bluuebunny/crossref_metadata_2025_split huggingface dataset:
Construction and Expression of Oligonucleotide-Based Antisense Cassettes: FIGURE 1.
Authors: Amarzguioui Mohammed 
Published: February 2011

INTRODUCTIONThis protocol describes simple and robust methods for the construction and cloning of expression constructs that can be used to deliver antisense effectors, exemplified by the hammerhead ribozyme and short hairpin RNA (shRNA), into cultured cell lines. The protocol also describes the construction of reporter vectors to be used for target validation. Due to the variable efficacy of the antisense effectors, it is advisable to design multiple constructs targeting different sites. Once the different constructs have been generated, their relative efficacy can be readily determined through reporter cotransfection experiments, in which a stretch of cDNA encompassing all target sites is cloned directionally into the 3′ UTR (untranslated region) of a reporter (psiCheck-2; Promega). Successful cleavage of the target site results in degradation of reporter mRNA, with concomitant decrease in translated product, which is detected by a luminescence-based assay system.
Preparation of Mice for Long-Term Intravital Imaging of the Mammary Gland: FIGURE 1.
Authors: Ewald Andrew J. , Werb Zena , Egeblad Mikala 
Published: February 2011

INTRODUCTIONGenetic studies and tumor biopsies have shown the importance of stromal components for cancer progression, but much remains to be learned about the dynamic interactions among the distinct tumor components within live animals. One challenge of studying cell behavior in progressively developing tumors has been the difficulty of maintaining live mice on the microscope stage. To prepare mice for long-term intravital imaging, auxiliary equipment is necessary to enable and to control anesthesia (such as the anesthesia gas mixer itself, a gas humidifier, indwelling lines for saline, and heat blanket). The other important component is to gain optical access to the mammary gland. This protocol describes a surgical technique that creates a skin flap with the mammary gland. The method is relatively easily taught, does not compromise the peritoneal cavity or any major blood vessels, and is generally well tolerated by the mice. There is minimal inflammatory response to the surgery itself if the solutions and tools are sterile, the surgical work area is clean, and aseptic techniques are used. This protocol works well for a single long-term image session, but does not enable repeated imaging sessions. For such approaches, methods for implanting imaging windows over the inguinal mammary gland should be used instead.
General Approach to Adoptive Transfer and Cell Labeling for Immunoimaging
Authors: Matheu Melanie P. , Cahalan Michael D. , Parker Ian 
Published: February 2011

INTRODUCTIONImaging the single-cell dynamics of the immune system within an intact environment requires the ability to look deep inside tissues and organisms with spatial and temporal resolutions adequate to track cell morphology, motility, and signaling processes, all while minimizing perturbation of the system under study. Fluorescence techniques are highly suited for this purpose, permitting both labeling of specific cells, organelles, or proteins and functional readout of physiological events, and two-photon microscopy allows these processes to be visualized within native tissue environments. Adoptive transfer, as described here, is the generally preferred method for introducing labeled cells of interest into a host animal for immunoimaging. Cells are derived from a donor animal with a genetic background identical to that of the host and can either be endogenously fluorescent (e.g., isolated from a transgenic mouse expressing the fluorescent protein) or can be labeled before transfer. Typically, transferring 2-6 × 106 labeled cells of a given type results in an appropriate cell density for two-photon imaging.
In Vivo Lymph Node Imaging: FIGURE 1.
Authors: Matheu Melanie P. , Cahalan Michael D. , Parker Ian 
Published: February 2011

INTRODUCTIONImaging the single-cell dynamics of the immune system within an intact environment requires the ability to look deep inside tissues and organisms with spatial and temporal resolutions adequate to track cell morphology, motility, and signaling processes, all while minimizing perturbation of the system under study. Fluorescence techniques are highly suited for this purpose, permitting both labeling of specific cells, organelles, or proteins and functional readout of physiological events, and two-photon microscopy allows these processes to be visualized within native tissue environments. Although intravital immunoimaging requires invasive surgery to access the tissue or organ of interest, these preparations have the advantage of keeping blood and lymphatic flow intact. In the procedure for in vivo imaging of the inguinal lymph node (LN) described here, a rubber O-ring is glued onto a skin flap so as to form a fluid chamber for the dipping microscope objective. This arrangement also helps stabilize the tissue mechanically without interrupting circulation.
Solution Protein Digest
Authors: Link Andrew J. , LaBaer Joshua 
Published: February 2011

INTRODUCTIONThis protocol describes the trypsin digestion of proteins in solution. Proteolytic digestion is critical for mass spectrometric sequencing because it generates peptides that have molecular weights within the mass range of the mass spectrometer. For tandem mass spectrometry, the digestion is typically performed using the protease trypsin, which cleaves at the carboxy-terminal side of lysine (K) and arginine (R) residues with the exception of K-P and R-P sites. There are numerous variations for digestion of proteins in solution. Because trypsin is a rather robust enzyme, tryptic digestions can be performed under various denaturing conditions (4 M urea, 2 M guanidine-HCl, 0.1% SDS, and >10% acetonitrile). Solubilization of proteins in 8 M urea and then dilution to 2-4 M urea before adding trypsin is commonly used to digest proteins that are difficult to solubilize. For most digestion protocols, including the protocol described in this article, cysteine residues are reduced and alkylated prior to digestion. The reduction and alkylation of disulfide bonds help to denature proteins, making their proteolytic sites more accessible to proteolysis. The The modified trypsin (TPCK-treated) used in this protocol is a serine endopeptidase prepared by treating trypsin with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inactivate any remaining chymotryptic activity. TPCK acetylates the ϵ-amino groups of lysine residues to limit autolysis. Modified trypsin cleaves at K-P and R-P bonds at a much slower rate than other amino acid residues.
Labeling Lysosomes in Live Cells with Neutral Red
Authors: Chazotte Brad 
Published: February 2011

INTRODUCTIONLysosomes, although diverse in shape and size, are membranous sacs containing more than 40 different acid hydrolases. The enzymes operate optimally at the acidic pH (~5) found in the lysosome. A number of the fluorescent approaches to visualizing lysosomes make use of their acidic pH. Commonly used probes include neutral red, DAMP (N-(3-[2,4-dinitrophenyl amino] propyl)-N-(3-aminopropyl)methylamine), and acridine orange (a DNA stain). This protocol describes the labeling of lysosomes in live cells with neutral red.
Microproteomics: Quantitative Proteomic Profiling of Small Numbers of Laser-Captured Cells
Authors: Roulhac Petra L. , Ward James M. , Thompson J. Will , Soderblom Erik J. , Silva Michael , Moseley M. Arthur , Jarvis Erich D. 
Published: February 2011

INTRODUCTIONDuring the last decade, significant progress in the analysis of whole genomes and transcriptomes has triggered efforts to analyze the proteome. Advancements in protein extraction, purification, and identification have been driven by the development of mass spectrometers with greater sensitivity and resolution. Nevertheless, comparative and quantitative proteomic technologies have not progressed to the extent of genomic and transcriptomic technologies for accessing gene expression differences. Unlike the genome, which is similar throughout all cells in a given organism, the proteome varies in different cells. Also, there is no self-replicating amplification mechanism for proteins such as the polymerase chain reaction (PCR) for DNA. Therefore, developing methods that extract, separate, detect, and identify proteins from extremely small samples are needed. The advent of laser capture microdissection (LCM) has expanded the analytical capabilities of proteomics. LCM has proven an effective technique to harvest pure cell populations from tissue sections. This protocol describes a microproteomic platform that uses nanoscale liquid chromatography/tandem mass spectrometry (nano-LC-MS/MS) to simultaneously identify and quantify hundreds of proteins from LCMs of tissue sections from small tissue samples containing as few as 1000 cells. The LCM-dissected tissues are subjected to protein extraction, reduction, alkylation, and digestion, followed by injection into a nano-LC-MS/MS system for chromatographic separation and protein identification. The approach can be validated by secondary screening using immunological techniques such as immunohistochemistry or immunoblots.
SL Trans-Splicing in a Caenorhabditis elegans In Vitro Extract
Authors: Lasda Erika L. , Kuersten Scott , Blumenthal Thomas 
Published: February 2011

INTRODUCTIONCaenorhabditis elegans is one of a number of organisms in which spliced leader (SL) trans-splicing contributes to the maturation of many pre-mRNA transcripts. C. elegans and related nematodes have two distinct classes of SL RNA—SL1 and SL2—that are used differentially for different classes of pre-mRNAs; namely, SL1 trans-splices non-operon genes and first genes in operons, while SL2 splices downstream operon genes. In vitro RNA splicing has previously been used to analyze substrate RNA sequences, required protein and RNA factors, and splicing mechanisms in a variety of systems. This protocol describes an in vitro splicing assay using C. elegans extract that promotes both cis- and trans-splicing of substrate RNAs. It has the ability to correctly specify and detect SL1 versus SL2 trans-splicing. Operon-derived substrates splice SL2, while substrates with an intron-like sequence followed by a 3′ trans-splice site (outron) splice SL1. Unlabeled T7-transcribed RNA substrates are incubated with crude embryonic extract and trans-spliced by endogenous SL small nuclear ribonucleoproteins (snRNPs). Spliced RNA is analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Specific RT and PCR primers ensure detection of trans-spliced substrate RNA and allow a means to distinguish between SL1 and SL2 trans-spliced products. Substrates containing introns are also cis-spliced. All reaction products are ATP-dependent.
Family-Based Genetic Association Tests
Authors: Martin Eden R. , Rampersaud Evadnie 
Published: February 2011

INTRODUCTIONGenetic studies in families have historically focused on tests of linkage. These tests measure the concordance between the pattern of segregation of a genetic marker allele with the occurrence of a disease phenotype within families and provide information about the amount of genetic recombination between a marker locus and a putative disease locus. Thus, linkage is a statement about transmission of gametes from parents to offspring. In contrast, association is a statement about the population of gametes. Frequencies of marker alleles in disease and nondisease individuals give us information about the amount of association, or correlation, between marker allele and disease status. Family data not only provide us information about linkage by looking at transmissions within families, but also give us information about association by looking at allelic state and the presence of disease phenotype across families. This chapter describes the evolution of family-based tests of association as well as the variety of applications for these tests.
Dynamic, Long-Term In Vivo Imaging of Tumor–Stroma Interactions in Mouse Models of Breast Cancer Using Spinning-Disk Confocal Microscopy
Authors: Ewald Andrew J. , Werb Zena , Egeblad Mikala 
Published: February 2011

INTRODUCTIONTumors contain many components in addition to the cancer cells, including blood vessels, fibroblasts, and immune cells. Genetic studies and tumor biopsies have generated insights into the importance of these stromal components for cancer progression. However, it remains a challenge to reveal the dynamic interactions among the distinct tumor components within live animals. Studies involving multiphoton microscopy allow direct imaging of cellular movement in live mice, but multiphoton microscopy is expensive, complex, and usually relies on a single excitation wavelength for all fluorophores. This article describes a method for intravital imaging using a microlensed spinning-disk confocal microscope. Although tissue penetration with spinning-disk confocal microscopy is lower than with multiphoton microscopy, image acquisition with this method is very rapid, so artifacts from respiratory motion are avoided. Photobleaching and phototoxicity are low, and multicolor acquisition is cheaper and easier than with multiphoton microscopy. This article discusses various aspects of experimental setup, as well as methods for addressing technical barriers, such as generating and working with multiple tumor microenvironments within individual live mice, image collection, and long-term anesthesia.
High-Speed Optical Coherence Tomography Imaging of the Beating Avian Embryonic Heart
Authors: Gu Shi , Jenkins Michael W. , Watanabe Michiko , Rollins Andrew M. 
Published: February 2011

INTRODUCTIONCongenital heart defects (CHDs) affect thousands of newborns each year in the United States. Recent research using animal model systems indicates that the abnormal function of the early tubular heart precedes structural defects such as septal defects. Optical coherence tomography (OCT) is an imaging modality that can provide high spatial and temporal resolution to study both the structure and the function of the tubular heart. With technical advances in OCT imaging speed, especially with frequency domain OCT and image-based retrospective gating, it is now possible to image a beating avian embryonic heart in three dimensions under physiological conditions and follow morphogenesis over critical periods of developmental time. These technological advances have already revealed novel aspects of heart development. By expanding our understanding of heart development, research using OCT technology combined with other imaging modalities may eventually lead to strategies to predict, treat, and even prevent CHDs.
MYB46 Modulates Disease Susceptibility to Botrytis cinerea in Arabidopsis
Authors: Ramírez Vicente , Agorio Astrid , Coego Alberto , García-Andrade Javier , Hernández M. José , Balaguer Begoña , Ouwerkerk Pieter B.F. , Zarra Ignacio , Vera Pablo 
Published: February 2011

Abstract
In this study, we show that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis-element located in the 5′ promoter region of the pathogen-induced Ep5C gene, which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knockdown mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence, our results substantiate that defense-related signaling pathways and cell wall integrity are interconnected and that MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense.
Pollen Semi-Sterility1Encodes a Kinesin-1–Like Protein Important for Male Meiosis, Anther Dehiscence, and Fertility in Rice
Authors: Zhou Shirong , Wang Yang , Li Wanchang , Zhao Zhigang , Ren Yulong , Wang Yong , Gu Suhai , Lin Qibing , Wang Dan , Jiang Ling , Su Ning , Zhang Xin , Liu Linglong , Cheng Zhijun , Lei Cailin , Wang Jiulin , Guo Xiuping , Wu Fuqing , Ikehashi Hiroshi , Wang Haiyang , Wan Jianmin 
Published: February 2011

AbstractIn flowering plants, male meiosis produces four microspores, which develop into pollen grains and are released by anther dehiscence to pollinate female gametophytes. The molecular and cellular mechanisms regulating male meiosis in rice (Oryza sativa) remain poorly understood. Here, we describe a rice pollen semi-sterility1 (pss1) mutant, which displays reduced spikelet fertility (~40%) primarily caused by reduced pollen viability (~50% viable), and defective anther dehiscence. Map-based molecular cloning revealed that PSS1 encodes a kinesin-1–like protein. PSS1 is broadly expressed in various organs, with highest expression in panicles. Furthermore, PSS1 expression is significantly upregulated during anther development and peaks during male meiosis. The PSS1–green fluorescent protein fusion is predominantly localized in the cytoplasm of rice protoplasts. Substitution of a conserved Arg (Arg-289) to His in the PSS1 motor domain nearly abolishes its microtubule-stimulated ATPase activity. Consistent with this, lagging chromosomes and chromosomal bridges were found at anaphase I and anaphase II of male meiosis in the pss1 mutant. Together, our results suggest that PSS1 defines a novel member of the kinesin-1 family essential for male meiotic chromosomal dynamics, male gametogenesis, and anther dehiscence in rice.
Drug hypersensitivity: Flare‐up reactions, cross‐reactivity and multiple drug hypersensitivity
Authors: PICHLER Werner J. , DAUBNER Barbara , KAWABATA Tom 
Published: February 2011

AbstractIn drug hypersensitivity, change of drug treatment and continuation with a new drug may result in reappearance of drug hypersensitivity symptoms. This is not uncommon in patients with chronic infections requiring continued and long‐lasting antibiotic treatments. For the clinician, the question arises whether these symptoms are due to cross‐reactivity, are due to a new sensitization or are a reflection of a multiple drug hypersensitivity syndrome. Based on the p‐i concept (pharmacological interaction with immune receptors), we propose that the efficient stimulation of T cells by a drug is the sum of drug–T‐cell receptor affinity and readiness of the T cell to react, and therefore not constant. It heavily depends on the state of underlying immune activation. Consequently, drug hypersensitivity diseases, which go along with massive immune stimulations and often high serum cytokine values, are themselves risk factors for further drug hypersensitivity. The immune stimulation during drug hypersensitivity may, similar to generalized virus infections, lower the threshold of T‐cell reactivity to drugs and cause rapid appearance of drug hypersensitivity symptoms to the second drug. We call the second hypersensitivity reaction a “flare‐up” reaction; this is clinically important, as in most cases the second drug may be tolerated again, if the cofactors are missing. Moreover, the second treatment is often too short to cause a relevant sensitization.
Muscle transcriptome profiling in divergent phenotype swine breeds during growth using microarray and RT‐PCR tools
Authors: D’Andrea M. , Dal Monego S. , Pallavicini A. , Modonut M. , Dreos R. , Stefanon B. , Pilla F. 
Published: February 2011

SummaryUsing an array consisting of 10 665 70‐mer oligonucleotide probes, the longissimus dorsi muscle tissue expression during growth in nine pigs belonging to Casertana (CT), an autochthonous breed characterized by slow growth and a massive accumulation of backfat, was compared with that of two cosmopolitan breeds, Large White (LW) and a crossbreed (CB; Duroc × Landrace × Large White). The results were validated by real‐time PCR. All animals were of the same age and were raised under the same environmental conditions. Muscle tissues were collected at 3, 6, 9 and 11 months of age, and a total of 173 genes showed significant differential expression between CT and the cosmopolitan genetic types at 3 months of age. Time series cluster analysis indicated that the CT breed had a different pattern of gene expression compared with that of the LW and the CB. Four of the eight clusters highlighted the gene differences between CT and the other two breeds, which were further supported by statistical analyses: clusters 4 and 5 contained a total of 71 genes that were underexpressed at 3 months of age, and cluster 3 and cluster 7 included 28 and 42 genes respectively that were overexpressed at 3 months of age. As expected, differentially expressed genes belonged to the category of genes coding for contractile fibres and transcription factors involved in muscle development and differentiation. These findings highlight muscle expression genes during pig growth and are useful to understand the genetic meaning of the different developmental rates.
Transcription specificity of the class Ib genes SLA‐6, SLA‐7 and SLA‐8 of the swine major histocompatibility complex and comparison with class Ia genes
Authors: Kusza S. , Flori L. , Gao Y. , Teillaud A. , Hu R. , Lemonnier G. , Bősze Z. , Bourneuf E. , Vincent‐Naulleau S. , Rogel‐Gaillard C. 
Published: February 2011

SummaryOur aim was to analyse the transcription levels of the three non‐classical class Ib genes SLA‐6, SLA‐7 and SLA‐8 of the swine major histocompatibility complex in various tissues and conditions and to compare them to the transcription levels of classical class Ia genes. Twenty‐five adult tissues from two pig breeds, pig renal PK15 cells infected with the Pseudorabies virus, and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide or a mixture of phorbol myristate acetate and ionomycin were included in our study. Relative transcription was quantified by quantitative real‐time PCR. On average, in adult tissues and PBMCs and compared to SLA‐6, the transcription level of SLA‐Ia genes was 100–1000 times higher, the level of SLA‐8 was 10–20 times higher, and that of SLA‐7 was five times higher. Thus, SLA‐8 is the most transcribed SLA‐Ib gene, followed by the SLA‐7 and SLA‐6 genes. The highest transcription levels of SLA‐Ib transcripts were found in the lymphoid organs, followed by the lung and the digestive tract. The tissue variability of expression levels was widest for the SLA‐6 gene, with a 1:32 ratio between the lowest and highest levels in contrast to a 1:12 ratio for the SLA‐7 and SLA‐8 genes and a 1:16 ratio for the SLA‐Ia genes. During PK‐15 infection and PBMC stimulation, SLA‐Ia and SLA‐8 genes were downregulated, whereas SLA‐6 and SLA‐7 were upregulated, downregulated or not significantly modified. Our overall results confirm the tissue‐wide transcription of the three SLA‐Ib genes and suggest that they have complementary roles.