bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of Antisera Using Fusion Proteins
Authors: Dodson Sara E. , Heilman Craig J. , Kahn Richard A. , Levey Allan I. 
Published: July 2007

AbstractThis unit details the use of bacterially produced fusion proteins for the production of antisera, allowing for the large‐scale generation of affinity‐purified antibodies to specific, targeted epitopes. The use of pET vectors containing a polyhistidine (His) or glutathione‐S‐transferase (GST) tag to construct bacterial expression plasmids are provided as prototypical examples of fusion protein methodology. The basic protocols provided in this unit describe: (1) transformation of E. coli for high‐yield production of soluble fusion protein, (2) purification of soluble fusion proteins for use in immunization using chelated nickel or glutathione affinity chromatography (for His‐ and GST‐tagged fusion proteins, respectively), (3) immunization of rabbits with purified fusion protein and collection of antisera, and (4) characterization of antisera for antibody specificity using immunoblotting techniques. Support protocols describe the purification of His‐tagged insoluble fusion proteins for animal immunization and the construction and use of affinity columns for purifying antibodies using soluble fusion proteins. Curr. Protoc. Neurosci. 40:5.7.1‐5.7.26. © by John Wiley & Sons, Inc.
E. coli Genome Manipulation by P1 Transduction
Authors: Thomason Lynn C. , Costantino Nina , Court Donald L. 
Published: July 2007

AbstractThis unit describes the procedure used to move portions of the
E. coli
genome from one genetic variant to another. Fragments of ∼100 kb can be transferred by the P1 bacteriophage. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome. Curr. Protoc. Mol. Biol. 79:1.17.1‐1.17.8. © 2007 by John Wiley & Sons, Inc.
Paired‐End diTagging for Transcriptome and Genome Analysis
Authors: Ng Patrick , Wei Chia‐Lin , Ruan Yijun 
Published: July 2007

AbstractThe Paired‐End diTagging (PET) procedure enables one to obtain sequence information from both termini of any contiguous DNA fragment. This is achieved by a series of enzymatic manipulations that introduce MmeI sites directly flanking each DNA insert during the construction of a plasmid library. Subsequent MmeI digestion and self‐ligation results in the production of covalently‐linked paired‐end ditags (PETs) that can be extracted and then concatenated for efficient sequencing. By mapping the PET sequences to assembled genomes, the original DNA fragments from which the PETs were derived can be precisely localized. This unit details two applications of PET technology. In GIS‐PET, ditagging of mRNA converted to full‐length cDNA enables whole‐transcriptome analysis, including novel gene identification, gene prediction validation, and gene expression studies. In ChIP‐PET, ditagging of chromatin immunoprecipitation–enriched genomic DNA fragments enables the global mapping of transcription factor binding sites. A recent innovation (Multiplex Sequencing of Paired‐End ditags; MS‐PET) enables PETs to be sequenced using high‐throughput 454 sequencing, greatly increasing the amount of data that can be collected in each run. Curr. Protoc. Mol. Biol. 79:21.12.1‐21.12.42. © 2007 by John Wiley & Sons, Inc.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Bursik Steve 
Published: July 2007

AbstractThe pursuit of scientific knowledge has been considerably advanced by the use of biochemical molecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules, and/or the radiolabeled products that result from biochemical reactions in which the parent molecule was involved, can be traced using a variety of instruments that detect radioactivity. This appendix begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a molecular biology laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detection and imaging methods used in experimental analysis are reviewed. Finally, an outline of an orderly response to a spill of radioactive material is presented. Curr. Protoc. Mol. Biol. 79:A.1F.1‐A.1F.18. © 2007 by John Wiley & Sons, Inc.
Fosmid Libraries for Genomic Structural Variation Detection
Authors: Donahue William F. , Ebling Heather M. 
Published: July 2007

AbstractFosmid libraries have demonstrated their utility for a number of applications. These include filling gaps between BACs and small insert libraries in sequence assemblies, performing hybridization/screening studies to isolate functional elements within the genome (Vergin et al., 1998), and detecting insertions, deletions, and rearrangements in structural variation studies (Tuzun et al., 2005). This unit covers the basic methodologies for the construction of fosmid libraries with tight insert sizes suitable for these applications. Basic Protocol 1 covers the shearing, size selection, and recovery of DNA from a pulsed‐field gel. Basic Protocol 2 covers the cloning of insert DNA into the fosmid vector, packaging of DNA into infective phage particles, and the infection/transformation of bacteria. A commentary section is provided, which outlines many of the critical parameters involved in fosmid library construction, along with some additional background information and a section discussing anticipated results. Curr. Protoc. Hum. Genet. 54:5.20.1‐5.20.18. © 2007 by John Wiley & Sons, Inc.
Analysis of Expression Data: An Overview
Authors: Grewal Anoop , Lambert Peter , Stockton Jordan 
Published: July 2007

AbstractAfter providing a brief introduction to microarray chips and experimental details, this overview discusses analysis techniques. Data analysis from microarray experiments generally involves two parts: acquiring and normalizing the data, and interpreting it. This unit focuses mostly on the latter, as it is less technology‐specific.Curr. Protoc. Hum. Genet. 54:11.4.1‐11.4.14. © 2007 by John Wiley & Sons, Inc.
Quantification of Creatine and Guanidinoacetate Using GC‐MS and LC‐MS/MS for the Detection of Cerebral Creatine Deficiency Syndromes
Authors: Young Sarah , Struys Eduard , Wood Tim 
Published: July 2007

AbstractInherited defects in creatine biosynthesis and cellular uptake are neurometabolic disorders characterized by seizures, developmental delay, mental retardation, autistic‐like behavior, and creatine deficiency in the brain. Metabolic screening of these disorders is possible using analytical techniques that quantify creatine and its precursor guanidinoacetate in urine, plasma, or cerebrospinal fluid (CSF). Elevated creatine in urine is suggestive of a deficiency of the X‐linked creatine transporter, SLC6A8. Decreased or elevated levels of guanidinoacetate in urine, plasma, or CSF suggest deficiencies of the creatine biosynthetic enzymes, arginine:glycine amidinotransferase (AGAT) or guanidinoacetate methyltransferase (GAMT), respectively. This unit describes three stable isotope dilution–mass spectrometric methods for analyzing creatine and guanidinoacetate. Gas chromatography/mass spectrometry with negative‐ion chemical ionization is a highly sensitive technique, suitable for detection of low analyte levels resulting from AGAT deficiency and in CSF. The two liquid chromatography–tandem mass spectrometric approaches are amenable to high‐throughput screening and have simple sample preparation requirements. Curr. Protoc. Hum. Genet. 54:17.3.1‐17.3.18. © 2007 by John Wiley & Sons, Inc.
Polychromatic Analysis of Mitochondrial Membrane Potential Using JC‐1
Authors: Lugli Enrico , Troiano Leonarda , Cossarizza Andrea 
Published: July 2007

AbstractDissipation of the mitochondrial membrane potential (ΔΨm) has been accepted as a hallmark of some apoptotic processes. Depending on the model studied, it can occur during the early stages of cell death or later on, after loss of DNA integrity. Discordant data in the literature could be the consequence of using improper probes such as rhodamine 123 (R123) or DiOC6(3), which are not always appropriate for the study of ΔΨm. The lipophilic cation JC‐1, a specific probe for measuring ΔΨm, is currently the gold standard. Thanks to recently developed instruments and additional probes for cell surface and intracellular markers, it can be used in polychromatic flow cytometric assays to simultaneously detect ΔΨm along with other biological parameters.
High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining
Authors: Edwards Bruce S. , Ivnitski‐Steele Irena , Young Susan M. , Salas Virginia M. , Sklar Larry A. 
Published: July 2007

AbstractThis unit describes a system for the automated high‐throughput analysis of cell cytotoxicity in 96‐well and 384‐well microplates. Discrete cell cultures are analyzed at rates of 40/min (∼2.5 min/96 wells, ∼10 min/384 wells) and cytotoxicity is quantified on the basis of a combination of propidium iodide (PI) fluorescence analysis and cell counting performed by the flow cytometer. Only 2 µl is aspirated from a culture for analysis so that assays can be performed in small volumes to minimize reagent cost and usage.
Live‐Animal Imaging of Renal Function by Multiphoton Microscopy
Authors: Dunn Kenneth W. , Sutton Timothy A. , Sandoval Ruben M. 
Published: July 2007

AbstractIntravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high‐resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high‐speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis.
Proteomic Analysis of Pluripotent Stem Cells
Authors: Bendall Sean C. , Booy Aaron T. , Lajoie Gilles 
Published: July 2007

AbstractMass spectrometry (MS)–based proteomics has become one of the most powerful tools for identifying expressed proteins, providing quick insights into molecular and cellular biology. Traditionally, proteins isolated by either one‐ or two‐dimensional gel electrophoresis are digested with a site specific protease. The resulting peptides are subject to one of two forms of analysis: (1) matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS, where a “mass fingerprint” of all the peptides in a sample is generated, or (2) electrospray ionization tandem MS (ESI‐MS/MS), where a mass fragmentation spectra is generated for each peptide in a sample. The resulting mass information is then compared to that of a theoretical database created with available genomic sequence information. This unit provides protocols for this type of assessment in embryonic stem cells (ESCs). Curr. Protoc. Stem Cell Biol. 2:1B.1.1‐1B.1.33. © 2007 by John Wiley & Sons, Inc.
Differentiation of Embryonic Stem Cells into Cartilage Cells
Authors: Singh Khillan Jaspal 
Published: July 2007

AbstractEmbryonic stem (ES) cells have complete potential to form all types of cells. Although these cells have indefinite capacity for self‐renewal, the mechanisms that control their lineage‐restricted differentiation are not well understood. Due to their potential to form all types of cells, these cells are expected to have applications in regenerative medicine to cure human diseases. Osteoarthritis (OA) is a degenerative disease of articular cartilage of weight bearing joints. Approximately twenty million people suffer from this debilitating disease. Therefore, the induced differentiation of ES cells into cartilage‐producing cells will have potential application to cure OA. This unit describes a system to induce differentiation of a high percentage of ES cells into mesenchymal cells that differentiate into chondrocytes, the cartilage‐producing cells. A quantitative production of chondrocytes can be a powerful resource to alleviate the suffering of those patients with OA. Furthermore, this can be an excellent system to investigate the upstream events of cell‐restricted differentiation during the inaccessible period of development. Curr. Protoc. Stem Cell Biol. 2:1F.1.1‐1F.1.13. © 2007 by John Wiley & Sons, Inc.
Differentiation of Human Embryonic Stem Cells to Cardiomyocytes by Coculture with Endoderm in Serum‐Free Medium
Authors: Mummery Christine L. , Ward Dorien , Passier Robert 
Published: July 2007

AbstractMany of the applications envisaged for human embryonic stem cells (hESC) undergoing cardiomyogenesis require that the differentiation procedure is robust and high yield. For many hESC lines currently available this is a challenge; beating areas are often obtained but subsequent analysis shows only few (<1%) cardiomyocytes actually present. Here the authors provide a protocol based on serum‐free coculture with a mouse endoderm‐like cell line (END2), which yields cultures containing on average 25% cardiomyocytes for two widely available hESC lines, hES2 and hES3. The authors also provide a variant on the protocol based on growth of hESC aggregates/embryoid bodies in END2‐conditioned medium and a method for dissociating beating aggregates without compromising cardiomyocyte viability so that they can be used for transplantation into animals or further (electrophysiological) analysis. Curr. Protoc. Stem Cell Biol. 2:1F.2.1‐1F.2.14. © 2007 by John Wiley & Sons, Inc.
Magnetic and Transparent Composites by Linking Liquid Crystals to Ferrite Nanoparticles through Covalent Networks
Authors: Song H. M. , Kim J. C. , Hong J. H. , Lee Y. B. , Choi J. , Lee J. I. , Kim W. S. , Kim J.‐H. , Hur N. H. 
Published: July 2007

AbstractThe fabrication of transparent, flexible, and optically homogeneous magnetic composites containing ferrite (Fe3O4) nanoparticles, liquid crystals (LCs), and siloxane backbones is reported. The transparent magnets are achieved by covalently bonding LCs to the siloxane backbones and then linking them to dopamine‐functionalized ferrite nanocrystals. They exhibit simultaneous high transparency and strong magnetic properties. A remarkable feature of these films is that the surface morphology of the LC‐attached ferrite films can be tuned by an external magnetic field, demonstrating a striped surface in the direction of the field. We show that the LC‐attached film can act as an alignment layer to orient LCs, enabling the development of LC alignment surfaces on the basis of these nanomagnet–LC polymer composites.
Origin of the Reduced Fill Factor and Photocurrent in MDMO‐PPV:PCNEPV All‐Polymer Solar Cells
Authors: Mandoc M. M. , Veurman W. , Koster L. J. A. , de Boer B. , Blom P. W. M. 
Published: July 2007

AbstractThe photogeneration mechanism in blends of poly[2‐methoxy‐5‐(3′,7′‐dimethyloctyloxy)‐1,4‐phenylene vinylene] (MDMO‐PPV) and poly[oxa‐1,4‐phenylene‐(1‐cyano‐1,2‐vinylene)‐(2‐methoxy‐5‐(3′,7′‐dimethyloctyloxy)‐1,4‐phenylene)‐1,2‐(2‐cyanovinylene)‐1,4‐phenylene] (PCNEPV) is investigated. The photocurrent in the MDMO‐PPV:PCNEPV blends is strongly dependent on the applied voltage as a result of a low dissociation efficiency of the bound electron–hole pairs. The dissociation efficiency is limited by low carrier mobilities, low dielectric constant, and the strong intermixing of the polymers, leading to a low fill factor and a reduced photocurrent at operating conditions. Additionally, electrons trapped in the PCNEPV phase recombine with the mobile holes in the MDMO‐PPV phase at the interface between the two polymers, thereby affecting the open‐circuit voltage and increasing the recombination losses. At an intensity of one sun, Langevin recombination of mobile carriers dominates over trap‐assisted recombination.
Surface‐Enhanced Raman Scattering Using Microstructured Optical Fiber Substrates
Authors: Amezcua‐Correa A. , Yang J. , Finlayson C. E. , Peacock A. C. , Hayes J. R. , Sazio P. J. A. , Baumberg J. J. , Howdle S. M. 
Published: July 2007

AbstractMicrostructured optical fibers (MOFs) represent a promising platform technology for fully integrated next generation surface enhanced Raman scattering (SERS) sensors and plasmonic devices. In this paper we demonstrate silver nanoparticle substrates for SERS detection within MOF templates with exceptional temporal and mechanical stability, using organometallic precursors and a high‐pressure chemical deposition technique. These 3D substrates offer significant benefits over conventional planar detection geometries, with the long electromagnetic interaction lengths of the optical guided fiber modes exciting multiple plasmon resonances along the fiber. The large Raman response detected when analyte molecules are infiltrated within the structures can be directly related to the deposition profile of the nanoparticles within the MOFs via electrical characterization.
Multifunctional Hydrogels that Promote Osteogenic Human Mesenchymal Stem Cell Differentiation Through Stimulation and Sequestering of Bone Morphogenic Protein 2
Authors: Benoit D. S. W. , Collins S. D. , Anseth K. S. 
Published: July 2007

AbstractThe extracellular environment controls many cellular activities thereby linking external material cues to internal cell function. By better understanding these processes, synthetic extracellular material niches can be tailored to present cells with highly regulated physical and/or chemical cues that promote or suppress selected cell functions. Here, poly(ethylene glycol) (PEG) hydrogels are functionalized with fluvastatin‐releasing grafts and growth‐factor‐binding heparin domains to enable the dynamic exchange of information between the material and cells from the outside‐in and inside‐out (i.e., bidirectional signaling). By incorporating a fluvastatin‐releasing graft and carefully controlling the dose and temporal release, materials are designed to promote bone morphogenic protein 2 (BMP2) and alkaline phosphatase (ALP) production by human mesenchymal stem cells (hMSCs). When the release of fluvastatin is controlled to occur over 2 weeks, BMP2 and ALP production is increased 2.2‐fold and 1.7‐fold, respectively, at day 28 compared to hMSCs cultured in the absence of fluvastatin. By introducing a heparin functionality into the gel to sequester and localize the hMSC‐produced BMP2, the osteogenic differentiation of hMSCs is further augmented over fluvastatin delivery alone. Osteopontin and core‐binding factor α1 gene expression is sixfold and fourfold greater, respectively, for hMSCs exposed to fluvastatin in the presence of the heparin functionalities. These results demonstrate how multifunctional gels that interact with cells in a bidirectional manner can efficiently promote selected cell functions, such as the osteogenic differentiation of hMSCs.
Fluorescent, Carrier‐Trapping Dopants for Highly Efficient Single‐Layer Polyfluorene LEDs
Authors: Zhao Z. , Li J.‐H. , Lu P. , Yang Y. 
Published: July 2007

AbstractTwo fluorescent molecules with an alkynylanthracene core and pyrene end‐cappers have been synthesized and fully characterized. Carbazole moieties are introduced into one molecule at the C9 position of the fluorene linkages to enhance the hole‐transport ability of the molecule and to reduce intermolecular interactions. Both compounds exhibit high thermal stabilities and narrow energy bandgaps. Single‐layer polymer light‐emitting diodes (PLEDs) based on poly(9,9‐dioctylfluorene) (PFO) doped with the synthesized compounds exhibit excellent performance. A PLED with 0.2 % of dopant 7 had a high luminance efficiency of 10.7 ± 0.3 cd A–1 as well as a brightness of 1400 cd m–2 at a current density of 13 mA cm–2, and a low turn on voltage (3.1 V) at a brightness of 10  cd m–2. A maximum brightness of 20 500 ± 1400 cd m–2 at 7 V was also measured. The high efficiency of the device's performance is attributed to the good electron and hole trapping ability of the dopants, which possess suitable energy levels as compared to those of PFO.
TNF‐α inhibitor etanercept and hematologic malignancies: Report of a case and review of the literature
Authors: Nair Bijay , Raval Girindra , Mehta Paulette 
Published: July 2007

AbstractWe report here a 57‐year‐old man treated with etanercept for 6 months for psoriasis who developed myelodysplasia with acute myeloid leukemia. Leukemia cells had distinct karyotype associated with poor prognosis. The patient did not respond to cytosine arabinoside 100 mg/m2 continuous infusion over 7 days with daunorubicin 45 mg/m2 daily for 3 days. He also did not respond to salvage induction therapy with gemtuzumab (6 mg/m2 on day 1 and 4 mg/m2 on day 8) and intravenous continuous infusion cytosine arabinoside 200 mg/m2. We review other cases of lymphoma and leukemia associated with tumor necrosis factor inhibitors and suggest mechanisms by which inhibition of the TNF‐α family may predispose to cancer. We also suggest that all patients being considered for TNF‐α treatment be screened for hematologic malignancies or premalignancies with blood counts and bone marrow aspirates/biopsies if indicated. Am. J. Hematol., 2007. © 2007 Wiley‐Liss, Inc.
Pyruvate kinase deficiency associated with severe liver dysfunction in the newborn
Authors: Raphaël Martine F. , Van Wijk Richard , Schweizer Joachim J. , Schouten‐van Meeteren Netteke A.Y. , Kindermann Angelika , van Solinge Wouter W. , Smiers Frans J. 
Published: July 2007

AbstractSignificant hyperbilirubinaemia, anemia, and splenomegaly are common features in patients with severe haemolysis due to pyruvate kinase (PK) deficiency. Until now, severe neonatal PK deficiency has not been associated with fatal liver disease at this age. We present two neonatal cases of severe PK deficiency complicated with progressive fatal liver disease. The patients presented with severe haemolysis, progressive cholestasis, and hepatosplenomegaly, and both patients ultimately developed liver failure at a very young age. Despite extensive investigations, no specific explanation for liver disease and failure was found. We suggest that the PK deficiency itself directly led to liver dysfunction. Am. J. Hematol., 2007. © 2007 Wiley‐Liss, Inc.