pinecone/core-2020-05-10-deduplication huggingface dataset:
Isolation of Photosystem II Reaction Center Complexes from Plants

18 Pags. The definitive version is available at: http://www.springerlink.com/content/1064-3745/Methods to isolate and purify 6- and 5-Chl D1/D2/Cyt b559 photosystem II (PSII) reaction center (RC) complexes from plants are presented, and the advantages and disadvantages of each procedure are discussed. One of the simpler 6-Chl procedures and a procedure for isolating 5-Chl complexes are described in detail. Furthermore, a rapid procedure that produces relatively large amounts of less pure 6-Chl material (i.e., more nonpigmented protein) is also described. Criteria to assess the purity of PSII RC preparations are presented, and problems associated with each of the isolation procedures are discussed.This work was supported by the Chemical\ud
Sciences, Geosciences, and Biosciences Division, Office of Science, U.S. Department of Energy\ud
(MS) and by the MCYT of Spain (Grant BFU2005-04722-C02-01 and AGL2008-00377) to RP.Peer reviewe
Purification and Crystallization of Oxygen-Evolving Photosystem II Core Complex from Thermophilic Cyanobacteria

This chapter describes the purification and crystallization of oxygen-evolving photosystem II core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus. Procedures used for purification of photosystem II from the cyanobacterium involves cultivation of cells, isolation of thylakoid membranes, purification of crude and pure photosystem II core complexes by detergent solubilization, followed by differential centrifugation and column chromatography. The purified core dimer particles were successfully used for crystallization, and the methods and conditions used for crystallization are presented. These purification and crystallization procedures can be applied for another thermophilic cyanobacterium T. elongatus
Ribosome display: a technology for selecting and evolving proteins from large libraries

The selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins)
Cytological dissection of the triticeae chromosomes by the gametocidal system.

Triticeae species have a large and complex genome, which has made it difficult to obtain their sequence data. Some alien chromosomes called the gametocidal (Gc) chromosomes introduced into common wheat can induce chromosomal breakage resulting in the generation of deletions and translocations. The induced deletions have been established as deletion stocks in common wheat. This Gc system is also effective in inducing chromosomal breakages in Triticeae chromosomes added to common wheat. The induced aberrant chromosomes can be identified by chromosome banding and fluorescence in situ hybridization and can be established in common wheat as dissection lines. This Gs system will be useful to dissect the single chromosomes of Triticeae species
The Targeted Expression of Nucleotide Sugar Transporters to the E. coli Inner Membrane

The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards evaluating the relationship between their structure and function. We have therefore utilised the OmpA signal sequence to deliberately target the expression of a mammalian nucleotide sugar transporter, the murine CMP-sialic acid transporter, to the E. coli inner membrane. The functionality of the recombinant CMP-sialic acid transporter could then be evaluated either following the spheroplasting of E. coli cells or through the isolation of the E. coli inner membrane and the formation of mixed phosphatidylcholine-inner membrane proteoliposomes.Office of the Snr Dep Vice Chancellor, Institute for GlycomicsFull Tex
High-throughput analyses of gene functions on a cell chip by electroporation.

Fig.1は次の論文のFig.1を引用 Fujimoto, H., Kato, K., Iwata, H. (2008) Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Anal. Bioanal. Chem. 392, 1309–1316.Fig.2は次の論文のFigure 7を引用 Yamauchi, F., Kato, K., Iwata, H. (2004) Spatially and temporally controlled
electroporation of adherent cells on plasmid DNA-loaded electrode. Nucleic Acids Res.
32, e187.Fig.3は次の論文のFig.6を引用 Fujimoto, H., Kato, K., Iwata, H. (2008) Electroporation microarray for parallel transfer
of small interfering RNA into mammalian cells. Anal. Bioanal. Chem. 392, 1309–1316Genome-wide functional annotation of genes is one of the major challenges in current biology. Such investigation requires a high-throughput methodology for efficient and parallel overexpression or silencing of multiplexed genes in living cells. The transfection method described here employs an electric pulse and a cell-chip technology, and provides the possibility of analyzing gene functions in a high-throughput manner
The developmental vitamin D (DVD) model of schizophrenia

It is now widely acknowledged that exposure to adverse environmental factors in utero may not only affect how the brain develops but have long-lasting consequences for later brain function in the adult offspring. This idea has gained particular prominence amongst researchers interested in the etiology of neurodevelopmental disorders such as schizophrenia and autism. Approximately 10 years ago we proposed that developmental vitamin D (DVD) deficiency may explain several epidemiological features of this disease, most noticeably the winter/spring season of birth effect. In 2003 we published results from our first study indicating there were structural changes in how the brain develops in these offspring. Since then we have firmly established that DVD deficiency not only affects brain cell differentiation and gross anatomy but also produces alterations in behavior in these offspring as adults. In this chapter we describe how we came to construct the model we use today. Over the past 7 years the model has proved informative producing both structural brain changes (ventriculomegaly) and behavioral alterations (hyperlocomotion in response to NMDA antagonists) that are thought to be relevant to schizophrenia
Homeostatic regulation of glutamate neurotransmission in primary neuronal cultures

El pdf es la versión post-print.Glutamate is the mayor excitatory neurotransmitter in vertebrate nervous system. It has a crucial role in most brain functions under physiological conditions through the activation of both ionotropic and metabotropic glutamate receptors. In addition, extracellular glutamate concentration is tightly regulated through different excitatory amino acid transporters (EAAT). Glutamate neurotransmission is also involved in the neurotoxic effects of many environmental chemicals and drugs. Furthermore, homeostatic changes in glutamate neurotransmission appear in response to prolonged block / enhancement of electrical activity. Here we describe different approaches to evaluate alterations in glutamate neurotransmission regarding glutamate receptors and glutamate transporters by using primary cultures of neurons and of astrocytes. The methods are based on the increased fluorescence of calcium-sensitive probes in response to glutamate agonists, on radioligand binding to glutamate receptors and transport sites and on inmunocytochemistry visualization of glutamate receptors.This work was supported by grant PI 06/1212 (Spanish Ministry of Health) and SGR 2009/SGR/214 (Generalitat de Catalunya, Spain).Peer Reviewe
Fluorescence Correlation Spectroscopy

317 p.International audienceFluorescence correlation spectroscopy (FCS), implemented in microscopy, relies on performing an autocorrelation of the time fluctuating intensity arising from individual molecules diffusing through a confocal volume. It allows us to investigate a large variety of dynamic processes and to quantify photophysical, photochemical, interaction, diffusion, and transport properties of molecules. This tutorial chapter is intended to give an "hands on" view of FCS. After a brief introduction on the principle of FCS, the major theoretical assumptions are emphasized, and the main analytical expression are given. Then the key parameters that have to be considered when building a FCS system are discussed. The complete method of operation is described, including calibration, measurement, and data treatment. The major difficulties that are encountered when performing for the first time FCS are illustrated by examples of measurements, and possible solutions are proposed
Nanowire Biosensors for Label-Free, Real-Time, Ultrasensitive Protein Detection

Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Among recent research advances exploiting new nanomaterials for biomolecule analysis, silicon nanowires (SiNWs), which are configured as field-effect transistors (FETs), have emerged as one of the most promising and powerful platforms for label-free, real-time, and highly sensitive electrical detection of proteins as well as many other biological species. Here, we describe a detailed protocol for realizing SiNW biosensors for protein detection that includes SiNW synthesis, FET device fabrication, surface receptor functionalization, and electrical sensing measurements. Moreover, incorporating both p-type and n-type SiNWs in the same sensor array provides a unique means of internal control for sensing signal verification.Chemistry and Chemical Biolog
Diagnostic, Prognostic and Therapeutic Value of Gene Signatures

Gene expression studies have revealed diagnostic profiles and upregulation of specific pathways in\ud
many solid tumors. Some gene-expression signatures are already used as predictors of relapse in\ud
early breast cancer patients. The explosion of new information in gene expression profiling could\ud
potentially lead to the development of tailored treatments in many solid tumors. In addition, many\ud
studies are ongoing to validate these signatures also in predicting response to hormonal, chemotherapeutic,\ud
and targeted agents in breast cancer as well as in other tumors.\ud
This book has been carried out with the aim of providing readers a useful and comprehensive\ud
resource about the range of applications of microarray technology on oncological diseases.\ud
The book is principally addressed to resident and fellow physicians, medical oncologists, molecular\ud
biologists, biotechnologists, and those who study oncological diseases. The chapters have been\ud
written by leading international researchers on these topics who have prepared their manuscripts\ud
according to current literature and field experience with microarray technology
Analysis of mRNA abundance and stability by ribonuclease protection assay

Gene expression is a multi-step process, which proceeds from DNA through RNA to protein. The tight regulation of this process is essential for overall cellular integrity and physiological homeostasis. Regulation of the messenger RNA (mRNA) levels has emerged as a crucial event in the modulation of the expression of genetic information. The mechanisms by which this process occurs have been extensively studied and begin to be much better understood. They involve a network of complex pathways that use intrinsic features of the target mRNA, like stability, to control its relative abundance in the cytoplasm. Thus, the analysis of the mRNA stability and abundance is essential to properly undertake gene expression studies. This chapter describes the ribonuclease protection assay, a widely accepted approach to evaluate the quality and amount of a target mRNA. This technique displays a higher sensitivity than classical Northern blot analysis and may be used either individually or in combination with other quantitative methods, such as quantitative reverse-transcription PCR, as complementary procedures rendering more complete and reliable information on gene expression.Peer Reviewe
The KEGG databases and tools facilitating omics analysis: latest developments involving human diseases and pharmaceuticals.

In this chapter, we demonstrate the usability of the KEGG (Kyoto encyclopedia of genes and genomes) databases and tools, especially focusing on the visualization of the omics data. The desktop application KegArray and many Web-based tools are tightly integrated with the KEGG knowledgebase, which helps visualize and interpret large amount of data derived from high-throughput measurement techniques including microarray, metagenome, and metabolome analyses. Recently developed resources for human disease, drug, and plant research are also mentioned
A high-throughput microfluidic method for generating and characterizing transcription factor mutant libraries

Characterizing libraries of mutant proteins is a challenging task, but can lead to detailed functional insights on a specific protein, and general insights for families of proteins such as transcription factors. Challenges in mutant protein screening consist in synthesizing the necessary expression ready DNA constructs and transforming them into a suitable host for protein expression. Protein purification and characterization are also non-trivial tasks that are not easily scalable to hundreds or thousands of protein variants. Here we describe a method based on a high-throughput microfluidic platform to screen and characterize the binding profile of hundreds of transcription factor variants. DNA constructs are synthesized by a rapid two-step PCR approach without the need of cloning or transformation steps. All transcription factor mutants are expressed on-chip followed by characterization of their binding specificities against 64 different DNA target sequences. The current microfluidic platformcan synthesize and characterize up to 2400 protein–DNA pairs in parallel. The platform method is also generally applicable, allowing high-throughput functional studies of proteins
Modulatory effects of proteoglycans on proteinase activities.

International audienceProteoglycans (PGs), composed of a core protein and one or more covalently attached sulfated glycosaminoglycan (GAG) chains, interact with a wide range of bioactive molecules, such as growth factors and chemokines, to regulate cell behaviors in normal and pathological processes. Additionally, PGs, through their compositional diversity, play a broad variety of roles as modulators of proteinase activities. Interactions of proteinases with other molecules on the plasma membrane anchor and activate them at a specific location on the cell surface. These interactions with macromolecules other than their own protein substrates or inhibitors result in changes in their activity and/or may have important biological effects. Thus, GAG chains induce conformational changes upon their binding to peptides or proteins. This behavior may be related to the ability of GAGs to act as modulators for some proteins (1) by acting as crucial structural elements by the control of proteinase activities, (2) by increasing the protein stability, (3) by permitting some binding to occur, exposing binding regions on the target protein, or (4) by acting as coreceptors for some inhibitors, playing important roles for the acceleration of proteinase inhibition. Understanding the modulatory effects exerted by PGs on proteinase activities is expected to lead to new insights in the understanding of some molecular systems present in pathological states, providing new targets for drug therapy
Proteoglycans and osteolysis.

International audienceOsteolysis is a complex mechanism resulting from an exacerbated activity of osteoclasts associated or not with a dysregulation of osteoblast metabolism leading to bone loss. This bone defect is not compensated by bone apposition or by apposition of bone matrix with poor mechanical quality. Osteolytic process is regulated by mechanical constraints, by polypeptides including cytokines and hormones, and by extracellular matrix components such as proteoglycans (PGs) and glycosaminoglycans (GAGs). Several studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial: some studies showed a repressive effect of GAGs on osteoclastic differentiation, whereas others described a stimulatory effect. The controversy also affects osteoblasts which appear sometimes inhibited by polysaccharides and sometimes stimulated by these compounds. Furthermore, long-term treatment with heparin leads to the development of osteoporosis fueling the controversy. After a brief description of the principal osteoclastogenesis assays, the present chapter summarizes the main data published on the effect of PGs/GAGs on bone cells and their functional incidence on osteolysis
Gene knockdown in Paracoccidioides brasiliensis using antisense RNA

Paracoccidioides brasiliensis is a thermal dimorphic fungus which at host environment exhibits a multinucleated and multibudding yeast form. The cellular and molecular mechanisms underlying these phenotypes
remain to be clarified, mostly due to the absence of efficient classical genetic and molecular techniques. Here we describe a method for gene expression knockdown in P. brasiliensis by antisense RNA (aRNA)
technology taking advantage of an Agrobacterium tumefaciens-mediated transformation (ATMT) system.
Together, these techniques represent a reliable toolbox that can be employed for functional genetic analysis of putative virulence factors and morphogenic regulators, aiming to the identification of new potential drug targets.Fundação para a Ciência e a Tecnologia, Portugal (FCT) - Ref.
PTDC/BIA-MIC/108309/2008, Ref. SFRH/BD/33446/2008)
Single molecule FRET characterization of lage ribozyme folding

A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg2+ and Ca2+) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments
Exploiting multisite gateway and pENFRUIT plasmid collection for fruit genetic engineering

El pdf es la versión post-print.MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gateway Entry vectors dedicated to genetic engineering in fruits. It comprises a number of fruit-operating promoters as well as C-terminal tags adapted to the Gateway standard. In this way, flanking regulatory/labeling sequences can be easily Gateway-assembled with a given gene of interest for its ectopic expression or silencing in fruits. The resulting gene constructs can be analyzed in stable transgenic plants or in transient expression assays, the latter allowing fast testing of the increasing number of combinations arising from MultiSite methodology. A detailed description of the use of MultiSite cloning methodology for the assembly of pENFRUIT elements is presented.This work was supported by the Spanish Ministry of Science and Education (Ramón y Cajal programme\ud
BIO2005-01015 and BIO2008-03434 projects), the European Commission (programme I3P and EU-SOL project) and Fundación Genoma España (ESP-SOL project).Peer reviewe
Probability, Statistics, and Computational Science

In this chapter, we review basic concepts from probability theory and computational statistics that are fundamental to evolutionary genomics. We provide a very basic introduction to statistical modeling and discuss general principles, including maximum likelihood and Bayesian inference. Markov chains, hidden Markov models, and Bayesian network models are introduced in more detail as they occur frequently and in many variations in genomics applications. In particular, we discuss efficient inference algorithms and methods for learning these models from partially observed data. Several simple examples are given throughout the text, some of which point to models that are discussed in more detail in subsequent chapters
To detect and analyze sequence repeats whatever be their origin

International audienceThe development of numerous programs for the identification of mobile elements raises the issue of the founding concepts that are shared in their design. This is necessary for at least three reasons. First, the cost of designing, developing, debugging and maintaining software could present a danger of distracting biologists from their main bioanalysis tasks that require a lot of energy. Some key concepts on exact repeats are always underlying the search for genomic repeats and we recall the most important ones. All along the chapter, we try to select practical tools that may help the design of new identification pipelines. Second, the huge increase of sequence production capacities requires to use the most efficient data structures and algorithms to scale up tools in front of the data deluge. This paper provides an up-to-date glimpse on the art of string indexing and string matching. Third, there exists a growing knowledge on the architecture of mobile elements built from literature and the analysis of results generated by these pipelines. Besides data management which has led to the discovery of new families or new elements of a family, the community has an increasing need in knowledge management tools in order to compare, to validate or simply to keep trace of mobile elements models. We end the paper with first considerations on what could help the near future of such research on models
Analytic methods for modeling stochastic regulatory networks

The past decade has seen a revived interest in the unavoidable or intrinsic
noise in biochemical and genetic networks arising from the finite copy number
of the participating species. That is, rather than modeling regulatory networks
in terms of the deterministic dynamics of concentrations, we model the dynamics
of the probability of a given copy number of the reactants in single cells.
Most of the modeling activity of the last decade has centered on stochastic
simulation of individual realizations, i.e., Monte-Carlo methods for generating
stochastic time series. Here we review the mathematical description in terms of
probability distributions, introducing the relevant derivations and
illustrating several cases for which analytic progress can be made either
instead of or before turning to numerical computation
In vitro studies on odontogenic tumors

Ameloblastomas are uncommon benign neoplasms of the jaws. They originate from dental epithelial cells, but they are not capable of mineralizing or forming enamel. The study of these tumors is limited to live tissue collected from patients during scheduled surgery. Ameloblastomas grow slowly in vivo and this property is translated to their behavior in vitro. Here, we describe the methods to culture ameloblastomas in organotypic cultures, as well as to isolate stem/progenitor cells from these tumors