bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combined 3C-ChIP-Cloning (6C) Assay: A Tool to Unravel Protein-Mediated Genome Architecture
Authors: Tiwari Vijay K. , Baylin Stephen B. 
Published: March 2009

INTRODUCTIONProgress in technologies to address long-range chromosomal interactions in vivo has extensively revised concepts about different aspects of transcriptional regulation. These methods allow probing physical proximities between chromatin elements without specifically identifying the protein components that mediate such interactions. Here we describe a detailed protocol for Combined 3C-ChIP-Cloning (6C) technology, which combines multiple techniques to identify the proteins that bridge distant genomic regions, while simultaneously identifying such physical proximities. This method is also useful for determining if a candidate protein might mediate long-range interactions, both in cis and in trans in the nucleus. We discuss how the 6C technique can be incorporated with other techniques to discover all the chromatin regions in the nucleus that interact with a given gene or chromatin region of interest in a specific protein-dependent manner. Such information allows complete, cell-type-specific mapping of all the chromatin interactions mediated by specific proteins. The 6C assay advances our understanding of the three-dimensional aspects of the higher-order folding of chromatin and provides an important tool to examine the role of specific proteins in nuclear organization. In addition to providing a detailed protocol of the 6C technique, we discuss how this technology can be used by investigators working in the area of chromatin biology, with special interest in chromatin long-range interactions.
Alcian Blue Staining of the Mouse Fetal Cartilaginous Skeleton: Figure 1.
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard 
Published: March 2009

INTRODUCTIONThe vertebrate skeleton forms by endochondral and intramembranous bone formation. During endochondral bone formation, mesenchyme condensations give rise to cartilages that are eventually replaced by bone. However, there are some permanent cartilages that do not ossify, such as the cartilage of the trachea and articular cartilage of the joints, and intramembranous bone formation occurs directly without a cartilage template. This protocol is ideal for revealing the cartilaginous skeleton of mouse embryos between 12.5 and 16.5 days post-coitum (dpc). The cartilaginous skeleton is stained dark blue by alcian blue, whereas the other tissues of the embryo are made transparent by a clearing solution, benzyl alcohol/benzyl benzoate (BABB).
Alizarin Red Staining of Post-natal Bone in Mouse
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard 
Published: March 2009

INTRODUCTIONThe vertebrate skeleton forms by endochondral and intramembranous bone formation. During endochondral bone formation, mesenchyme condensations give rise to cartilages that are eventually replaced by bone. However, there are some permanent cartilages that do not ossify, such as the cartilage of the trachea and articular cartilage of the joints, and intramembranous bone formation occurs directly without a cartilage template. This article describes an abbreviated outline of a technique for staining the bone of newborn and adult mice. Because the ossified skeleton is a robust tissue, even the skeletons of dead animals can be processed using this method.
Regulating Protein Stability in Mammalian Cells Using Small Molecules
Authors: Hagan Emily L. , Banaszynski Laura A. , Chen Ling-chun , Maynard-Smith Lystranne A. , Wandless Thomas J. 
Published: March 2009

INTRODUCTIONRegulating protein stability using small molecules provides a rapid, reversible, and tunable method to study a protein of interest’s (POI) role in cells. We recently designed a small protein domain based on the 12-kDa FKBP (FK506 binding protein) that can be fused at either the carboxyl or amino terminus of a protein of interest. This destabilization domain (DD) confers instability to fusion protein partners. The method described here explains how to use a DD fusion to control the biological activity of a POI. In the absence of a small molecule ligand, the DD is unstable and directs the fusion protein for degradation. Addition of the ligand stabilizes the DD, allowing the fusion protein to accumulate in cells and the POI to exert its biological effect. The ligand is specific for the DD and has no detectable off-target effects. By utilizing the specificity of genetic fusion and the speed of small molecule binding, this technique provides an alternative to RNA interference to study a POI’s role in cells.
A General Method for Conditional Regulation of Protein Stability in Living Animals
Authors: Sellmyer Mark A. , Thorne Steve H. , Banaszynski Laura A. , Contag Christopher H. , Wandless Thomas J. 
Published: March 2009

INTRODUCTIONThe ability to rapidly and reversibly perturb protein levels in living animals is a powerful tool for researchers to determine protein function in complex systems. We recently designed a small protein domain based on the 12-kDa FKBP (FK506 binding protein) that can be fused at either the carboxyl or amino terminus of a protein of interest. This destabilization domain (DD) confers instability to fusion protein partners, allowing targeted degradation of the protein of interest. A small molecule called Shield-1 binds to the DD and protects the fusion protein from degradation. Small-molecule-mediated post-translational regulation of protein stability affords this system rapid, reversible, and tunable control of protein levels and functions in a variety of model systems. Theoretically, a number of transgene delivery methods (e.g., viral, liposomal, or stem cell) can be used for the analysis of a DD fusion protein in an animal model. This protocol uses tumor xenografts in mice as one such mechanism for delivering the fusion protein and presents a method for delivering Shield-1 to regulate the fusion proteins in vivo.
In Situ Hybridization Analysis of Embryonic Beak Tissue from Darwin’s Finches
Authors: Abzhanov Arhat 
Published: March 2009

INTRODUCTIONThe beaks of Darwin’s finches develop their distinct shapes during embryogenesis. To visualize where and when mRNA transcripts of genes involved in beak development are present during embryogenesis, this protocol describes how to analyze embryonic tissue samples using in situ hybridization. The principle of this technique is to use probes specific to mRNAs coding for proteins of interest to reveal how the corresponding genes are expressed in the beaks of different species of Darwin’s finches.
Microarray Analysis of Embryonic Beak mRNA from Darwin’s Finches
Authors: Abzhanov Arhat 
Published: March 2009

INTRODUCTIONIn this protocol, microarray technology is used as a very sensitive and rapid method to identify genes that are potentially involved in beak development and morphology in Darwin’s finches. The method allows for the direct comparison between cDNA targets from two different species (each labeled with a different dye). The prevalence of one of the dyes for any of the genes on the resulting scan indicates a higher level of accumulation of transcripts from that gene in a particular beak morphology/species. The obtained expression profiles can be clustered to identify transcripts that are expressed in a species- and/or size-specific manner.
Preparation of Recombinant Baculoviruses with the BVboost System
Authors: Airenne Kari J. , Laitinen Olli H. , Mähönen Anssi J. , Ylä-Herttuala Seppo 
Published: March 2009

INTRODUCTIONWe have developed an improved transposition-based system (BVboost) for the generation of recombinant baculoviruses. This system bypasses the disadvantages of the original transposition-based generation of baculoviral genomes in Escherichia coli while remaining a simple, rapid, and convenient viral production technique. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculoviral genomes (bacmids) in E. coli with a mutated sacB gene. Recombinant bacmids can be generated at a frequency of ~107/μg of donor vector with a negligible background. The BVboost system also allows efficient setups for high-throughput screening and gene expression purposes. After cloning the desired gene/cDNA/library into a BVboost system-compatible donor plasmid, the recombinant baculoviral genome is prepared simply by transforming electrocompetent DH10BacΔTn7 E. coli cells with the donor. Transfer from the donor vector into the baculoviral genome (bacmid) occurs via a Tn7-mediated site-specific transposition reaction in E. coli cells. The selection scheme guarantees that virtually all colonies are correct. The recombinant baculoviral genome is subsequently extracted from E. coli culture using a modified isolation procedure for large plasmids. To generate the recombinant viruses, insect cells are transfected with the isolated recombinant bacmid. This protocol provides instructions on how to prepare recombinant baculoviruses by the BVboost system in order to express the desired gene(s) in insect and/or vertebrate cells.
Pancreatic adenocarcinoma up‐regulated factor (PAUF), a novel up‐regulated secretory protein in pancreatic ductal adenocarcinoma
Authors: Kim Sun A. , Lee Yangsoon , Jung Dawoon E. , Park Kyung Hwa , Park Jeong Youp , Gang Jingu , Jeon Sun Bok , Park Eui Chul , Kim Young‐Gun , Lee Bogman , Liu Qing , Zeng Wen , Yeramilli Subramanyam , Lee Soojin , Koh Sang Seok , Song Si Young 
Published: March 2009

The identification of novel tumor‐specific proteins or antigens is of great importance for diagnostic and therapeutic applications in pancreatic cancer. Using oligonucleotide microarrays, we identified a broad spectrum of differentially expressed pancreatic cancer‐related genes. Of these, we selected an overexpressed expressed sequence taq and cloned a 721‐bp full‐length cDNA with an open reading frame of 196 amino acids. This novel gene was localized on the Homo sapiens 16p13.3 chromosomal locus, and its nucleotide sequence matched the Homo sapiens similar to common salivary protein 1 (LOC124220). We named the gene pancreatic adenocarcinoma up‐regulated factor. The pancreatic adenocarcinoma up‐regulated factor was secreted into the culture medium of pancreatic adenocarcinoma up‐regulated factor‐overexpressing Chinese hamster ovary cells, had an apparent molecular mass of ~25 kDa, and was N‐glycosylated. The induction of pancreatic adenocarcinoma up‐regulated factor in Chinese hamster ovary cells increased cell proliferation, migration, and invasion ability in vitro. Subcutaneous injection of mice with Chinese hamster ovary/pancreatic adenocarcinoma up‐regulated factor cells resulted in 3.8‐fold greater tumor sizes compared to Chinese hamster ovary/mock cells. Reverse transcription–polymerase chain reaction and western blotting with antirecombinant human pancreatic adenocarcinoma up‐regulated factor antibodies confirmed that pancreatic adenocarcinoma up‐regulated factor was highly expressed in six of eight pancreatic cancer cell lines. Immunohistochemical staining of human pancreatic cancer tissues also showed pancreatic adenocarcinoma up‐regulated factor overexpression in the cytoplasm of cancer cells. Transfection with pancreatic adenocarcinoma up‐regulated factor‐specific small‐interfering RNA reduced cancer cell migration and invasion in vitro. Treatment with antirecombinant human pancreatic adenocarcinoma up‐regulated factor in vitro and in vivo reduced proliferation, migration, invasion, and tumorigenic ability. Collectively, our results suggest that pancreatic adenocarcinoma up‐regulated factor is a novel secretory protein involved in pancreatic cancer progression and might be a potential target for the treatment of pancreatic cancer. (Cancer Sci 2009; 100: 828–836)
Review article: future therapies for management of metastatic gastroenteropancreatic neuroendocrine tumours
Authors: SRIRAJASKANTHAN R. , TOUMPANAKIS C. , MEYER T. , CAPLIN M. E. 
Published: March 2009

SummaryBackground  Gastroenteropancreatic neuroendocrine tumours (GEP NETs) are relatively uncommon tumours that occur anywhere within the gastrointestinal tract. The prevalence of GEP NETs is estimated to be 35 per 100 000 population. Patients often present with metastatic disease and consequently, palliative treatments form the mainstay of therapy.Aim  To review the current and novel therapeutic options for management of GEP NETs.Methods  Searches for all studies related to GEP NETs, NETs and carcinoid tumours in Medline and abstracts from international meetings.Results  Somatostatin analogues remain the first line therapy for management of symptoms of GEP NETs and may have anti‐proliferative action. New somatostatin analogues with different somatostatin receptor affinity have been developed. Radionuclide peptide receptor therapy is established in patients with positive somatostatin scintigraphy. A number of new agents and targeted therapies are currently being evaluated in a phase I and II studies and these include angiogenic inhibitors, mammalian target of rapamycin inhibitors and immune therapies.Conclusions  A number of nonsurgical therapies are available for management of gastroenteropancreatic neuroendocrine tumours. It is hoped, the development of some of these promising novel therapies will expand the therapeutic armamentarium.
Clinical trial: factors associated with freedom from relapse of heartburn in patients with healed reflux oesophagitis ‐ results from the maintenance phase of the EXPO study
Authors: LABENZ J. , ARMSTRONG D. , ZETTERSTRAND S. , EKLUND S. , LEODOLTER A. 
Published: March 2009

SummaryBackground  Ability to predict freedom from heartburn relapse during maintenance therapy for healed reflux oesophagitis may facilitate optimal treatment choices for individual patients.Aim  To determine factors predicting freedom from heartburn relapse during maintenance proton pump inhibitor therapy in patients with healed reflux oesophagitis.Methods  This post‐hoc analysis used data from the maintenance phase of the EXPO study (AstraZeneca study code: SH‐NEG‐0008); 2766 patients with healed reflux oesophagitis and resolved heartburn received once‐daily esomeprazole 20 mg or pantoprazole 20 mg for 6 months. Multiple logistic regression analysis determined factors associated with freedom from heartburn relapse.Results  Heartburn relapse rates were lower with esomeprazole than pantoprazole in all subgroups analysed. Esomeprazole treatment was the factor most strongly associated with freedom from heartburn relapse (odds ratio 2.08; P < 0.0001). Other factors significantly associated with freedom from heartburn relapse were Helicobacter pylori infection, greater age, non‐obesity, absence of epigastric pain at baseline, pre‐treatment nonsevere heartburn and GERD symptom duration ≤5 years.Conclusions  Several factors predict freedom from heartburn relapse during maintenance proton pump inhibitor therapy for healed reflux oesophagitis, the strongest being choice of proton pump inhibitor. These findings outline the importance of optimizing acid control and identifying predictors of relapse for effective long‐term symptom management in reflux oesophagitis patients.
Tectonic vs. climate forcing in the Cenozoic sedimentary evolution of a foreland basin (Eastern Southalpine system, Italy)
Authors: Mancin N. , Di Giulio A. , Cobianchi M. 
Published: March 2009

ABSTRACTThis paper discusses the Cenozoic interaction of regional tectonics and climate changes. These processes were responsible for mass flux from mountain belts to depositional basins in the eastern Alpine retro‐foreland basin (Venetian–Friulian Basin). Our discussion is based on the depositional architecture and basin‐scale depositional rate curves obtained from the decompacted thicknesses of stratigraphic units. We compare these data with the timing of tectonic deformation in the surrounding mountain ranges and the chronology of both long‐term trends and short‐term high‐magnitude (‘aberrant’) episodes of climate change. Our results confirm that climate forcing (and especially aberrant episodes) impacted the depositional evolution of the basin, but that tectonics was the main factor driving sediment flux in the basin up to the Late Miocene. The depositional rate remained below 0.1 mm year−1 on average from the Eocene to the Miocene, peaking at around 0.36 mm year−1, during periods of maximum tectonic activity in the eastern Southern Alps. This dynamic strongly changed during the Pliocene–Pleistocene, when the basin‐scale depositional rate increased to an average of 0.26 mm year−1 (Pliocene) and 0.73 mm year−1 (Pleistocene). This result fits nicely with the long‐term global cooling trend recorded during this time interval. Nevertheless, we note that the timing of the observed increase may be connected with the presumed onset of major glaciations in the southern flank of the Alps (0.7–0.9 Ma), the acceleration of the global cooling trend (since 3–4 Ma) and climate variability (in terms of magnitude and frequency). All these factors suggest that combined high‐frequency and high‐magnitude cooling–warming cycles are particularly powerful in promoting erosion in mid‐latitude mountain belts and therefore in increasing the sediment flux in foreland basins.
The imbalance between Survivin and Bim mediates tumour growth and correlates with poor survival in patients with multiple myeloma
Authors: Romagnoli Mathilde , Séveno Céline , Wuillème‐Toumi Soraya , Amiot Martine , Bataille Régis , Minvielle Stéphane , Barillé‐Nion Sophie 
Published: March 2009

SummarySurvivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock‐down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3‐only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult‐to‐treat cancer.
Methotrexate and aminopterin exhibit similar in vitro and in vivo preclinical activity against acute lymphoblastic leukaemia and lymphoma
Authors: Kang Min H. , Harutyunyan Narine , Hall Connor P. , Papa Rachael A. , Lock Richard B. 
Published: March 2009

SummaryDue to the development of neurological toxicity and resistance to methotrexate (MTX), other antifolates have been evaluated for its potential replacement in the treatment of childhood acute lymphoblastic leukaemia (ALL). Aminopterin (AMT) has been suggested to provide clinical advantages over MTX and other antifolates. AMT activity, compared with MTX, was evaluated in ALL and lymphoma preclinical models. The minimum survival fraction at the range of concentrations tested was lower with AMT than with MTX in 3 out of 15 cell lines. Both AMT and MTX significantly extended the event‐free survival of mice bearing 3 out of 4 xenografts with equivalent activity.
MicroRNA‐27 enhances differentiation of myeloblasts into granulocytes by post‐transcriptionally downregulating Runx1
Authors: Feng Jue , Iwama Atsushi , Satake Masanobu , Kohu Kazuyoshi 
Published: March 2009

SummaryWe investigated the regulation of the transcription factor Runx1 by microRNA (miR)‐27 and the resulting effects upon the differentiation of myeloblasts into granulocytes. When 32D.cl3 cell differentiation was induced using granulocyte colony‐stimulating factor (CSF3), Runx1 transcription was moderately downregulated, while Runx1 protein levels were completely inhibited, suggesting an involvement of post‐transcriptional regulation. Simultaneously, levels of miR‐27 and its precursor increased substantially. Reporter assays revealed that miR‐27 targets the 3′UTR of the Runx1 transcript. Furthermore, introduction of pre‐miR‐27 alone into 32D.cl3 cells resulted in downregulation of Runx1 protein, thereby allowing the cell differentiation even in the absence of CSF3. Conversely, transduction of anti‐miR‐27 caused upregulation of Runx1 protein, thereby antagonizing the CSF3‐mediated granulocyte differentiation. Finally, the CSF3‐induced transcription factor C/EBPalpha enhanced transcription of a host gene of miR‐27, C9orf3, via activation of its promoter. Thus, miR‐27 enhances differentiation of myeloblasts into granulocytes via post‐transcriptional downregulation of Runx1.
Chronic rhinosinusitis with and without nasal polyps is associated with decreased expression of glucocorticoid‐induced leucine zipper
Authors: Zhang X‐H. , Lu X. , Long X‐B. , You X‐J. , Gao Q‐X. , Cui Y‐H. , Liu Z. 
Published: March 2009

SummaryBackground
Chronic rhinosinusitis without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP) is characterized by persistent inflammation of sinonasal mucosa. Glucocorticoid‐induced leucine zipper (GILZ) is a recently described anti‐inflammatory mediator.Objective
Here we analysed the expression of GILZ in CRSsNP and CRSwNP, its association with response to surgery, and its cytokine‐driven expression regulation in the upper airways.Methods
The messenger RNA (mRNA) and protein expression of GILZ in 33 CRSsNP, 32 CRSwNP, and 11 control samples was assessed by means of a quantitative RT‐PCR and immunohistochemistry, respectively. Nasal explant culture was used to investigate the effect of IFN‐γ, IL‐4, IL‐13, IL‐1β, and TNF‐α on GILZ mRNA expression in normal sinonasal mucosa.Results
The GILZ mRNA and protein expression was significantly suppressed in both CRSsNP and CRSwNP patients compared with controls. No significant difference in GILZ expression was found between CRSsNP and CRSwNP patients. Comparing patients responsive and patients recalcitrant to surgery, a significant further decrease of GILZ expression was found in recalcitrant patients both in the CRSsNP and in the CRSwNP group. IL‐1β, TNF‐α, IL‐4, and IL‐13 reduced, whereas IFN‐γ enhanced GILZ mRNA levels in the sinonasal mucosa.Conclusion
Down‐regulated expression of GILZ may contribute to the pathogenesis of CRSsNP and CRSwNP and associate with response to surgery. GILZ expression in the upper airways can be regulated differentially by different cytokines.