koch’s postulates he and a colleague were awarded the Nobel Prize in Medicine. After H. pylori was established as a gastric pathogen, it took several years before a model was developed in mice, opening the study of the organism and its pathogenicity to the general scientific community. However, while the model is widely utilized, there are a number of difficulties that can arise and need to be overcome. The purpose of this chapter is to raise awareness regarding the problems, and to offer reliable protocols for successfully establishing the H. pylori mouse model.available in PMC 2013 January 1
Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells.

Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45(-)CD49f(+/-)Thy1(+)gp38(+) mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell-cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity
Applications of Microfluidics for Molecular Diagnostics

Diagnostic assays implemented in microfluidic devices have developed rapidly over the past decade and are expected to become commonplace in the next few years. Hundreds of microfluidics-based approaches towards clinical diagnostics and pathogen detection have been reported with a general theme of rapid and customizable assays that are potentially cost-effective. This chapter reviews microfluidics in molecular diagnostics based on application areas with a concise review of microfluidics in general. Basic principles of microfabrication are briefly reviewed and the transition to polymer fabricated devices is discussed. Most current microfluidic diagnostic devices are designed to target a single disease, such as a given cancer or a variety of pathogens, and there will likely be a large market for these focused devices; however, the future of molecular diagnostics lies in highly multiplexed microfluidic devices that can screen for potentially hundreds of diseases simultaneously
Techniques in Neuroscience

Microdialysis cerebral technique has been widely employed in order to study neurotransmitter release. This technique presents numerous advantages such as it allows work with sample in vivo from freely moving animals. Different drugs in different points implanted probes in several brain areas can be infused simultaneously by means of microdialysis. Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopamine (DA) neurons in the nigrostriatal system, which in turn produces profound neurochemical changes within the basal ganglia, representing the neural substrate for Parkinsonian motor symptoms. Over the years, a broad variety of experimental models of the disease have been developed and applied in diverse animal species. The two most common toxin models used employ 6-hydroxydopamine (6-OHDA) and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenilpyridinium ion (MPTP/MPP+), either given systemically or locally applied into the nigrostriatal pathway, to resemble PD features in animals. Both neurotoxins selectively and rapidly destroy catecholaminergic neurons, although with different mechanisms. Since in vivo microdialysis coupled to high-performance liquid chromatography (HPLC) is an established technique for studying physiological, pharmacological, and pathological changes of a wide range of low molecular weight substances in the brain extracellular fluid, here we describe a rapid and simple microdialysis technique that allows the direct quantitative study of the damage produced by 6-OHDA and MPP+ toxins on dopaminergic (DAergic) striatal terminals of rat brain.peer-reviewe
Prospects for a sustainable increase in the availability of long chain omega 3s: lessons from the Antarctic krill fishery.

•	The global summit on nutrition, health and human behaviour (GSNHHB) identified the objective of increasing “the availability of long chain Omega‐3 (especially docosahexaenoic acid) for human consumption in a sustainable, environmentally responsible way”. \ud
•	The objectives of management for sustainability include maintaining continuity of supply and limiting negative impacts. These objectives have associated challenges which are best illustrated using a case study.\ud
•	Marine fisheries are likely to remain the main source of docosahexaenoic acid (DHA) for the foreseeable future. I use the example of the Antarctic krill fishery, which is a minor but high value source of DHA, to illustrate the issues, processes, actors and risks involved in attempting to manage natural resources in a sustainable, environmentally responsible way.  \ud
•	One of the key issues is uncertainty: The natural state of ecosystems, how they respond to exploitation, and how these responses will be affected by environmental change are not clearly understood. The solution is to use “precautionary” measures, which often means catching less than is theoretically possible, and additional restrictions on where the fishery can operate.\ud
•	The “sustainability” of the Antarctic krill fishery has been questioned in a way that has impacted the delivery of Antarctic krill products to consumers. In reality, the fishery is one of only 3% of fisheries worldwide that the Food and Agriculture Organisation of the United Nations considers “under-exploited” and one of the few that have a management approach committed to limiting impacts on the both the target stock and the wider ecosystem.  Disagreement arises partly because there is no universal agreement about the definitions of “sustainable” and “environmentally responsible” or about the standards of evidence required to support a claim.  A potential solution is to identify the different objectives that people have for the ecosystem, and to agree acceptable trade-offs, levels of risk, and standards of evidence. This approach is compatible with the ecosystem approach to fisheries recommended by the 2002 World Summit on Sustainable Development.\ud
•	The GSNHHB’s commitment to sustainability is a positive step which should be followed by engagement with suppliers to support the appropriate management and recovery of fished ecosystems.\ud
•	GSNHHB’s objectives imply an increase in supply beyond the capacity of marine fisheries and therefore the development of alternative sources. \ud
•	The steps towards sustainability are similar for all sources. They include identifying the different objectives that people have for the source ecosystem, evaluating the risks of not achieving these objectives, establishing trade-offs between objectives, and ensuring appropriate monitoring. It is essential for groups with an interest in source ecosystems to work with each other, and with scientists and managers, to achieve these steps.\u
Promoter independent abortive transcription assays unravel functional interactions between TFIIB and RNA polymerase.

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiation. X-ray crystallography of TFIIB-RNAP II complexes shows that the TFIIB linker region penetrates the RNAP active center, yet the impact of this arrangement on RNAP activity and underlying mechanisms remains elusive. Promoter-independent abortive initiation assays exploit the intrinsic ability of RNAP enzymes to initiate transcription from nicked DNA templates and record the formation of the first phosphodiester bonds. These assays can be used to measure the effect of transcription factors such as TFIIB and RNAP mutations on abortive transcription
Interfollicular epidermal stem cells: boosting and rescuing from adult skin

Publicado na série "Methods in Molecular Biology", vol. 989 (2013), ISSN 1064-3745Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to
accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent
separation from the remaining primary human keratinocytes culture. Those include rapid adherence
of freshly isolated human keratinocytes to collagen type IV through the β 1-integrin ligand and Rho-
Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic
separation to obtain populations based in the combined CD49f bri /CD71 dim expression. Flow
cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early
epidermal markers keratins19/5/14 and in correlation to CD49f bri /CD71 dim subpopulations. The stepby-
step methodology herein described indulges the boosting and consecutive puri fi cation and separation
of interfollicular epidermal stem cells from human keratinocytes cultures
An in silico model for interpreting polypharmacology in drug-target networks.

<Book Title> In Silico Models for Drug DiscoveryRecent analysis on polypharmacology leads to the idea that only small fragments of drugs and targets are a key to understanding their interactions forming polypharmacology. This idea motivates us to build an in silico approach of finding significant substructure patterns from drug-target (molecular graph-amino acid sequence) pairs. This article introduces an efficient in silico method for enumerating, from given drug-target pairs, all frequent subgraph-subsequence pairs, which can then be further examined by hypothesis testing for statistical significance. Unique features of the method are its scalability, computational efficiency, and technical soundness in terms of computer science and statistics. The presented method was applied to 11, 219 drug-target pairs in DrugBank to obtain significant substructure pairs, which can divide most of the original 11, 219 pairs into eight highly exclusive clusters, implying that the obtained substructure pairs are indispensable components for interpreting polypharmacology
Unbiased stereological method to assess proliferation throughout the subependymal zone

The subependymal zone (SEZ), frequently named as adult subventricular zone (SVZ), is a niche of adult neural stem and progenitor cells that lines a large extension of the lateral ventricles of the brain. The majority of the studies do not analyze the SEZ throughout its entire extension. Instead, studies of cell populations within the SEZ typically focus their analysis on a narrow space between specific bregma coordinates that provides a perspective of only a small portion of the SEZ. We have previously proposed a standard division for the SEZ at the anterior-posterior and dorsal-ventral axes based on external brain anatomical hallmarks (Falcao et al., PLoS One 7:e38647, 2012). Herein, we describe in detail the procedure and a stereological approach that can be used to obtain an unbiased estimation of the SEZ cell proliferation under physiological and pathological conditions. This approach takes into consideration clear SEZ anatomical divisions, both on the anterior-posterior and dorsal-ventral axes, which will standardize future studies on the SEZ
Microscopic analysis of chromatin localization and dynamics in C. elegans

During development, the genome undergoes drastic reorganization within the nuclear space. To determine tridimensional genome folding, genome-wide techniques (damID/Hi-C) can be applied using cell populations, but these have to be calibrated using microscopy and single-cell analysis of gene positioning. Moreover, the dynamic behavior of chromatin has to be assessed on living samples. Combining fast stereotypic development with easy genetics and microscopy, the nematode C. elegans has become a model of choice in recent years to study changes in nuclear organization during cell fate acquisition. Here we present two complementary techniques to evaluate nuclear positioning of genes either by fluorescence in situ hybridization in fixed samples or in living worm embryos using the GFP-lacI/lacO chromatin-tagging system
Genetic transformation of wheat via Agrobacterium-mediated DNA delivery

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium’s natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.)
Gene reporter assay to validate microRNA targets in drosophila S2 cells

Bioinformatics programs have helped tremendously in identifying the targets of microRNAs, which are small noncoding RNAs that regulate gene expression posttranscriptionally. However, the partial complementarity between miRNAs and their targets hinders the accuracy of target prediction, necessitating the use of experimental validation procedures. Here, we describe a gene reporter assay typically used in our lab to validate putative miRNA-mRNA interactions in Drosophila S2 cells.Scientific and Technical Research Council of Turkey (104T144 to BA
Master regulators of posttranscriptional gene expression are subject to regulation

MicroRNAs (miRNAs) are small noncoding RNAs of 17-25 nt in length that control gene expression posttranscriptionally. As master regulators of posttranscriptional gene expression, miRNAs themselves are subject to tight regulation at multiple steps. The most common mechanisms include miRNA transcription, processing, and localization. Additionally, intricate feedback loops between miRNAs and transcription factors result in unidirectional, reciprocal, or self-directed elegant control mechanisms. In this chapter, we focus on the posttranscriptional regulatory mechanisms that generate miRNAs whose sequence might be slightly different from the miRNA-coding sequences. Hopefully, this information will be helpful in the discovery of novel miRNAs as well as in the analysis of deep-sequencing data and ab initio prediction of miRNAs. © Springer Science+Business Media New York 2014.the Scientific and Technical Research Council of Turkey (104T144, 107T475, and 210T006 to BA
Introduction to machine learning

The machine learning field, which can be briefly defined as enabling computers make successful predictions using past experiences, has exhibited an impressive development recently with the help of the rapid increase in the storage capacity and processing power of computers. Together with many other disciplines, machine learning methods have been widely employed in bioinformatics. The difficulties and cost of biological analyses have led to the development of sophisticated machine learning approaches for this application area. In this chapter, we first review the fundamental concepts of machine learning such as feature assessment, unsupervised versus supervised learning and types of classification. Then, we point out the main issues of designing machine learning experiments and their performance evaluation. Finally, we introduce some supervised learning methods
Cryo-electron microscopy of membrane proteins

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base
Synthesis and Application of Dye-Ligand Affinity
Adsorbents

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity towards a large number of proteins. They are commercially available, are inexpensive, and can easily be immobilized. Important factors that contribute to the successful operation of a dye-ligand chromatography include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions used in the adsorption and elution stages, and contacting parameters like flow rate and column geometry. In general, with dye-ligand affinity chromatography, the specificity is provided by the adsorption and elution conditions employed in a particular purification, and these must often be worked out by trial and error. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris crude extract on Cibacron Blue 3GA-Sepharose is given as an example
Assisting Target Acquisition in Perspective Views

International audienceThis article introduces an interaction technique designed to assist target acquisition in flat documents that are visualized in perspective. One reason to allow camera tilts in graphical interfaces is that perspective views provide users with a gradual variation of scale, allowing them to see local detail in the context of a general overview. Our analysis, however, shows that the non-linearity of scale variation in a perspective view jeopardizes the acquisition of very remotely located objects. We introduce and experimentally evaluate a solution in which: 1. viewing angle is automatically coupled with tilt angle; and 2. the tilt is constrained so that the virtual camera stays at a constant altitude and remains pointed to a fixed spot on the document. Our results show that with our enhanced perspective navigation technique targets are easy to reach even for extremely high levels of difficulty. Target acquisition time obeys Fitts' Law and performance becomes as rapid as with the familiar pan and zoom technique