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We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 μm from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.
20 Related JoVE Articles!
Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells
Institutions: Tel Aviv University.
The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo
evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g.,
bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e.,
mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.
Cancer Biology, Issue 93, Matrigel plug, angiogenesis, microbubbles, ultrasound, fibered-confocal endomicroscopy, conditioned media.
An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse
Institutions: Tulane University, Tulane University.
Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called ‘angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.
Medicine, Issue 86, Alkali-burn Injury, Corneal Neovascularization (NV), Corneal Blindness, Angiogenesis, Inflammation, Hemangiogenesis, Lymphangiogenesis
Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles
Institutions: Stanford University , Stanford University .
Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo
. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ
using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo
. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro
, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.
Empty Value, Issue 79, Stem Cells, animal models, bioengineering (general), angiogenesis, biodegradable, non-viral, gene therapy
Rat Mesentery Angiogenesis Assay
Institutions: University of Gothenburg.
The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the
study of sprouting angiogenesis in vivo
[see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor
tissues, as discussed in invited review papers1,2
. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network
to be assessed microscopically in situ;
(ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect
relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4
. The model demonstrates a high level of
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins,
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e.
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5
); (ii) natural iron-unsaturated human lactoferrin, which stimulates
, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7
; and (iii)
low-molecular-weight heparin fractions produced by various means8,9
. Moreover, the assay is highly suited to studies of the combined
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo
models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).
Physiology, Issue 52, angiogenesis, mesentery, objective variables, morphometry, rat, local effects, systemic effects
Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model
Institutions: Medical College of Georgia.
Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.
Immunology, Issue 45, Metastasis, CTL adoptive transfer, Lung, Tumor Immunology
Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
Institutions: Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center.
It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2
. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo
for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3
. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4
. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo
expanded T cells is determined.
Immunology, Issue 47, Adoptive T cell therapy, Breast Cancer, HER-2/neu, common gamma chain cytokines, Bryostatin 1, Ionomycin
A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells
Institutions: University of Massachusetts, University of Massachusetts, University of Massachusetts.
Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5
. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8
. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9
. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13
. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11
. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.
Cancer Biology, Issue 55, tumor, vascular, endothelial, tube formation, Matrigel, in vitro
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Institutions: Cedars-Sinai Medical Center.
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Chemistry, Issue 88, Cancer treatment, personalized medicine, polymalic acid, nanodrug, biopolymer, targeting, host compatibility, biodegradability
A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions
Institutions: Torrey Pines Institute for Molecular Studies, Cascade LifeSciences Inc..
Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro
technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2
tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo
behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.
Bioengineering, Issue 77, Cellular Biology, Biophysics, Physiology, Molecular Biology, Biomedical Engineering, Immunology, Cells, Biological Factors, Equipment and Supplies, Cell Physiological Phenomena, Natural Science Disciplines, Life Sciences (General), circulating cells, extravasation, physiological shear stress, endothelial cells, microenvironment, chemokine gradient, flow, chamber, cell culture, assay
The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye
Institutions: Boston Children's Hospital, The Hebrew University of Jerusalem, Harvard Medical School.
The mouse corneal micropocket assay is a robust and quantitative in vivo
assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.
Neuroscience, Issue 90, Angiogensis, neovasculatization, in vivo assay, model, fibroblast growth factor, vascular endothelial growth factor
Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2
. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5
. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6
. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7
. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9
. Bevacizumab however failed to prolong overall survival in a recent phase III trial26
. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12
, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer
Institutions: Watson School of Biological Sciences, Cold Spring Harbor Laboratory, University of Oslo and Oslo University Hospital.
The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo
in the intact microenvironment cannot be completely replicated in these in vitro
settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.
Cancer Biology, Issue 73, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Genetics, Oncology, Pharmacology, Surgery, Tumor Microenvironment, Intravital imaging, chemotherapy, Breast cancer, time-lapse, mouse models, cancer cell death, stromal cell migration, cancer, imaging, transgenic, animal model
Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo
Institutions: Newcastle University , University College, London.
Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.
Developmental Biology, Issue 77, Neurobiology, Neuroscience, Biomedical Engineering, Cellular Biology, Molecular Biology, Medicine, Anatomy, Physiology, Ophthalmology, Angiogenesis Modulating Agents, Growth and Development, Lymphangiogenesis, Angiogenesis, Mouse Neonatal Retina, Immunofluorescent-Staining, confocal microscopy, imaging, animal model
Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis
Institutions: American University, National Cancer Institute, NIH.
Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro
in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro
angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.
Cancer Biology, Issue 91, Angiogenesis, tube formation, fibroblast, endothelial cell, matrix, 3B-11, basement membrane extract, tubulogenesis
Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy
Institutions: École Polytechnique Fédérale de Lausanne, Oregon Health & Science University.
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.
Bioengineering, Issue 86, Intravital imaging, epifluorescence, two-photon imaging, Tumor matrix, Matrix remodeling
An Orthotopic Model of Murine Bladder Cancer
Institutions: Tulane University, Tulane University.
In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.
Medicine, Issue 48, Bladder tumor, orthotopic, mouse, ultrasound
In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum
Institutions: Caliper Life Sciences.
4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo
evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.
Cellular Biology, Issue 26, tumor, mammary, mouse, bioluminescence, in vivo, imaging, IVIS, luciferase, luciferin
Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging
Institutions: Caliper Life Sciences.
Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.
Medicine, Issue 50, osteolytic lesions, micro CT, tumor, bioluminescence, in vivo, imaging, IVIS, luciferase, low dose, co-registration, 3D reconstruction


text:
Outlander Sport continues to be Mitsubishi’s top seller in the U.S., and the month’s sales total includes the initial sales of the 2013 Outlander Sport, which began production in Normal, Illinois in July.
“Customers continue to be attracted to the Outlander Sport’s mix of style, performance, and fuel efficiency,” said MMNA President & CEO Yoichi Yokozawa. “We expect sales of the Outlander Sport to be further fueled by our ‘Unpretentious’ marketing campaign, which began over the Labor Day weekend.”
Mitsubishi Motors North America, Inc., (MMNA) is responsible for all manufacturing, sales and marketing operations for Mitsubishi Motors in the United States. MMNA sells cars and light trucks through a network of approximately 400 dealers. For more information, contact the Mitsubishi Motors News Bureau at (888) 560-6672 or visit media.mitsubishicars.com.
SOURCE Mitsubishi Motors North America

domain: news


Every year, two cities from two of the EU’s member states are chosen and designated as the European Capitals of Culture, where they organise series of cultural events with a strong European dimension.
The initiative was developed in 1985 and has, to date, been awarded to more than 50 cities across the EU, supported by the European Commission. The initiative was designed with four aims, which were to:
- Highlight the richness and diversity of cultures in Europe;
- Celebrate the cultural and historical features Europeans share;
- Increase European citizens’ sense of belonging to a common cultural area;
- Foster the contribution of culture to the development of cities.
It has been proven to show enormous success and provided opportunities for regenerating cities, raising the international visibility and profile of cities, enhancing the image of cities in the eyes of their own inhabitants, breathing new life into a city’s culture, and of course boosting tourism. Therefore, the benefits of socio-economic development and the impact for the chosen city are now also considered in determining the chosen cities.
This year, 2014, saw Riga (capital city of Latvia) and Umeå (a town in northern Sweden) becoming the European Capitals of Culture. Each of the two cities will be holding 200-300 exciting cultural events through the year, while Umeå will be running the year under the motto “Curiosity and Passion”. Let’s have a look at what they are offering!
The Official Video
Know more about Umeå
The Official Video (1)
The Official Video (2)
Know more about Riga
European Capitals of Culture have now been designated until year 2019:
- 2015 – Mons (Belgium) and Plzeň (Czech Republic)
- 2016 – Donostia-San Sebastián (Spain) and Wrocław (Poland)
- 2017 – Aarhus (Denmark) and Paphos (Cyprus)
- 2018 – Leeuwarden (Netherlands) and Valetta (Malta)
- 2019 – Matera (Italy) and Plovdiv (Bulgaria)
Have you been to any of them? Share your thoughts and experiences with us in our section “Europe and You“!


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I always get good feedback when I blog about poodle wallpapers from the post-war period. They seem to trigger an emotional response, and people either love them or hate them. I’ve pretty much exploited all the poodle wallpapers in the collection, but fear not, I’ve found a substitute. This paper features a black sheep, primping...
Many of the most iconic products of the 1920s—perfume bottles, vanities, and jewelry—are feminine objects like those seen in this sidewall, now on view in The Jazz Age: American Style in the 1920s.

domain: artistic


Power News We love to talk!
Rural Electrification Corporation (REC) has received Rs 42,700 crore as repayment from state distribution companies under Ujjawal Discom Assurance Yojana (UDAY) after discoms raised Rs 2.3 lakh crore through bonds.
For the quarter ended March 31, 2017, REC’s net profit grew 13.7% to Rs 1,319 crore, while total revenue fell 1.74% to Rs 5,978 crore. The company made an impairment provision of Rs 616 crore for two power projects, one in Tamil Nadu and one in Sikkim. For Fy17, the financier’s net profit rose 11% to Rs 6,246 crore while the total income fell 1.7% to Rs 23,350.79 crore.
The power sector lender had lent around Rs 78,000 crore to various states. The UDAY scheme provided for 75% of the loans being converted into state government bonds. The remaining 25% rests on the books of discoms and will be paid as regular debt.
Not all states entered UDAY for financial restructuring. The exposure to those states will be serviced by them as debt,” said PV Ramesh, chairman and managing director of REC. “We have already disbursed the repayments received from discoms as part of our total disbursements of Rs 58,000 crore in financial year 2016-17. We borrowed only Rs 28,000 crore in FY17,” Ramesh said.
In FY18 the company plans to raise around Rs 55,000 crore of which Rs 7,000 crore to Rs 10,000 crore will be through foreign currency loans. The company will look at raising green dollar denominated bonds, Masala bonds and a niche new category of social impact bonds. In the domestic market the company would look at issuing commercial paper, 54 EC bonds and tax free bonds. “Around Rs 30,000 crore would be raised through rupee denominated bonds including commercial papers and 54 EC bonds or tax saving bonds. The company had not issued tax saving bonds in the last 3 years and plan to do one in this financial year,”said Ramesh.
“The company has the mandate to raise around $300 million in foreign currency loans by June end, which the company wants to take up to $450 million. These would be 10 year bonds with a market driven coupon rate. REC has a total loan book of Rs 201,929 crore and outstanding borrowings of Rs 167,517 crore as of March 31, 2017. The cost of borrowings has gone down by 37 basis points to 6.85% in 2016-17.
- Centre has given Uttar Pradesh Rs 11,500 crore for electrification: Piyush Goyal Read more
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- Govt to roll out Rs17,000 crore rural household electrification scheme Read more


George Strait's next tour will be his last
Updated 10:38 am, Thursday, September 27, 2012
George Strait announced on Wednesday plans for a two-year tour that will be his last.
Strait will continue to record new music and said he won't cease performing altogether. But "as far as a structured tour goes," he said, "after the last date in 2014, that's gonna be it for touring."
Strait said "this was a hard decision for me to make. … Don't think I'm retiring because I'm not. I'm still gonna make records."
Looking every bit the country music king in his signature black Stetson, Strait was cheerful at the news conference, held at the Country Music Hall of Fame in Nashville, Tenn., and streamed live on his website.
Strait said he'd thought about pulling off the road when he reached 60 years old.
The singer, who became a grandfather earlier this year, turned 60 in May.
He also said he didn't want to stay on the road so long that one day he would "book a tour and nobody came."
The Poteet native has recorded 59 No. 1 singles in his career, which took off in 1981 with the album "Strait Country" and the single "Unwound."
In addition to spending more time with his grandson, Strait said he intends to work in some more golfing and fishing.
The first leg of his "Cowboy Rides Away" tour includes a June 1 stop in his backyard, San Antonio. He'll also play a March 17 date at Reliant Stadium to close out the Houston Livestock Show and Rodeo.
Strait will begin recording a new CD in October in Nashville.
And he held out the possibility that he might play a concert or two after 2014 - but there won't be any more structured tours.
Is there a chance he'll change his mind?
"In 2016 I might say, 'What a dummy.' I might reconsider," Strait said. "But at this particular time, I'm pretty sure I won't."
Hector Saldana of the San Antonio Express-News contributed to this report.


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Emile Heskey turned down chance to join Carlisle as free agent, reveals manager Graham Kavanagh
Carlisle manager Graham Kavanagh has revealed former England striker Emile Heskey has turned down his offer to join the club.
Heskey is a free agent after a spell in Australia with Newcastle Jets.
Kavanagh said: 'I tried to get Emile in. Unfortunately, he turned it down as I think he wants to be playing in the Championship or going back abroad.'
Free agent: Emile Heskey is looking for a new club after leaving Australian side Newcastle Jets
The former England international, who has played for Leicester, Liverpool, Birmingham, Wigan and Aston Villa, making over 500 appearances in English top-flight football was recently spotted out on the town with ex-teammate Stephen Ireland.
Ireland and Heskey played together at Villa but neither particularly enjoyed their time in the Midlands. Heskey endured long dry spells and Ireland struggled to hold down a regular place under successive managers.
Back in the swing of things: Emile Heskey was pictured on the town with former teammate Stephen Ireland
Been around: The former England striker made over 500 appearances for English clubs including Aston Villa
The 36-year-old has rediscovered his form down-under. Heskey has scored 10 goals in 42 A-League matches - more than he scored in double the amount of games at Aston Villa.
It’s not too late to play MailOnline Fantasy Football… There’s £1,000 to be won EVERY WEEK by the highest scoring manager
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A furious, last-inning comeback fell just short for the Bayfield High School baseball team Saturday.
The Wolverines, in the Class 2A District Tournament at Cedaredge, fell to Legend High School of Parker 10-7.
Bayfield, trailing 10-3, pushed four runs across in the seventh, boosted by an RBI double by Brenden Harrison.
The Wolverines, who cut the lead to 10-5, scored two more runs with two outs before the Titans closed out the game.
BHS took an early 2-0 lead in the bottom of the first on a two-RBI single by Shea Conrad.
Strasburg upset host Cedaredge 5-2 in the opening game Saturday, a contest delayed for two hours by rain.
Strasburg (11-9) and Legend (10-10) played for the District title later Saturday in Cedaredge.
The Wolverines, champions of the Intermountain League tournament and regular season, ended the seaon 13-5.

domain: game


text:
0:00 - John is going to Summer League! Hydration tips?
5:30 - MoneyBall ProAm Pairings Announced - WHO YA GOT?
12:00 - NBA Draft - Jaren and Miles go lotto. Better Fit?
20:00 - Reviewing our mock - Spolier: it wasn’t GREAT
31:00 - RANK EM - Non-MSU B1G player ready to make an immediate impact
40:00 - FOOTBALL CROOTIN - Bye, Dwan. Hi Devontae.
50:00 - Crootin’ Predictions - more to come...and soon?

domain: hobby


text:
It's perfectly natural to be nervous during a job interview. In fact, it can even help you to perform at your best and come across as animated and enthusiastic. But, it's never helpful to be overwhelmed with nerves. If you find yourself shaking, sweating and short of breath, you need to learn and employ some techniques to control your nervousness.
The basis of controlling your nerves comes from a couple of useful ideas you can put into practice before the big event. For several days before the interview, rehearse the scene in your head, mentally visualizing the first few minutes of the interview. Imagine yourself walking in calmly and confidently, greeting your interviewers with a firm handshake and smiling pleasantly. Set an expectation for yourself in your head that things will go well, and imagine a positive reaction from your interviewers. Positive visualization of this type works for controlling anxiety in all kinds of high-pressure situations.
There's really no substitute for practicing when it comes to controlling nerves. Ask a friend or mentor to simulate an interview with you. Walk into the room and practice your greeting and how you will sit. Have him ask you several of the questions that you anticipate, including some of the difficult ones. Practice your answers out loud. If there are questions that have stumped you in previous interviews, practice those. The more you practice, the more fluent you will become, and the less your nerves will overwhelm you.
Odd things that may go wrong on your way to the interview site can throw you, and set off your nerves. So a couple of days ahead, lay out the outfit you plan to wear. Check that everything is clean and in good repair, and that it fits comfortably. Drive to the interview site, preferably at the same time of day as you will have to do it on the day of your interview. Check how long it takes you to get there, and whether there are any holdups on the way. Find where you're going to park and exactly which door you'll use to enter the building. If you have all of this knowledge already in your head, you can focus more confidently on your performance.
Once you're at the interview, waiting in the lobby, be aware of your breathing. Slow it down and take regular, medium-deep breaths. Notice if your neck and shoulders are tense, or if you have any tics such as a tapping foot. Roll your shoulders back to relax them and let the tension out of your body. Stand up tall as you enter the interview room and smile confidently as you shake hands with the interviewer and make eye contact. A confident exterior can help you to feel more confident inside.
- Jupiterimages/Goodshoot/Getty Images

domain: psychology


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Cole Winters is one of the first African-American lawman of the Old West. In the 19th century, the Indian Territory was a place where there was a thin line between lawmen and outlaws. After being raised by Native Americans, Cole Winters was hired by the U.S. Marshals Service to track down and bring back outlaws from the Territories. Dead or alive. When his gun fighter brother, Joseph Two Guns, becomes wanted for a crime he did not commit, Cole does the only thing he can to get his brother justice... He hunts him down.
To the Gallows, is a classic Western with fast-paced gunfights and martial arts action of the time. It is the beginning chronicle of a man the Native Americans called The Buffalo Ghost and the legend that followed.
G. S. Luckett is a screenwriter and indie author of dark fantasy and supernatural action thrillers.

domain: library


text:
93%(14)13 out of 14 people found this document helpful
This preview shows page 3 out of 3 pages.
GroupRole, if any, played in theRevolutionEffect of the Revolution on thegroupPolitical, economic, or socialeffects of the war on thegroupMen colonistsMen were asked to fight for the cause of freedom from the BritishThough many had given their lives,they did so because of the cause for freedom.The new government was forming, but there was not much change on this group of men. Women colonistsWithout the men around, the womenhad to take some of the roles men had, such as tending to farming, and taking care of everything around their homesMany women made widows, also many had lost their eldest children to the war. Laws did change and woman also started gaining new freedoms from woman's oppression. Some new rights were set into place for woman, but nothing significant had changed until many years later.Black slaves and freed BlacksAsked to fight for both sides to gain freedom, after a certain amount of time, they were free, but if they stayed they would be paid a certain amount.Many had died, but those that did live long enough some were set free, other sent back to their masters, and then many escaped to the north where they could be free.Nothing changed in the south for blacks, but in the north, theywere a free people, and startedto have some rights. Native AmericansThe natives picked a side and fought for that side.Many of them had died in this war, and they lost a lot of land.They lost a lot of land to the Americans and were forced further west as the Americans started to expand. Citeand reference all sourced material consistent with APA guidelines. Schultz, K. (NA). Hist2[University of Phoenix Custom Edition eBook]. NA, NA: Cengage Learning. Retrieved from University of Phoenix, HIS115 US History to 1865 website.

domain: history


Australia’s freshly crowned Independent Rock Artist of the Year, GRANDHOUR, has today released it’s much anticipated 5th single from the award winning album Bombs & Bullets.
Scar By Scar, a fan favourite track, has been released with a new music video which shows a different side of the band through the clip’s comic-book theme.
The song explores finding the strength to pick yourself up ‘after the storm’ – “when the fall is far,
that’s what makes you who you are, it’s how you face it – knock by knock, and take it scar by scar….”
The single will available on-line through iTunes, Spotify and all good music stores. Check out the official music video on YouTube, Facebook.com/grandhour or grandhourband.com
You can purchase the award-winning album Bombs & Bullets right now on CD from grandhourband.com/shop
Comic illustrations – John Franklin
Video footage – John Bigelow, Interactive Media Solutions
Additional footage – Nicole Hawkins, Infinity8 Multimedia
Editing & colour grading – Nicole Hawkins, Infinity8 Multimedia
Written by Nicole Hawkins
Performed by GRANDHOUR
℗ & © Rock Hawk Records, Pty Ltd


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Celebs are starting to arrive at the 2019 BAFTA Film Awards!
The annual ceremony, taking place at the Royal Albert Hall in London, honors the work of the talented artists in the film industry over the last year. Timothée Chalamet, Amy Adams, Lucy Boynton and Lily Collins are among the stars that have already started to arrive at the award show.
Among the nominees at Sunday's ceremony include Glenn Close, Lady Gaga, Melissa McCarthy, Olivia Colman and Viola Davis, who are all up for Leading Actress. Bradley Cooper, Christian Bale, Rami Malek, Steve Coogan and Viggo Mortensen all received Leading Actor nominations.
BlackkKlansman, The Favourite, Green Book, ROMA and A Star Is Born are all up for Best Film.
Let's take a look at all of the stars as they arrive on the red carpet ahead of the ceremony!

domain: celebrity


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Improvapella® is the beautiful love child of a cappella music and comedic improvisation. Using the songs from a live a cappella group as inspiration, the actors will create a montage of scenes never to be seen again. Only the a cappella group knows what songs will be sung. Will the next song be a tried and true rock anthem? A love song? A TV theme? We never know until show time! But why should our actors have all the fun? To keep our guest group on their feet as well, the audience chooses the song order.
Improvapella® is the product of Mike Amaral’s love of all things improv and a cappella, infused with his joyous sense of surprise, fair play, and whimsy.
What about musicians and instruments, you ask? Feel free to check out The Set List.


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These are the top 5 investing books that changed my life to become an investor like Warren Buffett and Charlie Munger. I consider these authors to be role models on the path toward financial freedom: Danielle Town, Phil Town, Mohnish Pabrai, and Guy Spier. I am super grateful to have these teachers to learn how to invest like the best in a close knit community of investors who try to buy wonderful companies at attractive prices. Their values definitely reflect my own and I try to implement these principles and methodologies in my own investing practice.
These top 5 books are:
1. Invested (2018) by Danielle Town and Phil Town
2. Education of a Value Investor (2014) by Guy Spier
3. PayBack Time (2010) by Phil Town
4. Dhandho Investor (2007) by Mohnish Pabrai
5. Rule #1 (2006) by Phil Town
Please support your local library and/or bookstore to check these out! 🙂
I’m excited to be on a path to FIRE (Financial Independence, Retire Early), and I look forward to making more investor friends. Connect with me on my Instagram @ michellemarki!

domain: finance


This topic provides step-by-step instructions to assist developers, including acquiring the LabKey Server source code, installing required components, and building LabKey Server from source. The instructions here are written for a Windows
machine. To set up development on a Mac/OSX/Linux
machine, use these instructions in conjunction with the topic: Notes on Setting up OSX for LabKey Development
Obtain the LabKey Source Files
The LabKey source files are stored in multiple GitHub repositories. To build LabKey Server, follow these steps.
Install a Git Client
You can use GitHub Desktop or another Git client.
- Install GitHub Desktop:
- Install a command-line git option for your platform:
Clone LabKey Source Files from GitHub
- Begin by cloning the LabKey server repository from git into a directory such as labkeyHome.
git clone https://github.com/LabKey/server.git labkeyHome
For example, on Windows this may be: C:\dev\labkey\labkeyHome. This directory is referred to as $LABKEY_HOME below.
- Clone the following core repositories. Note that the first two go into $LABKEY_HOME\server\modules, while the third goes into $LABKEY_HOME\server.
$LABKEY_HOME\server\modules> git clone https://github.com/LabKey/platform.git
$LABKEY_HOME\server\modules> git clone https://github.com/LabKey/commonAssays.git
$LABKEY_HOME\server> git clone https://github.com/LabKey/testAutomation.git
An abbreviated map of your finished enlistment, showing the main folder structure (with many other elements omitted):
LABKEY_HOME (i.e. C:\dev\labkey\labkeyHome)
- If you will be using a branch other than develop (the default branch when you clone a repository), you can check out all repositories to a given branch, by using the following commands from the root of your enlistment. First recursively check out all the subdirectories, then check out the root on the same branch. For example, to switch from develop to the release21.11-SNAPSHOT branch:
$LABKEY_HOME> gradlew gitCheckout -Pbranch=release21.11-SNAPSHOT
$LABKEY_HOME> git checkout release21.11-SNAPSHOT
- You could also choose to check out each repository, including the server repository separately. For example, to change the platform repo to the release21.11-SNAPSHOT branch:
$LABKEY_HOME\server\modules> cd platform
$LABKEY_HOME\server\modules\platform> git checkout release21.11-SNAPSHOT
Note: If you are subscribed to a premium edition of LabKey Server, you will need to set up an Artifactory account in order to access premium modules. Follow the steps in this topic:
Install Required Prerequisites
To update the JDK in the future, you will:
- 1. Repeat the steps above (installing the new version in a location parallel to the old).
- 2. Update your system variable JAVA_HOME to point to the new location.
- 3. Update the IntelliJ > File > Project Structure > SDK > labkey home path to point to the new location. Rename the existing 'labkey' to it's version number. Add the new JDK, then rename the new version "labkey".
- 4. If necessary, add any additional flags to the VM options section. For example, when updating to Java 16, you'll add these flags to your JVM switches:
--add-opens=java.base/java.lang=ALL-UNNAMED --add-opens=java.base/java.io=ALL-UNNAMED --add-opens=java.base/java.util=ALL-UNNAMED --add-opens=java.desktop/java.awt.font=ALL-UNNAMED --add-opens=java.base/java.text=ALL-UNNAMED
Platform-specific installation instructions:
- PostgreSQL: Install using your Linux machine's native tools or download a PostgreSQL binary package. Follow the instructions in the downloaded package to install PostgreSQL.
Consider installing the database using docker
, facilitating launch with a command like:
$ docker run -p 5432:5432 --name postgres -e POSTGRES_PASSWORD=[password] -d postgres
Node.js and npm
The LabKey build depends on Node.js and the node package manager (npm). The build process automatically downloads and installs the versions of npm and node that it requires. You should not install npm and node yourself. For details on the Node.js dependency and build-prod script requirement, see Node.js Build Dependency
Note that you do not
need to install Gradle. LabKey uses the gradle wrapper to make updating version numbers easier. The gradle wrapper script (either gradlew or gradlew.bat) is included in the enlistment and is already in the $LABKEY_HOME directory.
Create a gradle properties file to contain information about global settings for your gradle build following these steps:
- Create a ".gradle" directory in your home directory.
- On OSX and Linux, typically: /Users/<you>/.gradle.
- On Windows, typically: C:\Users\<you>\.gradle). Note: the Windows file explorer may not allow you to create a folder beginning with a period. To solve this navigate to C:\Users\<you>\ in the command prompt and type mkdir .gradle.
- Create a gradle.properties file in the .gradle directory using the following process:
- Copy the file $LABKEY_HOME/gradle/global_gradle.properties_template to C:\Users\<you>\.gradle. Rename the file to create a file named gradle.properties.
- In the gradle.properties file, substitute your <CATALINA_HOME> directory (the location of your Tomcat installation), including the specific version number, for the value after systemProp.tomcat.home. Use forward slashes, not backslashes, for the Tomcat path, even on Windows. For example:
Environment Variables and System PATH
- Create or modify the system environment variable JAVA_HOME so it points to your JDK installation location (for example, C:\labkey\apps\jdk-##). Note: If you've already set the JAVA_HOME variable to point to your installation of the JRE, you should modify it to point to the JDK.
- Create or modify the system environment variable CATALINA_HOME so that it points to your Tomcat installation (for example, C:\labkey\apps\tomcat-#.#.##).
- Add the JDK to your system PATH. Using the JAVA_HOME variable will simplify upgrades later
- Add the following location to your system PATH. This directory won't exist yet, but add it to the path anyway. The Gradle build downloads binaries used by the server, such as graphvis, dot, and proteomics tools, to this directory.
- For example, C:\dev\labkey\labkeyHome\build\deploy\bin.
On OSX, for example, you would place the environment variables in your .bash_profile:
export JAVA_HOME=`/usr/libexec/java_home -v 1.11`
GWT Browser Settings (Optional)
The default developer build is optimized for Chrome but the target browser can be controlled through either a command line parameter or by setting the 'gwtBrowser' property in the gradle.properties file. Available settings are: gwt-user-chrome (the default value), gwt-user-firefox, or gwt-user-ie.
Open the LabKey Project in IntelliJ
The LabKey development team develops LabKey using IntelliJ IDEA. You can use the license-free Community Edition if you are planning on modifying or extending the LabKey source code.
Below we describe how to configure the IntelliJ development environment; we recommend employing the same general principles if you are using a different development environment. Some developers have experimented with Eclipse as the IDE and you can find some set up details on the Developer Message Board
- Download and install the latest version of IntelliJ IDEA. Either the Community or Ultimate Edition will work.
Over time, you will want to update IntelliJ to stay current with features and enhancements. You can use the Help > Check for updates...
option in the application. Watch for installation alerts about any .idea files you may have edited. Note that updates may be incremental and after updating, a second more recent update may be available.
After updating, recheck your settings described in the following section, particularly your JDK and CATALINA_HOME settings.
Configure the LabKey Project in IntelliJ
- Run the following gradle commands to configure the workspace and other default settings:
- Build the application from the command line
- Open the LabKey project in IntelliJ:
- Launch IntelliJ.
- If your IntelliJ install is brand new, you will see the "Welcome to IntelliJ" pop up screen. Click Open.
- If you have previously installed IntelliJ, select File > Open.
- Select the LabKey IntelliJ project directory: $LABKEY_HOME
- Set Path Variables
- Select File > Settings > Appearance & Behavior > Path Variables. (On a Mac, the menu path is IntelliJ IDEA > Preferences > Appearance & Behavior > Path Variables).
- Click the plus icon in the upper right. Set the CATALINA_HOME path variable to <CATALINA_HOME>, the root directory of your Tomcat installation, for example, C:\labkey\apps\apache\apache-tomcat-#.#.##.
- If developing with TypeScript, add a NODE_PATH variable as well and set it to the path to the node binary that is installed by the build, for example, $LABKEY_HOME\build\.node\node-v#.#.##\bin\node
- Click OK to close the Settings window.
- Configure the Target JDK
- In IntelliJ, select File > Project Structure.
- Under Project Settings, click Project.
- Under Project SDK click New and then click JDK.
- Browse to the path of your JDK, for example, (C:\java\jdk-##), and click OK.
- Click Edit. Change the name of the JDK to "labkey".
- Click OK to close the Project Structure window.
- Open the Gradle tool window at View > Tool Windows > Gradle.
- Click the refresh icon. This will take as much as 5-20 minutes. You should start seeing messages about its progress. If not, something is probably hung up. Wait for this sync to complete before progressing with further IntelliJ configuration steps.
Edit configuration options as follows:
- Select Run > Edit Configurations. (If the menu is greyed-out, wait until IntelliJ finishes indexing the project files.)
- Open the Application node in the left panel and select LabKey Dev.
- The following instructions apply to IntelliJ version 2020.3. You can update now, or for earlier releases of IntelliJ, follow this section in the documentation archives
- From the Modify options... menu, check that both Add VM Options and Use classpath of module are checked.
- Hover over the entry panels in the upper part of the Run panel to see a description of what to select or enter in each one.
- VM options:
- Confirm that the path separators are appropriate for your operating system. On Windows, ensure that the paths to the jar files are separated by semicolons. For example: "./bin/bootstrap.jar;./bin/tomcat-juli.jar;C:/Program Files (x86)/JetBrains/IntelliJ IDEA 20##.#.#/lib/idea_rt.jar". For Macs, the paths should be separated by a colon (:) instead.
- Check that any JVM memory settings (like "-Xms###" and "-Xmx###") in this line are sufficient (if present at all). Determine the correct values in the topic Configure Tomcat Settings. If these settings are not present, the system allocations for memory will be used.
- To run your instance in development mode include the argument -Ddevmode=true
- For JDK 16 and up, include the arguments --add-opens=java.base/java.lang=ALL-UNNAMED --add-opens=java.base/java.io=ALL-UNNAMED --add-opens=java.base/java.util=ALL-UNNAMED --add-opens=java.desktop/java.awt.font=ALL-UNNAMED --add-opens=java.base/java.text=ALL-UNNAMED
- Select classpath:
- Hover over the upper right panel and set the classpath to "...platform.api.main" (or "api_main" or "org.labkey-api_main" in previous releases).
- Working directory:
- Confirm that this points to your current Tomcat installation (i.e. to the same location as you set CATALINA_HOME).
- Click OK, to close the Run/Debug Configurations window.
Be sure that IntelliJ has enough heap memory. The default max is OK if you’re just dealing with the core modules, but you will likely need to raise the limit
if you’re adding in custom modules, 3GB seems sufficient.
You may also want to increase the number of open tabs intelliJ will support. The default is 10, and depending on your working process, you may see tabs disappear unexpectedly. Select Settings > Editor > General > Editor Tabs
and scroll down to the Closing Policy
section to increase the number of tabs you can have open in intelliJ at one time.
Build and Run LabKey
Configure the pg.properties File
The LabKey source includes a configuration file for use with PostgreSQL (pg.properties
), specifying JDBC settings, including URL, port, username, password, etc.
- Open the file $LABKEY_HOME/server/configs/pg.properties
- Edit the file, adding your values for the jdbcUser and jdbcPassword. (This password is the one you specified when installing PostgreSQL. If your password contains an ampersand or other special XML characters, you will need to escape them, as the value will be substituted into an XML template without encoding. For example, if your JDBC password is "this&that", then use the escaped version "this&that".)
- In a command window, go to $LABKEY_HOME
- Run "gradlew pickPg" to configure labkey.xml with the corresponding database settings.
- You may need to manually create the directory <CATALINA_HOME>/conf/Catalina/localhost.
When you build LabKey, the values that you've specified in the properties file are copied into the LabKey configuration file, labkey.xml, overwriting previous values. This file is then copied into <CATALINA_HOME>/conf/Catalina/localhost
To learn more about the build process, the various build targets available, and how the source code is transformed into deployed modules, see Build LabKey from Source
- On the command line, go to the $LABKEY_HOME directory, and invoke the gradle build target:
To control which modules are included in the build, see Customize the Build
Run LabKey Server
To run and debug LabKey:
- Select Run > Debug 'LabKey Dev' in IntelliJ.
- If Tomcat starts up successfully, navigate your browser to http://localhost:8080/labkey to begin debugging (assuming that your local installation of Tomcat is configured to use the Tomcat default port 8080).
While you are debugging, you can usually make changes, rebuild, and redeploy LabKey to the server without stopping and restarting Tomcat. Occasionally you may encounter errors that do require stopping and restarting Tomcat.
Run the Basic Test Suite
- Install Geckodriver to run tests on Firefox.
- Run the command within your $LABKEY_HOME directory, to initiate automated tests of LabKey's basic functionality:
gradlew :server:testAutomation:uiTest -Psuite=DRT
Other automated tests are available. For details, see Run Automated Tests
. Note that 'R' must first be configured
in order to run some tests.
Optional Modules on GitHub
Many optional modules are available from the LabKey repository on GitHub
. To include these modules in your build, install a Git client and clone individual modules into the LabKey Server source.
Clone Modules from LabKey's GitHub Repository
- To add a GitHub module to your build, clone the desired module into labkeyHome/labkey/server/modules. For example, to add the 'reagent' module:
...labkeyHome\server\modules>git clone https://github.com/LabKey/reagent.git
Note that you can get the URL by going to the module page on GitHub (for example, https://github.com/LabKey/reagent), clicking Clone or Download
, and copying the displayed URL.
Manage GitHub Modules via IntelliJ
Once you have cloned a GitHub module, you can have IntelliJ handle any updates:
To add the GitHub-based module to IntelliJ (and have IntelliJ generate an .iml file for the module):
- Edit your settings.gradle file to include the new module
- In IntelliJ, open the Gradle tool window at View > Tool Windows > Gradle.
- Refresh the Gradle window by clicking the arrow circle in the upper left of the Gradle window
To update the GitHub-based module using IntelliJ:
- To have IntelliJ handle source updates from GitHub, go to File > Settings (or Intellij > Preferences).
- Select Version Control.
- In the Directory panel, scroll down to the Unregistered roots section, select the module, and click the Plus icon in the lower left.
- In the Directory panel, select the target module and set its VCS source as Git, if necessary.
- Note that IntelliJ sometimes thinks that subdirectories of the module, like module test suites, have their sources in SVN instead of Git. You can safely delete these SVN sources using the Directory panel.
- To sync to a particular GitHub branch: in IntelliJ, go to VCS > Git > Branches. A popup menu will appear listing the available Git modules. Use the popup menu to select the branch to sync to.
If you have added a new module to your enlistment, be sure to customize the build
to include it in your Gradle project and then refresh the Gradle window to incorporate it into IntelliJ, as described above.
To generate documentation, such as the XML reference and Javadoc, clone the tools repository into the enlistment root (to create a sibling of the /server directory):
$LABKEY_HOME>git clone https://github.com/LabKey/tools.git
Then you can run the documentation generation tasks, such as:
Premium Resources Available
Subscribers to premium editions of LabKey Server can learn more about developing with LabKey with the developer topics and code examples listed on this page:
Premium edition clients also have the option to use Microsoft SQL Server instead of PostgreSQL as the database. Learn more in this topic:
Learn more about premium editions


“Long time no see, Haruyuki onii-chan. You’re as fat as ever!”
Monthly Archive: August 2012
Unfortunately the day as finally come when Bandai Entertainment has announced they will be discontinuing sales for DVD/BD, manga, and novels. The warning signs came in last December when they announced that they would not be licensing/releasing...
“I guess it’s not as easy as it is in cartoons…” -Ao Fukai
“I’ll update my blog where I please!” -Fleur Blanc
*TEARS* AngryAnimeBitches has officially reached the start of the third year of its Seasonal Anime Coverage! Man the years sure fly by!!!! Without any further ado, lets get started on the FALL ANIME 2012...
“Holy Shit!” -Han Juno
“Still, it’s human nature to wait. Waiting for the dove to return with a branch of hope in its beak” –Ivica Tanović


text:
Mexborough and Swinton Times, September 28.
Mr A.W.Black at Denaby and Conisborough.
Mr A.W.Black, the Liberal candidate for the Doncaster Division, on Wednesday evening, paid a visit to the populous mining district of Denaby Main and Conisborough. The meeting at the former was held in the open – air on a piece of ground nearly opposite the new Denaby Main Hotel. The weather, however, was anything but favourable to nominate gathering, yet, notwithstanding this a very considerable number of miners and their wives assembled to hear. Mr Blacks address. He was, accompanied by Alderman J.F. Clark and Councillor Theobald of Doncaster. Mr Wray, the son of the ex major of Barnsley presided.
Mr Black, who, on rising, received a most cordial and hearty reception, expressed his pleasure at seeing so larger in number present, and incidentally remarked upon the crowded and enthusiastic meeting held in the Guild Hall, Doncaster, on the previous evening, when hundreds were unable to gain admission.
The indications as to the results of the poll on Monday week were, he was glad to say, very hopeful, but still he behold them to do their utmost meanwhile, and particularly on the following day, to ensure success. The chief matter which was being brought before the electors was the war. The Tories had no right to make party capital out of it. (Hear, hear). Some of their opponents said if the Liberals came into power, they would give up the two conquered States in South Africa. Such statements were all moonshine; there was not a word of truth in it. No Liberal government or Tory government would think of altering the state of things back to what they were. Although the war could not have been avoided – and he had consistently maintained its – yet it was very unfortunate that the negotiations were in the hands of Mr Chamberlain (hear, hear).
He (Mr Black) happened to be in South Africa at the same time. Mr Chamberlain made his notorious speech, after the South African Committees enquiry had proved; Mr Cecil Rhodes was the prime mover and instigator of the Jameson Raid. Mr Chamberlain went out of his way to say, notwithstanding the fact of the proved complicity of Mr Rhodes, that he had done nothing inconsistent with his character as a man of honour. (Shame).
He (Mr Black) was in Pretoria on the day that speech was published there, and he was able to learn, not only from members of the government, but from ordinary workingmen, what their opinions was of Mr Chamberlain. There existed so much prejudice, and so much ill feeling against him, that it was easy for anyone to see that, when the difficult and delicate negotiations were placed in his hands, they had not a very good chance of arriving at the very best and most peaceful solution. (Hear, hear).
He (Mr Black) and always contended that the Boers themselves were responsible for the war. It was their narrow, intolerant, short-sighted, un Liberal policy that brought it about. Stop when the ultimatum was received – certainly an insulting ultimatum – then of course, the British nation was left with absolutely no alternative but to drive back the Boers from British territorial, and to go on with the conflict until they had ensured that he would never again be possible for the Boers to accumulate arms to be used against us – (hear, hear) – and that result could be brought about only by the incorporation of the two countries in the British Empire. (Cheers).
Both countries had already been annexed. He wished to be most emphatic on this point; there could be no possible will draw from that policy. (Cheers)
No Liberal candidate before the country was in favour of a reversal of the policy of annexation. Peace might be proclaimed now any day, and then the Government would have to take in hand. The peaceful settlement of those States, on lines calculated to secure peace, and contend with equality for the white races and justice for the native races. He contended that under British rule they would enjoy liberty, freedom, and equality as they are never enjoy before (hear, hear).
With regard to the conduct of the war, he agreed with Lord Roseberry that it had been bungled and mismanaged, the bungles being unparalleled. It costs thousands of precious lives and millions of money. Captain Lambton, of H.M.Ship “powerful” and only just arrived in time to save the fall of Ladysmith. When the government call for Vaughan Cheers at the outset of the war, they asked for one mounted men, where is one mounted a man was equal to 3. Amount, and asked for the guns, as Capt. Lambton it said, they were merely “ridiculous toys.”
Mr Winston Churchill, the war correspondent, who was himself a Tory, said that the Lee-Metford rifles used by our troops were not nearly so good as the Mauser rifles use by the enemy – and this statement had been borne out by facts.
The trust themselves were as brave as of all, but that was no reason why the weapons they used should be as of old. With regard to the costs of the war, Sir Michael Hicks Beach at first estimated that the cost of the war would be 10 million; in February last at a cost 23 million, a little later 60 million, in August (last month) it had risen to 70,000,000, and they will find that in the end it will cost about 100 million.
There was a most urgent need for reform in the War Office, and also reform in the army too. One thing he considered more than just, and that was that it was practically impossible for a private soldier to become an officer unless he had money or influence at his back. This state of things was a shame. (Hear, hear). This too is mine, was a scandal, they ought to give “brains” a chance. (Hear, hear).
The late government had had the question of Old Age Pensions before them. For nearly 5 years, and what had they done? Nothing tangible. They were as far away from it as ever. He (Mr Black) was strongly in favour of Old Age Pensions, and would support such a scheme under enable a man who spent the greater part of his life in honest toil, to receive something better than the Workhouse. (Hear, hear) and moreover than that, he should receive his pension as his right, and not as a kind of charity. If judges, civil servants, and Cabinet Ministers were entitled to receive pensions, the working man ought. Referring to the Workmen’s Compensation Act, he said it had been called the “Lawyers Relief Bill “and he thought that was a fitting name for it, seeing that the lawyers and no doubt got more out of it than the working classes. Only about one half of the working classes were included in the Act. If the workmen happened to be working on a building less than 30 feet high and accidentally fell and was injured fatally or otherwise, there would be no compensation.
When a memo was proposed, which would remove this disability, Mr Fison voted against it. He (Mr Black) was in favour of a legal eight hour day for mine, the taxing of Royalties and the better housing of the working classes.
In number of questions were then put to Mr Black, which were apparently answered to the satisfaction of those asking them.
The resolution of confidence in him (Mr Black) as a Liberal candidate was proposed by Ald J.F. Clark (Doncaster), seconded by councillors Theobald and supported by Mr Butcher was carried without opposition.
Another meeting was subsequently held in the Board School, Conisborough, where a very large number of the electors were in attendance, and gave Mr Black a most cordial and hearty reception. This meeting was under the presidency of Mr Brocklesby, Conisborough, who was supported by Alder man Clark, councillors Theobald, of Doncaster, and others. Mr Black gave a most interesting address on similar lines. The one given at Denaby, he remarked me frequently applauded, and were evidently highly appreciated by the audience. The usual “fit and proper” resolution was moved by Mr Charles Holmes, seconded by Mr Joseph March, and supported by Ald Clark and councillors Theobald. The resolution was passed without opposition.

domain: politics


text:
Classic Bollywood movies always strike a chord deep within, it could be because of their high emotional quotient, their dramatized situations, or just the personal nostalgia value. Read these brilliantly crafted lines and be instantly transported to the Golden era of movie-making:
With dialogues being an intricate part of every movie, these classic movies are a treasure trove of deeply connected writing.
Catch the best of these iconic movies only on ShemarooMe.
Click here to download the app now.

domain: entertainment


text:
Leah Hager Cohen, critically acclaimed nonfiction writer and novelist, has been appointed Distinguished Writer in Residence at the College of the Holy Cross. Cohen has served as the Jenks Chair of Contemporary American Letters at the College for the last five years. She will assume the three year position on July 1, 2014, and her responsibilities include teaching two classes per year, advising and mentoring students in the Creative Writing Concentration, and contributing to the overall literary life of the College.
“We are delighted that Leah will remain a member of the Holy Cross community. Her strengths as a writer, teacher and mentor make her a perfect fit for this position,” says Margaret Freije, vice president for academic affairs and dean of the College. “Students across disciplines at Holy Cross will benefit from her talents whether they are writing an analytical paper, a fellowship narrative or a work of fiction.”
“I’m moved and immensely pleased that the college has extended this opportunity,” says Cohen. “Working at Holy Cross turned out to be a greater gift than I ever might have anticipated, and being offered a chance to continue that work feels both inspiring and affirming.”
Cohen’s latest novel “No Book but the World” (April 3, 2014) is a powerful exploration of familial love and responsibility. Ava and Fred are siblings, raised in a “free” environment by their progressive parents, who gave little guidance and overlooked Fred’s serious developmental and social impairments. Now adults, Fred is accused of a horrific crime and Ava feels impelled to try to piece together the story of what actually happened. As she is drawn deeper into the unsettling details of the crime, she becomes obsessed with learning the truth, convinced that she and she alone will be able to explain her brother in a way that allows the rest of the world to regard him with sympathy.
The New York Times Book Review declares, “Leah Hager Cohen is one of our foremost chroniclers of the mundane complexities, nuanced tragedies and unexpected tendernesses of human connection.” The San Francisco Chronicle raves, “Cohen’s empathy is sure-footed and seemingly boundless; her writing gifts its characters with glints of ordinary human radiance. It is the possibility of this glinting that ultimately becomes Cohen’s most powerful gift to us, her readers, as well.”
Cohen is the author of five novels, including “The Grief of Others,” which was long-listed for the Orange Prize; selected as a New York Times Notable Book; and named one of the best books of the year by The San Francisco Chronicle, Kirkus Reviews, and Globe and Mail. She is also the author of five nonfiction titles, including “Train Go Sorry” and “I Don’t Know.” She is a frequent contributor to The New York Times Book Review.
Her book “The Grief of Others” (Riverhead, 2011) will soon hit the big screen. Production for the movie has started and will shoot in April in Nyack, N.Y. The cast includes Rachel Dratch (“Saturday Night Live”), Wendy Moniz and Trevor St. John (“One Life to Live”); and is directed by Patrick Wang, whose first film, “In the Family,” was nominated for the 2012 Independent Spirit Award for Best First Feature.
Cohen will give a reading from “No Book but the World” on Thursday, April 24 at 7:30 p.m. in Rehm Library at the College.
Comments are closed.


text:
We stayed here for two nights in September with four friends from USA and England and we had a memorable holiday! The views from the hotel overlook the sea towards Mull and it is only a few minutes walk from the ferry. Our weather was superb with sunshine and continual blue skies and we all loved Iona. Our hotel rooms... More
- Reservation Options:
- TripAdvisor is proud to partner with TripOnline SA so you can book your Argyll Hotel reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Argyll Hotel Isle Of Iona
- Argyll Hotel Scotland/Isle Of Iona

domain: travel


text:
Andra Gillespie Professor of Political Science, Emory University :
I really can't name 4 plausible GOP presidential nominees right now, especially not for 2012. I think that there are people to watch for 2016 and 2020. Despite his poor national debut earlier this year, I suspect that we haven't heard the last from Bobby Jindal. I would also expect the short list to include a Latino, whether it's Marco Rubio of Florida or George P. Bush. If there is a breakout candidate this year (I.e. If Chris Christie or Bob McDonnell wins their respective gubernatorial bids), then they would make the short list, too.
In general, though, if I were designing the ultimate GOP candidate, here's my prototype: I would look for a young, dynamic, telegenic politician with extensive executive experience who can take credit for innovative reforms in his/her jurisdiction while maintaining a sense of integrity. It seems to me that the GOP is saddled with the perception that they oppose policy for the sake of opposition; that they focus too much on social issues; and that they don't live up to their moral ideals. If a Republican big city mayor or governor can show that they practice what they preach and that they can translate their worldview into practical reforms that improve communities, then voters may be more receptive.


TAJIMA Tape Measure - 33 ft / 10 m x 1 inch G-Series Measuring Tape
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WARNING: This product can expose you to chemicals which is [are] known to the State of California to cause cancer and birth defects or other reproductive harm. For more information go to www.P65Warnings.ca.gov.
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text:
GRAND FORKS, ND (June 25, 2019) – Brendan Mullen and the Auto Finance Super Center/Express Employment Professionals of Grand Forks No. 11M team had a busy four-race weekend lined up with the NOSA Sprints this past weekend. Unfortunately, mother nature rained out two of the four shows, but instead of staying home on Saturday, the team loaded up and made the pull east to Ogilvie Raceway for the Midwest Power Series event.
The weekend started at Norman County Raceway in Ada, Minn. on Thursday, June 20th where Brendan showed vast improvement from a year ago, finishing second in his heat race. Mullen started the feature on the outside pole, but was under attack by NOSA all-time wins leader, Mark Dobmeier. Hotshoe Thomas Kennedy also moved up and took a position from Brendan, relegating him back to fourth. The No. 11M showed great speed and control the entire race and finished in sixth.
On Friday night, the team unloaded at River Cities Speedway during the Greater Grand Forks Fair special. Brendan finished ninth in his heat race, lining him up in 12th for the feature. Mullen moved up two positions in a caution-plagued feature, but finished the race with a top 10 finish.
With Brown County Speedway in Aberdeen, SD. raining out on Saturday, the CL Linfoot team made the haul to Ogilvie, Minn. to compete against the Midwest Power Series 360s. The heat race led off with a bang as Mullen traded slide jobs for the lead lap after lap with Thomas Kennedy, and took the checkers in second. In the feature, Brendan started eighth, ran a nice clean race, and finished in the same position.
This weekend, Brendan will be competing in weekly competition at River Cities Speedway.
Brendan and the entire Mullen Motorsports team would like to thank all their sponsors for their support!
Forks Finest Auto Body
Top Fives: 0
Top Tens: 5
Photo by Speedway Shots


text:
You may find more results for this query on our sister sites: GenomeWeb and Precision Oncology News.
The company had total revenues of $193.5 million during the quarter, driven by continued increases in revenue and test volume for its more recently launched and acquired tests.
HTG Molecular will provide Firalis with technology and components to support its commercialization of a rheumatoid arthritis theranostic assay.
The effort, which relied on Oxford BioDynamics' EpiSwitch platform, resulted in an assay that researchers believe could be developed into a test to guide therapy selection for patients.
Biogen will now be able to supply Lisa Tracker monitoring kits in the competitive tenders in which it participates in France and other parts of Europe.
The 18-month study will assess whether Vectra DA enables better patient outcomes and reduces costs compared to conventional approaches.
The partners will use Immunovia's antibody array platform for the discovery of diagnostic biomarkers associated with four autoimmune diseases.
The firm's CEO said that its assay is applicable in other indications where immunosuppressive therapies are used, such as rheumatoid arthritis.
Compiling results from six previous studies, the analysis found Vectra DA scores are associated with worsening disease but didn't address clinical utility.
The company said that it can distinguish patients suffering from lupus from those with other autoimmune diseases using its antibody array platform.
Contractor Palmetto GBA issued a draft local coverage determination proposing ending coverage of the test based on factors including a recent critical study.


text:
Published on March 31st, 2010 | by Kev Adams
I LOVE Music and I’m not afraid to have an opinion either haha! Just a shame too many confuse opinions with facts.
I’ve been a successful professional musician but didn’t fall out of love with music, even during the times I fell out of love with performing.
It’s often been the sound track to my life and there are plenty of songs which stop me in my tracks, make the hair stand up on the back of my neck and make me tingle. All the usual cliches apply.
I even have trouble passively listening and am so easily distracted when listening to good music it makes it difficult to concentrate on my work. And why listen to “bad” music? I’m not one for musical wall paper.