bluuebunny/crossref_metadata_2025 huggingface dataset:
The Wnt agonist R-spondin1 regulates systemic graft-versus-host disease by protecting intestinal stem cells
Authors: Takashima Shuichiro , Kadowaki Masanori , Aoyama Kazutoshi , Koyama Motoko , Oshima Takeshi , Tomizuka Kazuma , Akashi Koichi , Teshima Takanori
Published: February 2011

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT), and damage to the gastrointestinal (GI) tract plays a critical role in amplifying systemic disease. Intestinal stem cells (ISCs) play a pivotal role not only in physiological tissue renewal but also in regeneration of the intestinal epithelium after injury. In this study, we have discovered that pretransplant conditioning regimen damaged ISCs; however, the ISCs rapidly recovered and restored the normal architecture of the intestine. ISCs are targets of GVHD, and this process of ISC recovery was markedly inhibited with the development of GVHD. Injection of Wnt agonist R-spondin1 (R-Spo1) protected against ISC damage, enhanced restoration of injured intestinal epithelium, and inhibited subsequent inflammatory cytokine cascades. R-Spo1 ameliorated systemic GVHD after allogeneic BMT by a mechanism dependent on repair of conditioning-induced GI tract injury. Our results demonstrate for the first time that ISC damage plays a central role in amplifying systemic GVHD; therefore, we propose ISC protection by R-Spo1 as a novel strategy to improve the outcome of allogeneic BMT.
Construction and Expression of Oligonucleotide-Based Antisense Cassettes: FIGURE 1.
Authors: Amarzguioui Mohammed
Published: February 2011

INTRODUCTION

This 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

INTRODUCTION

Genetic 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

INTRODUCTION

Imaging 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

INTRODUCTION

Imaging 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

INTRODUCTION

This 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

INTRODUCTION

Lysosomes, 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

INTRODUCTION

During 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

INTRODUCTION

Caenorhabditis 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

INTRODUCTION

Genetic 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

INTRODUCTION

Tumors 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

INTRODUCTION

Congenital 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

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

In 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.
TrimethylamineN-oxide as a versatile cryoprotective agent in macromolecular crystallography
Authors: Mueller-Dieckmann Christoph , Kauffmann Brice , Weiss Manfred S.
Published: February 2011

The surge of macromolecular crystallography is intimately linked to the advent of methods for cryoprotecting macromolecular crystals. Only if crystals are kept cold during data collection can they withstand the effects of radiation damage during a diffraction experiment, especially at third-generation synchrotron sources. While a number of different cryoprotective agents and procedures have been described in the literature over the past three decades, it is still a time- and crystal-consuming process to establish and optimize a good cryo-condition for a specific crystal. In this study, trimethylamineN-oxide (TMAO) has been identified as a very versatile cryoprotectant for macromolecular crystals. In a few test cases it was shown that diffraction data collected from crystals treated with TMAO are of very good quality.
A flow-through reaction cell that couples time-resolved X-ray diffraction with stable isotope analysis
Authors: Wall Andrew J. , Heaney Peter J. , Mathur Ryan , Post Jeffrey E. , Hanson Jonathan C. , Eng Peter J.
Published: February 2011

A non-metallic flow-through reaction cell is described, designed forin situtime-resolved X-ray diffraction coupled with stable isotope analysis. The experimental setup allows the correlation of Cu isotope fractionation with changes in crystal structure during copper sulfide dissolution. This flow-through cell can be applied to many classes of fluid–mineral reactions that involve dissolution or ion exchange.
The structure factor in small-angle scattering and the effect of deviation from spherical symmetry
Authors: Hansen Steen
Published: February 2011

The effect of deviation from spherical symmetry is studied for the structure factor. This is done by combining the analytical expression for the excluded volume of an ellipsoid of revolution with the expression for the excluded volume correlation function for a sphere. This approach makes it possible to estimate the effect of small deviations from spherical symmetry as a function of axial ratio and volume fraction for relatively low volume fractions. The calculations are relevant for the case of short-range potentials where the Percus–Yevick formula is frequently applied, and indicate that even minor deviations from spherical symmetry may lead to significant effects on the structure factor at low scattering angles.
X-ray diffracted intensity for double-reflection channel-cut Ge monochromators at extremely asymmetric diffraction conditions
Authors: Ferrari Claudio , Germini Fabrizio , Korytár Dusan , Mikulík Petr , Peverini Luca
Published: February 2011

The width and integrated intensity of the 220 X-ray double-diffraction profile and the shift of the Bragg condition due to refraction have been measured in a channel-cut Ge crystal in an angular range near the critical angle of total external reflection. The Bragg angle and incidence condition were varied by changing the X-ray energy. In agreement with the extended dynamical theory of X-ray diffraction, the integrated intensity of the double diffraction remained almost constant, even for the grazing-incidence condition very close to the critical angle for total external reflection. A broadening of the diffraction profile not predicted by the extended theory of X-ray diffraction was observed when the Bragg condition was at angles of incidence lower than 0.6°. Plane wave topographs revealed a contrast that could be explained by a slight residual crystal surface undulation of 0.3° due to etching to remove the cutting damage and the increasing effect of refraction at glancing angles close to the critical angle. These findings confirm that highly asymmetric channel-cut Ge crystals can also work as efficient monochromators or image magnifiers at glancing angles close to the critical angle, the main limitation being the crystal surface preparation.
Drug hypersensitivity: Flare‐up reactions, cross‐reactivity and multiple drug hypersensitivity
Authors: PICHLER Werner J. , DAUBNER Barbara , KAWABATA Tom
Published: February 2011

In 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.
Ectopic expression of MECA‐79 as a novel prognostic indicator in gastric cancer
Authors: Okayama Hirokazu , Kumamoto Kensuke , Saitou Katsuharu , Hayase Suguru , Kofunato Yasuhide , Sato Yu , Miyamoto Kotaro , Nakamura Izumi , Ohki Shinji , Koyama Yoshihisa , Ishii Yoshimasa , Takenoshita Seiichi
Published: February 2011

The aim of this study was to clarify the clinical implications of a unique carbohydrate determinant, MECA‐79, in gastric cancer specimens and cells. Immunohistochemical analysis showed that 62 of 225 (27.6%) cases were defined as positive for MECA‐79. MECA‐79 expression was correlated with depth of invasion, venous invasion, TNM stage, and distant metastasis. In survival analyses, patients with MECA‐79 expression had worse prognosis by the log–rank test. Multivariate analysis of the Cox proportional hazard model showed that MECA‐79 expression was an independent factor of a worse cancer‐specific survival. Among 11 gastric cancer cells, MECA‐79 was observed in only MKN7 cells, which also expressed GlcNAc6ST‐2 transcript. A knockdown of GlcNAc6ST‐2 in MKN7 cells showed a markedly reduced expression of MECA‐79, suggesting that GlcNAc‐sulfation of MECA‐79 is mainly synthesized by GlcNAc6ST‐2. Furthermore, real‐time RT‐PCR analysis revealed that GlcNAc6ST‐2 was significantly increased in cancer tissues compared with paired normal mucosa. In conclusion, the expression of MECA‐79 could be a useful marker for the prognosis of gastric cancer. Our results might also provide novel perspectives on the biology of MECA‐79 and GlcNAc6ST‐2 in cancer progression and metastasis. (Cancer Sci 2011; 102: 1088–1094)
HLA‐A2‐restricted glypican‐3 peptide‐specific CTL clones induced by peptide vaccine show high avidity and antigen‐specific killing activity against tumor cells
Authors: Yoshikawa Toshiaki , Nakatsugawa Munehide , Suzuki Shiro , Shirakawa Hirofumi , Nobuoka Daisuke , Sakemura Noriko , Motomura Yutaka , Tanaka Yukie , Hayashi Shin‐Ichi , Nakatsura Tetsuya
Published: February 2011

Glypican‐3 (GPC3) is an onco‐fetal antigen that is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we identified an HLA‐A2‐restricted GPC3144–152 (FVGEFFTDV) peptide that can induce GPC3‐reactive CTLs without inducing autoimmunity in HLA‐A2 transgenic mice. In this study, we carried out a phase I clinical trial of HLA‐A2‐restricted GPC3144–152 peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivoγ‐interferon enzyme‐linked immunospot assay. The frequency of GPC3144–152 peptide‐specific CTLs after vaccination (mean, 96; range, 5–441) was significantly larger than that before vaccination (mean, 6.5; range, 0–43) (P < 0.01). An increase in the GPC3144–152 peptide‐specific CTL frequency was observed in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3144–152 peptide‐specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3144–152 peptide‐specific CTL clones from PBMCs of patients vaccinated with GPC3144–152 peptide by single cell sorting using Dextramer and CD107a antibody. These CTL clones had high avidity (the recognition efficiency showing 50% cytotoxicity was 10−10 or 10−11 M) and could recognize HCC cell lines expressing GPC3 in an HLA‐class I‐restricted manner. These results suggest that GPC3144–152 peptide vaccine can induce high avidity CTLs capable of killing HCC cells expressing GPC3. This trial was registered with University Hospital Medical Information Network number 000001395. (Cancer Sci 2011; 102: 918–925)
RNA aptamer against a cancer stem cell marker epithelial cell adhesion molecule
Authors: Shigdar Sarah , Lin Jia , Yu Yan , Pastuovic Mile , Wei Ming , Duan Wei
Published: February 2011

The lack of a specific targeting strategy against cancer stem cells in current cancer treatment regimens is at least partly responsible for life‐threatening cytotoxicity for patients undergoing traditional chemotherapy. An effective cancer stem cell targeting system is urgently required for the next generation of cancer medicine. Epithelial cell adhesion molecule (EpCAM) is overexpressed in most solid cancers and it has recently been identified as a cancer stem cell marker. In this study, we isolated a 40‐base RNA aptamer that binds to EpCAM from a random oligonucleotide library using systematic evolution of ligands by exponential enrichment. The aptamer was further truncated to 19 bases. This 19‐nt RNA aptamer interacts specifically with a number of live human cancer cells derived from breast, colorectal, and gastric cancers that express EpCAM, but not with those not expressing EpCAM, as analyzed using flow cytometry and confocal microscopy. The binding affinity of the EpCAM RNA aptamer to human cancer cells is approximately 55 nM. Importantly, this EpCAM RNA aptamer is efficiently internalized after binding to cell surface EpCAM. To our knowledge, this is the first RNA aptamer against a cancer stem cell surface marker being developed. Such cancer stem cell aptamers will greatly facilitate the development of novel targeted nanomedicine and molecular imaging agents for cancer theranostics. (Cancer Sci 2011; 102: 991–998)
A novel oncoprotein Pirh2: rising from the shadow of MDM2
Authors: Wang Zhihao , Yang Bo , Dong Lanlan , Peng Biwen , He Xiaohua , Liu Wanhong
Published: February 2011

Pirh2 (p53‐induced RING‐H2) is an E3 ubiquitin ligase that can target p53 for degradation and thereby repress a diverse group of biological activities regulated by p53. Notably, Pirh2, rather than MDM2, is the primary degrader of active p53 under conditions of DNA damage. Moreover, Pirh2 is highly expressed in multiple cancer cell lines regardless of p53 status. Recent research has shown that Pirh2 is involved in many signalling pathways related to the genesis and evolution of cancer. This review aims to summarize a comprehensive picture of the role of Pirh2 in cellular processes and its significance to tumorigenesis. Furthermore, this review focuses on its potential role as a cancer therapeutic target. (Cancer Sci 2011; 102: 909–917)
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

Using 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

Our 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.
Androgens modulate osteocalcin release by human visceral adipose tissue
Authors: Foresta Carlo , Strapazzon Giacomo , De Toni Luca , Gianesello Lisa , Bruttocao Andrea , Scarda Alessandro , Plebani Mario , Garolla Andrea
Published: February 2011

Objective  Androgens inhibit adipogenic differentiation through an androgen receptor (AR)‐mediated pathway, increase lipolysis and reduce lipid accumulation in adipocytes. Undercarboxylated osteocalcin (ucOCN) regulates insulin and adiponectin secretion and is released by adipose tissue (AT). Our objective was to investigate, ex vivo and in vivo, the role of androgens on osteocalcin (OCN) modulation in human AT.

Design, Patients, Setting  Omental AT (OAT) for in vitro study and blood samples from 91 male patients of Padova University Hospital were used.

Measurements  Omental AT was treated with dihydrotestosterone (DHT) in presence and in absence of flutamide. cOCN and ucOCN release by AT in a simple growth medium was evaluated by ELISA. OCN, both undercarboxylated (ucOCN) and carboxylated (cOCN) forms, was measured in serum by ELISA.

Results  After 24‐h DHT stimulation, the release of both cOCN and ucOCN by OAT was statistically increased (P < 0·05). Co‐incubation with flutamide blunted OCN production. Overweight and obese patients had lower total and free testosterone (T), associated with lower ucOCN and ucOCN/OCN ratio. Free T was negatively correlated to BMI (ρ = −0·706, P < 0·05) and positively correlated to ucOCN/OCN ratio (ρ = 0·223, P < 0·05).

Conclusions  Our data suggest that androgens modulate OCN release by OAT in vitro. In addition to the anti‐adipogenic role of androgens, they support a novel mechanism by which androgens could exert a protective effect in energy metabolism. This hypothesis appears even more significant considering that sexual hormones’ levels are greatly altered in obesity and that AT is both highly involved in their clearance and able to produce OCN.
Predictors of neonatal hypothalamic–pituitary–adrenal axis activity at delivery
Authors: Smith Alicia K. , Newport D. Jeffrey , Ashe Morgan P. , Brennan Patricia A. , LaPrairie Jamie L. , Calamaras Martha , Nemeroff Charles B. , Ritchie James C. , Cubells Joseph F. , Stowe Zachary N.
Published: February 2011

Objective  Clinical and preclinical studies indicate that maternal stress during pregnancy may exert long‐lasting adverse effects on offspring. This investigation sought to identify factors mediating the relationship between maternal and neonatal hypothalamic–pituitary–adrenal (HPA) axes in pregnant women with past or family psychiatric history.

Patients  Two hundred and five pairs of maternal and umbilical cord blood samples from a clinical population were collected at delivery.

Measurements  Maternal and neonatal HPA axis activity measures were plasma adrenocorticotrophic hormone (ACTH), total cortisol, free cortisol and cortisol‐binding globulin concentrations. The effects of maternal race, age, body mass index, psychiatric diagnosis (DSM‐IV), birth weight, delivery method and estimated gestational age (EGA) at delivery on both maternal and neonatal HPA axis measures were also examined. Incorporating these independent predictors as covariates where necessary, we evaluated whether neonatal HPA axis activity measures could be predicted by the same maternal measure using linear regression.

Results  Delivery method was associated with umbilical cord plasma ACTH and both total and free cord cortisol concentrations (T = 10·53–4·21; P < 0·0001–0·010). After accounting for method of delivery and EGA, we found that maternal plasma ACTH concentrations predicted 23·9% of the variance in foetal plasma ACTH concentrations (T = 6·76; P < 0·0001), and maternal free and total plasma cortisol concentrations predicted 39·8% and 32·3% of the variance in foetal plasma free and total cortisol concentrations (T = 5·37–6·90; P < 0·0001), respectively.

Conclusion  These data suggest that neonatal response is coupled with maternal HPA axis activity at delivery. Future investigations will scrutinize the potential long‐term sequelae for the offspring.
Limits to exploitation: dynamic food web models predict the impact of livestock grazing on Ethiopian wolves Canis simensis and their prey
Authors: Vial Flavie , Macdonald David W. , Haydon Daniel T.
Published: February 2011

1. Anticipating the consequences of, and sustainable limits to, human exploitation of ecosystem resources requires a quantitative understanding of food web dynamics. While dynamic food web models are commonly used to investigate the impact of human exploitation on marine ecosystem structure and dynamics, they are less commonly applied to human‐induced disturbances in terrestrial food webs.

2. The intensifying and expanding use of rangelands for domestic livestock production creates a potential conflict with the conservation of extensive populations of wildlife. The Bale Mountains National Park (BMNP) is an example of an area that increasingly has been used as an open‐access resource by pastoralists and their herds. Livestock grazing, through its impact on herbivorous rodents, is suspected to negatively affect the critically endangered Ethiopian wolf Canis simensis, a rodent specialist.

3. We developed a series of simple dynamic food chain models to explore the interactions between these trophic levels and how they might be affected by livestock grazing. We also explored how predictions made about these trophic dynamics are affected by the type of functional response linking the different trophic levels.

4. If rodent and/or wolf populations exhibit extreme type 2 responses to resource availability, they can both remain at densities close to their ungrazed equilibrium as livestock density increases, but will rapidly crash once vegetation biomass collapses as a result of the increased grazing pressure. The model predicts the maximum sustainable livestock density to lie between 32 and 117 TLU·km−2, above which populations of wolves are expected to become locally extinct.

5. Synthesis and applications. We show that dynamic trophic modelling is an informative approach when investigating the response of terrestrial ecosystems to human‐induced disturbance. In particular, the models described in this paper provide a first step towards enabling managers to predict the implications of changing patterns of human impact on rangelands and support the identification of sustainable grazing grounds for livestock. The models also reveal that monitoring primary productivity alone may be a simple and effective way to detect stresses on food chains and predict the impact of increased livestock grazing on higher trophic levels. In the context of the BMNP, the models predict that some areas of high conservation value appear to be exploited unsustainably, thereby possibly affecting long‐term conservation goals for the Park’s flagship species.
Litter inputs and plant interactions affect nectar sugar content
Authors: Baude Mathilde , Leloup Julie , Suchail Séverine , Allard Béatrice , Benest Danielle , Mériguet Jacques , Nunan Naoise , Dajoz Isabelle , Raynaud Xavier
Published: February 2011

1. Declines in availability of plant resources to pollinators are a major cause of pollinator loss. The management of plant communities to enhance floral resources is often proposed as a way to sustain pollinator populations. Nectar, the main energetic resource for pollinators, plays a central role in behaviour and composition of pollinator communities. Abiotic and biotic factors are known to influence nectar traits at both the species and community levels, but the impact of plant community composition itself has never been investigated.

2. Below‐ground interactions in plant communities can induce changes in plant development through (i) plant‐derived litter amendment of the soil and (ii) competition for soil resources between plants. We tested how plant below‐ground interactions affect above‐ground nectar traits involved in plant attractiveness to pollinators.

3. A short‐term pot experiment was carried out with three temperate grassland species Mimulus guttatus, Lamium amplexicaule, and Medicago sativa, showing distinct litter stoichiometry and competitive abilities for soil resources. Litter amendment (none, mono and tri‐specific litter) and plant interaction treatments (monocultures, two‐ and three‐species mixtures) were crossed in a factorial design.

4. Litter amendment to the soil led to an increase in total nectar sugar content in L. amplexicaule plants but not in the two other species. We also found that the presence of M. guttatus, a competitive species, reduced the total nectar sugar content in L. amplexicaule through a concomitant decrease in nectar volume per flower and in floral display size, but not in other species. Species‐specific responses of nectar traits to variation in soil nitrogen availability were thus observed, suggesting consequences for plant species and community attractiveness to pollinators. However, we did not find evidence that the legume M. sativa affected nectar traits of any neighbouring plants.

5. Synthesis. Our results demonstrate that litter inputs and competition between plants for soil resources can alter nectar traits linked to plant attractiveness to pollinators. This supports the idea that below‐ground plant–plant interactions for soil resources can influence above‐ground plant–plant interactions for pollination services. This offers promising perspectives in studying the role of below‐ground–above‐ground interactions on higher trophic levels.
Ecology’s cruel dilemma, phylogenetic trait evolution and the assembly of Serengeti plant communities
Authors: Anderson T. Michael , Shaw Joey , Olff Han
Published: February 2011

1. Ecologists debate the importance of neutral versus niche‐based explanations for patterns of species coexistence and whether small‐scale data can inform ecological understanding of communities, referred to by McNaughton [Ecological Monographs, 1983, 53, 291] as ‘ecology’s cruel dilemma.’ Research on phylogenetic relationships, traits and species co‐occurrence has attempted to address this topic, with results considerably mixed.

2. We address the hypothesis that plant community assembly is influenced by trait similarity across ecological gradients and this affects mean phylogenetic distance (MPD) of species within sites. We analysed specific leaf area (SLA), maximum plant height and phylogenetic relationships among Serengeti grasses, a system ideally suited to study community assembly because of an ecological gradient in which the dominant plant stress shifts from drought to light competition.

3. Phylogenetic community assembly theory predicts that MPD would be lowest (under‐dispersed) at dry sites and greatest (over‐dispersed) at sites with higher rainfall. Similarly, theory predicts that low soil nutrient concentrations should filter intolerant species, so that MPD is expected to be under‐dispersed at infertile, low‐elevation sites and over‐dispersed at fertile, higher‐elevation sites. However, as gradients of rainfall and soil fertility run counter to one another across the Serengeti, it was unclear how this covariation would influence MPD.

4. Surprisingly, traits showed different evolutionary patterns: SLA displayed convergent evolution while maximum plant height displayed Brownian evolution across the phylogeny. As predicted, statistically under‐dispersed assemblages occurred at lower rainfall, infertile sites while statistically over‐dispersed assemblages occurred at higher rainfall, fertile sites. However, the pattern across all plots was weak, with most plots showing no statistical pattern of MPD.

5. Multivariate analyses using structural equation modelling, which statistically controlled for covariation among environmental effects, revealed complex direct and indirect effects of environmental variation on MPD, including offsetting direct effects of SLA and maximum plant height due to their different patterns of trait evolution.

6. Synthesis. Spatially counteracting gradients of moisture and soil fertility across the Serengeti, combined with contrasting patterns of trait evolution, obscured the relationship between MPD and any single environmental variable. Our study shows that integrating trait and phylogenetic relationships across ecological gradients yields considerable insight into the ecological mechanisms that determine community composition, but that multivariate techniques may be required to appropriately reveal such patterns.