text:
Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.
21 Related JoVE Articles!
A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies
Institutions: University of Bern.
Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.
Medicine, Issue 91, tissue microarray, biomarkers, prognostic, predictive, digital pathology, slide scanning
Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules
Institutions: Ohio State University.
Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.
Bioengineering, Issue 92, adhesion, aggregation, Fc-fusion, cadherin, protocadherin
High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT
Institutions: Weizmann Institute of Science, Weizmann Institute of Science, Weizmann Institute of Science.
Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (μCT).
High resolution μCT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2
. As opposed to electron tomography, the μCT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, μCT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4
, which are both time consuming and destructive. Sample conditioning and positioning in μCT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180° or 360°between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7
. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.
Bioengineering, Issue 52, 3D imaging, tomography, x-ray, non invasive, ex-vivo
Analytical Techniques for Assaying Nitric Oxide Bioactivity
Institutions: University of Texas Health Science Center at Houston , Baylor College of Medicine .
Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1
. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo
NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo
. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-
) and nitrate (NO3-
) may only represent part of NO's effects in vivo
. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo
experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro
ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo
with organ bath myography.
Medicine, Issue 64, Molecular Biology, Nitric oxide, nitrite, nitrate, endothelium derived relaxing factor, HPLC, chemiluminscence
A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
Institutions: UMDNJ-Robert Wood Johnson Medical School.
Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1
or in biomaterial scaffolds2
. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.
Bioengineering, Issue 51, 3D, hanging drop cultures, cell sorting-out, differential adhesion
Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Institutions: London Health Sciences Centre, Western University, London Health Sciences Centre, Lawson Health Research Institute, Western University.
The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo
preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.
Medicine, Issue 84, Metastasis, circulating tumor cells (CTCs), CellSearch system, user defined marker characterization, in vivo, preclinical mouse model, clinical research
Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies
Institutions: Ottawa Hospital Research Institute, University of Ottawa, University of Ottawa, The Second Hospital of Shandong University, University of Tabuk, Ottawa General Hospital.
Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.
Medicine, Issue 85, mouse, tumor model, surgical stress, immunosuppression, perioperative immunotherapy, metastases
A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide
Institutions: Queen's University, Ask Science Products Inc..
In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner. Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.
Molecular Biology, Issue 92, Electroporation, Indium-Tin oxide, signal transduction, gap junctional communication, peptides, Stat3
A Protocol for the Production of KLRG1 Tetramer
Institutions: Brown University.
Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+
-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton.
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.
Microbiology, Issue 35, Immunology, Basic Protocols, Tetramer, Inclusion Bodies, Refolding, Monomer, Flow Cytometry, KLRG1, Cadherins
Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration
Institutions: State University of New York, Buffalo, Roswell Park Cancer Institute, State University of New York, Buffalo.
Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1
. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.
Medicine, Issue 93, substernal thyroid, retrosternal thyroid, intra-thoracic thyroid, goiter, endobronchial ultrasound, EBUS, transbronchial needle aspiration, TBNA, biopsy, needle biopsy
Detection of Alternative Splicing During Epithelial-Mesenchymal Transition
Institutions: Northwestern University Feinberg School of Medicine.
Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and γ-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.
Cellular Biology, Issue 92, alternative splicing, EMT, RNA, primer design, real time PCR, splice isoforms
Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow
Institutions: Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam.
During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e.,
through the cell-cell junctions and the transcellular route, i.e.,
through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro
assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.
Immunology, Issue 90, Leukocytes, Human Umbilical Vein Endothelial Cells (HUVECs), transmigration, VE-cadherin, PECAM-1, endothelium, transcellular, paracellular
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
Institutions: The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine, Yale School of Medicine, The Johns Hopkins University School of Medicine, The Johns Hopkins University School of Medicine.
B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2
. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10
. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11
. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8
, but staining on paraffin-embedded slides had been a challenge until recently13-18
. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.
Cancer Biology, Issue 71, Medicine, Immunology, Biochemistry, Molecular Biology, Cellular Biology, Chemistry, Oncology, immunohistochemistry, B7-H1 (PD-L1), pancreatic adenocarcinoma, pancreatic cancer, pancreas, tumor, T-cell immunity, cancer
From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia
Institutions: IRCCS, San Raffaele Scientific Institute, King's College London, IFOM, FIRC Institute of Molecular Oncology, Università Vita-Salute San Raffaele.
The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease’s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as “spots” on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e.
migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.
Medicine, Issue 92, Lymphocytes, Chronic Lymphocytic Leukemia, 2D Electrophoresis, Mass Spectrometry, Cytoskeleton, Migration
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1
, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2
(Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3
. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5
. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8
. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9
, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8
. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy
Institutions: Sainte-Justine University Hospital Research Center, Université de Montréal.
This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells' responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.
Medicine, Issue 80, Blood Cells, Lymphocytes, Spinal Diseases, Diagnostic Techniques and Procedures, Clinical Laboratory Techniques, Dielectric Spectroscopy, Musculoskeletal Diseases, Idiopathic scoliosis, classification, prognosis, G proteins, cellular dielectric spectroscopy, PBMCs
Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay
Institutions: University of California, Irvine (UCI).
Developmental Biology, Issue 3, embryo, Xenopus, organizer, animal pole, dissection

domain: medical


text:
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domain: design


text:
NORTH ANDOVER — Two newcomers are vying for an open School Committee seat in Tuesday’s election, with a 19-year-old not far removed from being in high school up against a mother with two young girls in the school system.
Mark Mezzina, 19, and Zora Warren, 38, will bring different perspectives to the School Committee when one or the other succeeds Christine Allen, who decided in January not to seek a third, three-year term.
“I think it’s good to have as many different perspectives on the School Committee as possible,” Mezzina said Friday. “Mine’s different and unique, but I think it would add to the discussion. I don’t want everyone to have same voice, same opinion. You need a student. Coming from a different perspective as the rest of the School Committee members, I think that would add value to the School Committee.”
Mezzina is a 2011 graduate of Austin Preparatory School. He attended Covenant Christian Academy in Peabody during his middle school years. Before that, he was home-schooled. In the fall, he attended the University of South Carolina, but transferred to the University of Massachusetts Lowell.
He has three sisters in North Andover public schools.
Last summer, he worked on former School Committee member Karin Rhoton’s effort to win a seat in the state House of Representatives. Rhoton, a Republican, lost in the general election to Methuen Democrat Diana DiZoglio.
Warren, the mother of two daughters, said a parent’s and a former teacher’s point of view is important for the committee.
“I bring to the table a variety of perspectives, teacher, parent, member of the community, taxpayer,” she said. “If elected to the School Committee, I’d be the only member who comes at it with an educational background, which I think adds value to the a variety of perspectives already on the School Committee.”
Warren has lived in North Andover for 10 years, she said, and her husband is from the town.
Both candidates want to improve the School Committee’s transparency and communication with the community. Mezzina said he thought better communication could have smoothed some of the controversy surrounding the switch to so-called standards-based grading.
“Had they made more of an effort to listen to the community earlier on, they could have avoided a lot of the problems they had with that,” he said.
Warren pointed to her effort to get the School Committee to create an email listserv, which allows residents to sign up to automatically receive emails with School Committee agendas and packets ahead of each meeting.
Selectman Rosemary Connelly Smedile, first elected to the board 12 years ago, is unopposed for a fifth three-year term. Smedile owns and operates Connelly Real Estate and served on the Finance Committee before her election to the Board of Selectmen.
This is the first time Smedile has not had an opponent.
Also unopposed is Housing Authority board member Mary Beth Soucy, who is running for a full five-year term. Soucy was appointed to the Housing Authority board to replace her mother, Helen Soucy, who died in May 2008. She was then elected to fill the remainder of the term.
Follow Douglas Moser on Twitter @EagleEyeMoser. To comment on stories and see what others are saying, log on to eagletribune.com.
Vote Tuesday North Andover polls are open 7 a.m. to 8 p.m. All voting takes place at North Andover High School, 430 Osgood St.

domain: news


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VILNIUS - Officials of the Vilnius city government on May 18 decided not to issue a permit for an EU-backed tolerance campaign rally in the capital saying they fear that the event could spark unrest in the country. The decision to ban the rally, scheduled for May 25, was made based on information that the event, which was to include the public display of a rainbow-colored flag, 's the symbol of the homosexual community, 's might be confronted by anti-gay protesters.
"Public safety and public order could be threatened, people's health and morals could be jeopardized," said an official letter from the Vilnius city government, signed by the head of the Public Order department Gintaras Tamosiunas.
"The city government was provided with data on possible violations of public order during the event," the letter sent to organizers of the rally added.
Anti-gay activists launched an Internet campaign calling on people to "resist the mandate of homosexuals" and gather to protest the planned rally.
"Democracy is a power of the majority and not the dictatorship of the minorities," a specialized Web site titled "Citizen's Linkup for Honor and Nation" said. It also calls on people to defend the family and values and "to stop the aggression of sodomy."
Vladimir Simonko, the chairman of Lithuanian Gay League, said that the city government's decision reflects the homophobic attitudes of Lithuanian society.
"It in fact shows that the majority of Lithuanian society does not accept us as fellow citizens and thinks we are not entitled to have the same rights as others," Simonko told The Baltic Times. (See interview, page 14)
The banned rally was meant to be part of a campaign called "For Diversity 's Against Discri-
mination" organized in cooperation with the European Union. It aimed at promoting tolerance towards minority groups, including gays and lesbians.
One of the highlights of the event was to have been the arrival of a so-called "anti-discrimination" bus, which is touring 19 EU member states and is designed to inform citizens of their rights under EU and national anti-discrimination legislation.
The decision by the Vilnius city government makes Vilnius the first capital in Europe to reject the campaign, which is now in its fourth season.
The European Union immediately criticized Vilnius' decision. The European Commission, the bloc's executive body issued a statement on May 21 saying that it "regrets the decision to cancel the planned event."
"It is the the first time in the four years the truck has been on tour that a stop has been canceled by local authorities," the statement said, adding that "the activities on board the truck also aim to raise awareness of anti-discrimination laws, which all Member States have signed up to."
"The decision by the city authorities shows how much still needs to be done to change behavior and attitudes towards discriminated groups and to promote awareness of diversity," the statement concluded.
The ban on the rally came days after trolleybus drivers in Vilnius and Kaunas refused to drive vehicles carrying advertisements calling for sexual tolerance.

domain: sociology


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Whether you prefer something sweeter, fruity, chocolatey, or lighter, we all have our weakness when it comes to cake. There’s also always a reason to have cake, so if you’re expecting guests, or celebrating a birthday; perhaps you’ve got a bake sale or you simply want to have one to hand for when friends pop by for afternoon tea, we’ve got the ideal recipe to suit every occasion.
Baking a beautiful cake doesn’t have to be difficult or stressful. The baking itself should be simple enough, and for a lot of the recipes if you’re looking for something more elaborate and show-stopping, then you can take some time on the decorating.
Give our best-ever chocolate fudge cake recipe
a go – it’s the ultimate in chocolate indulgence and very rich, so it’s perfect if you’re expecting guests over for dinner. Because it’s so rich, dish up in small portions, especially if it’s following a big meal. This cake’s topping sets very quickly so if you need to, use a hot, wet palette knife to smooth it and give you more time for decorating and make it easier.
You’ll find some traditional cake recipes in our collection, as well as some less common flavours and ingredients used like parsnips and courgettes. Yes, we know it’s a little unusual but trust us, they’re amazing! You can either play it safe or try something totally new, but whatever your preference, all our recipes are easy to make and won’t keep you in the kitchen too long. From show-stoppers to simple loaf cakes, we’ve got them all, so take your pick depending on the occasion and make a delicious cake that is sure to impress everyone!
Pick from easy but impressive cake recipes...

domain: food


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What was it like to attend the Olympics in 388 B.C.? Would the experience resemble Olympic festivals as we celebrate them today? This remarkable book transports us back to the heyday of the city-state and classical Greek civilization. It invites us to enter this distant, alien, but still familiar culture and discover what the Greeks did and didn't do during five thrilling days in August 2,400 years ago.
In the Olympic Stadium there were no stands, no shade-and no women allowed. Visitors sat on a grassy bank in the searing heat of midsummer to watch naked athletes compete in footraces, the pentathlon, horse and chariot races, and three combat sports-wrestling, boxing, andpankration,everyone's favorite competition, with virtually no rules and considerable blood and pain. This colorfully illustrated volume offers a complete tour of the Olympic site exactly as athletes and spectators found it. The book evokes the sights, sounds, and smells of the crowded encampment; introduces the various attendees (from champions and charlatans to aristocrats and prostitutes); and explains the numerous exotic religious rituals. Uniquely detailed and precise, this guide offers readers an unparalleled opportunity to travel in time, back to the excitement of ancient Olympia.
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domain: library


text:
Churchgoers chip in for fire victims
WITH just a few collections at St Josephs Church Tweed Heads, parishioners raised $8437 in cash to send to Victorian bushfire victims.
Saint Vincent de Paul, St Josephs Conference president Graham Murphy said the church decided to add an extra collection for the bushfire victims at each of its services two weeks ago.
He said many of the parishioners would have already donated to the appeal before, but the amount collected showed how generous they all were.
“A lot of people in the parish would have donated before that, but Australians are always most generous if their fellow Aussies are in need,” Mr Murphy said.
“Whether it is due to fire, flood or drought, we put our hand up and help out, which is great.”
He said St Vincent de Paul had encouraged cash donations because that was what was needed at the moment, rather than donations of clothes or furniture.
Mr Murphy used to live in Gippsland and experienced fires in 1965, but the damage they did was nothing compared to the current fires. He said it had shocked everyone at St Josephs.
“This last lot of fires in Victoria, I've never seen anything like that,” he said.
According to St Vincent de Paul, donations will be used to drive an effort to help the long-term recovery of communities affected by the tragedy and to help them rebuild their homes and, eventually, their lives.


text:
We don’t have a summary available yet.
The summary below was written by the Congressional Research Service, which is a nonpartisan division of the Library of Congress, and was published on Sep 26, 2017.
SNAP Vitamin and Mineral Improvement Act of 2017
This bill amends the Food and Nutrition Act of 2008 to allow Supplemental Nutrition Assistance Program (SNAP, formerly known as the food stamp program) benefits to be used to purchase multivitamin-mineral dietary supplements for home consumption.
The supplements: (1) must provide at least half of the vitamins and minerals for which the National Academy of Medicine establishes dietary reference intakes at 50% or more of the daily value for the intended life stage per daily serving, as determined by the Food and Drug Administration; and (2) may not exceed the tolerable upper intake levels for those nutrients for which an established tolerable upper intake level is determined by the National Academy of Medicine.


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Griffey Jr.UConn has advanced to 11 consecutive Final Fours.Louis has 1 hits and they have blocked 1 shots from the opposition, which is 19th in the league.To search for players who were born on a certain date, for example all players born on December 25, choose the month, day and year with the drop down boxes and then choose the ‘Full Date Search’ option.posted an assist and five shots in Sunday’s 6 win over the Blues in Game 3.
I don’t know if he will surpass Ohio State’s Dwayne Haskins, but teams will see what Mayfield, the 2017 Heisman Trophy winner from Oklahoma, did for the Browns, and believe Murray, the 2018 Heisman Trophy winner from Oklahoma, can do the same.South Florida has won the last eight meetings with the Blue Demons, the last an 83 triumph six years ago.Weekes was a young netminder who had played in 11 games prior to the trade with an .870 SV% and 3 GAA.It’s been a carousel all year long for the Flyers, as they’ve already set an NHL record by deploying eight goalies over the course of the season.A lot has to happen, including the Falcons winning their next four games.
To find all players born within a certain month and year, for example all players born in December of 1985, choose the month and year with the drop down boxes and then choose the ‘Month and Year Search’ option.A 10 win is just as much a winner as a 56 win.Guys like Michael Jordan.They have a team WHIP of 1 and their FIP as a unit is 2.
The Red Bull production line has slowed in recent years, but the team still have Carlos Sainz ‘on loan’ at Renault and are optimistic about their young drivers coming through the ranks.Last year was dismal and shows how quickly a 33-year-old’s stock can drop.The brave thing is not so Elite Womens Billy Turner Jersey much to support the person who does the revolutionary thing.We’re not going to trade a player because he asked to be traded, Casey said.Some deals are limited to specific regions, trim levels, or a particular percentage of dealer stock.1 running back for the Falcons.
But they will need to field a team, and it is conceivable that they land only one big-time star .We had to kick off to start the game, and our kicker missed the ball �?yes, missed the ball.In 12 games played, Game Youth Blake Wheeler Jersey he logged nine sacks and had a career-best five forced fumbles; he was especially dominant down the stretch, collecting six sacks and two forced fumbles over the team’s final six games of the regular season.Really tired of listening to the why didn’t they trade for this one or that one.
Seth designed the tattoos himself.Matt: Wow, Willis.And thinking http://www.packersauthenticsofficial.com/WOMENS-BILLY-TURNER-JERSEY.html solely in terms of league benefit, Chris Broussard can’t help but get excited about the possibility of Uncle Drew and KD teaming up in the Big Apple.
To find all players born within a certain month and year, for example all players born in December of 1985, choose the month and year with the drop down boxes and then choose the ‘Month and Year Search’ option.I should have sat out the ’99 season and then put myself on the market.To back up that brave assertion and to rid your thoughts of past TDI nastiness, the world’s second-largest carmaker has offered three pieces of evidence.Accordingly, they’re seeing the least amount of ice time on the team.According to Hockey-Graphs, the average NHLer stands six-foot-one and weighs 201 pounds .
Scouts and coaches won’t be assembled in Raleigh to see http://www.authenticshockeyjetsofficialstore.com/Blake_Wheeler_Jersey_Adidas him run, anyway — that day will be all about throwing for Finley.In his role as general manager, Kuharich made some awful trades, including dealing away future Hall of Famers Sonny Jurgensen and Tommy McDonald as well as Pro Bowl Eagles Timmy Brown, Maxie Baughan, and Irv Cross.Craig learned about a group of siblings, four African-American children — three boys, including Andrew, and a girl, at the time living in separate foster homes — who had all been exposed to alcohol while their mother was pregnant.So far, it’s been pretty easy �?easy as far as going from what we did last year, and the past few years, until now.
He’s been what we needed.On the other hand, rust proofing can be worthless if it’s not a proven system, fabric guard can be bought at your local supermarket, and paint sealant can be added later at a much lower price.

domain: sports


text:
Increased prevalence of scleroderma in southwestern Ontario: a cluster analysis.J Rheumatol. 2002 Sep; 29(9):1867-73.JR
To estimate the prevalence of scleroderma (systemic sclerosis, SSc) in 3 cities, Windsor, Sarnia, and Woodstock, Ontario, within our referral area, which has a referral base population of 1 million.
To compare the addresses and exposures of referrals with SSc, we performed a case control study using our patients with scleroderma and 2 age and sex matched controls from the same rheumatologist's practice.
Sixty-seven of 91 patients with SSc and 87 of 154 controls responded. The mean age of patients with SSc was 53.2 years versus 52.8 years in controls. There was no statistically significant increase in the number of SSc patients from Windsor (population 197,694): 14 patients (15.4%) with SSc versus 18 controls (11.6%) (p < 0.41); or Sarnia (population 72,738): 7 patients (7.7%) with SSc versus 7 controls (4.5%) (p < 0.31). However, there were 9 cases (9.9%) from Woodstock (population 32,086) versus one control (0.64%) (p < 0.0004). The point prevalence of scleroderma was at least 0.71/10,000 in Windsor, 0.96/10,000 in Sarnia, and 2.8/10,000 in Woodstock. There were no significant between-group differences in exposure to industrial toxins or chemicals including vinyl chloride, silica, and benzene, but exposure rates in both groups were low. Occupations and proportion of those who were work disabled were not different. Patients with SSc were not more likely to have smoked cigarettes (p < 0.43); however, they were more likely to drink at least 6 drinks of alcohol per week (p < 0.04) and had more dental fillings (p < 0.05). Patients with SSc knew on average 3.2 others with this disease, and controls knew only 0.25 others with scleroderma (p < 0.00001). Two patients with SSc knew someone with SSc in their workplace versus none of the controls.
Our a priori expected higher prevalence of scleroderma in Windsor and Sarnia did not reach significance, but the cluster in Woodstock seems statistically validated, and the exact reason for this cluster remains unclear. It is unlikely that all patients with SSc in Woodstock were seen by us, so the prevalence of scleroderma is at least 2.8/10,000, which is a medium to high prevalence compared to other studies. Associations with alcohol and dental fillings require further study.

domain: literature


A WOMAN who has been blind for almost 30 years can now see shapes again thanks to a revolutionary bionic eye.
Dianne Ashworth, 54, had electrodes fitted to her retina that send impulses to nerve cells, in the same way that occurs naturally in sighted people.
Speaking of the moment doctors switched on her prototype device, the mum-of-three said: “I didn’t know what to expect but all of a sudden I could see a little flash...
"Wow! It was amazing. Every time there was stimulation there was a different shape that appeared in front of my eye.”
Although other blind people have had bionic eyes fitted, including Brits Chris James, 54, and 60-year-old Robin Millar this year, scientists who developed Dianne’s say the way it was implanted makes it unique.
Dr Penny Allen led the Australian team of surgeons who performed the operation in Melbourne this May.
She said: “This is a world first. We implanted it behind the retina, demonstrating the viability of our approach.”
Dianne now hopes to see her son for the first time within 12 months as experts develop ways of improving the device while striving to create a complete bionic eye next year.
The implant’s electrodes are connected to a receptor fitted to the back of Dianne’s ear.
That is plugged in to a monitoring unit in a lab at the Australian Bionics Institute.
Two other Melbourne patients have had the device fitted and are waiting for them to be switched on.


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Dulles Expo Center
4320 Chantilly Shopping Center
Chantilly, VA 20153
Friday: 12:00 pm - 8:00 pm
Saturday: 10:00 am - 7:00 pm
Sunday: 11:00 am – 5:00 pm
Kids 12 and under: FREE
SAVE TIME - PURCHASE YOUR TICKETS
CASH ONLY AT BOX OFFICE
22nd Annual National Capital Boat Show
Only Boat Show For 2017!
Put aside the shovel, slow traffic, and heavy, wet snow this weekend and get out to the National Capital Boat Show. The snow will melt quickly, the cherry trees will be in full bloom in just a few short weeks, and you will be dreaming about being on the water in your new boat, fishing, swimming, and playing with friends and family so you'll want to be ready! Bring on Spring and Summer!
Find everything you need to hit the water this season at one of the largest boat shows in the region. Shop hundreds of new boats from dealers throughout Virginia and Maryland, including runabouts, saltwater fishing boats, ski boats, pontoons, deck boats, jet boats, jon boats, and PWCs.
In addition to the wide range of boats, exhibits will feature marinas, boat lifts, floating docks, outboard and inboard motors, marine electronics, waters sports and safety equipment, boating accessories and more. Plus, experts will also be on hand to answer questions and provide information on boat maintenance, repair, financing, insurance and boating safety.
There will be many dealers from all over the region displaying their boats! They are primed and ready to deal. Bring your trade in and make your best deal on a new boat. Don't miss this opportunity to get that new boat you have wanted at the best price around.
The National Capital Boat Show is your one-stop shop for the boating season. Stock up on all the essentials and gather amazing ideas for a great time on the water.
Dave Body 804-337-6479
For Booth Information
Marybeth Tedesco 804-337-8463

domain: hobby


text:
Umm, I wonder if anyone will actually see this?
Or bother to read it if they do... ¬_¬
Well anyway, I almost always end up on Livejournal for some reason or another, so I figured it was about time to join!
So I'm Jenny~ howdy!
I'm 17, quite English and I go to art college.
Basic info, you don't need anymore, not with all those interests on my profile o.O
Anyway, I hope my time here will be fun and I hope to meet some awesome people here!
If you actually read this, then cheers~! ^w^


text:
The record shows that on March 30, 2010, the trial court denied an identical petition to terminate her parental rights filed by the Children and Families. Principles of collateral estoppel dictate therefore that only such conduct as may have occurred between that date and the January 10, 2011 filing of the present petition may be considered. See Donahue v. Davis, 68 So.2d 163 (Fla.1953); Gordon v. Gordon, 59 So.2d 40 (Fla.1952); Gray v. Gray,91 Fla. 103 (Fla.1923).
Our examination of the record shows beyond question that no valid grounds for termination occurred during that time. Indeed, it demonstrates that the only change in the very favorable circumstances of the mother, including her successful efforts to break her drug dependency, to secure and hold full-time employment and to find a satisfactory home, was an arrest for an alleged offense which was later nolle prossed, and as to the circumstances of which there was no evidence, but which caused her to miss a single parenting class, part of a case plan which was otherwise fully complied with. Obviously, this far from justifies the termination of her parental rights.
D.C. v. Department of Children and Families, — So.3d —-, 2012 WL 716127 (Fla. 3rd DCA 2012).
As predicted, the fathers’ TPRs were affirmed and the pro se mother’s TPR was reversed. (There was no mention of one-parent TPR findings or why the fathers’ rights needed to be terminated if the mother’s weren’t.) The seemingly strict application of collateral estoppel in the case doesn’t sit well with me, but that’s for another post.

domain: law


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For Sale 1960 Chevrolet Impala
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This is one of the finest, most original and beautiful 1960 Impala's you will ever see. It has had a complete nut and bolt restoration and everything from head to toe has been refreshed or replaced. The big block 348 cu. in. motor and Powerglide transmission were professionally rebuilt less than 1,000 miles ago and perform flawlessly. The frame and undercarriage are like new. The paint is flawless and was done by Bill Miller (one of the best). Everything works on this beauty....even the clock & the radio! There is no bondo on this car. Any panel repairs that were made was done with metal. Pictures of the restoration go with the car along with all of the receipts. The chrome, glass, trim and stainless are all perfect. This car needs absolutely nothing, just a new owner to enjoy it. NADA "high retail" is $65,000.00.


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The Marathons have finished, the football season has ended, the summer has begun and so have a few more sports seasons such as Tennis; a sport that many more Brits have grown to love in the recent years thanks to our very own Andy Murray stealing titles left right and centre! GO ON ANDY!
Just like any other sport, tennis takes its own specific tolls on the body thus resulting in its own specific injuries. What else can you expect with such a high speed, high power, and high impact game; making full use of all the rotating joints (hips/shoulders), maximising muscle/limb extension, and placing massive pressure through the spine. Therefore we can expect our ‘specific injuries’ to resonate in the back, hips, knees and shoulders most often.
So, whether you will be enjoying the tennis from your couch this year or if you have been inspired to take up the sport for the summer, let’s have a look at how Pilates has helped these top players become the athletes they are today.
Tennis requires such immense strength, power, endurance and speed which can be obvious when watching the top players. However, their mobility, flexibility, control and balance are vital for all these requirements. Pilates is the only answer; improving range of movement, producing the ability to expend more power, therefore improving your all-round game. Not only will Pilates help to ‘improve’ aspects of Tennis training but it will reduce the risk of injuries too. It’s a No brainer!
Avoiding injuries is a very difficult task when training/playing tennis as it’s quite the one-sided sport and players can easily develop heavy muscular imbalances. Through restoration of balance and alignment this can be helped.
All body parts (muscles and bones) are linked together so what happens to one area of the body will have a certain impact/force/reaction on other areas of the body. Joint mobility (hips/knees/shoulder) and muscle flexibility are therefore crucial to allow the impact to travel through the body safely and without any loss of power.
“approximately 50% of the energy needed to hit a forehand is generated from the legs and trunk and transferred through the body to the racket”.
“When rotation stops, the arm is released in full extension and rotates around the shoulder girdle”.
“if the trunk does not rotate to provide force to the shoulder, it requires a 34% increase in the shoulder velocity to achieve the same ball velocity”.
These are all facts and actions that are made possible, avoiding injury, through Pilates training.
Some advice from Alexandra Prigent for the Pilates Foundation on ‘Pilates & Tennis Performance’:
‘A targeted Pilates programme should focus specifically on recruitment and strength of the deep and superficial muscles responsible for stabilising pivot points (pelvis and shoulders) and accelerating rotation velocity (spine, arm and wrist)’.
Here is a list of some of the Mat Pilates exercises that you know already (some we haven’t touched on yet in the blog) that will compliment certain areas of tennis training:
For: Hip mobility, glute recruitment and strength to transfer energy from the legs into hip rotation…
– SINGLE/DOUBLE LEG KICKS
For: Pelvic stability, spine stabilisation, internal oblique work for trunk rotation…
– SINGLE LEG CIRCLES
For: Centre, balance and alignment
– MOST EXERCISES
– EQUIPMENT PILATES
For: Trunk rotation to assist with acceleration and deceleration…
– SHOULDER BRIDGE
– CRISS CROSS
For: Spine extension/rotation…
– SWAN PREP/DIVE
– SPINE STRETCH FORWARD
– ROLL UP
For: Abdominal recruitment and strength in trunk
– ROLLING LIKE A BALL
– SPINE STRETCH FORWARD
– SINGLE LEG STRETCH
For: Shoulder girdle stability…
– SERRATUS PUSH UPS/PLANK
– SHOULDER BRIDGE
– Tennis players need strong stabilising muscles to help their bodies cope with the impact as mentioned previously; key stabilisers here are the glutes and Pilates is the most effective way to isolate and strengthen the muscles.
– Stretches out and loosens tight chest muscles, shoulders and upper back.
– Strengthens the core and obliques (muscles around the waist; trunk rotation).
– Increases flexibility in the lower back/hips; ‘help you get down lower for the drop shots and up higher for the lobs’.
So if you have decided to take tennis up this summer or you already play, you will undoubtedly suffer the same endurances whilst playing the game: ‘stretching forwards, sideways or overhead and frequently off-balance’. Reduce the risk of injury when you’re off balance, and recover your balance faster by including Pilates in your training!
In Andy Murray’s case, he uses Pilates not only for recovery and strength but for mindfulness and focusing:
‘I started Pilates a few weeks ago which has already helped. My body feels good compared to the last few years, hopefully I’m good to go for the next two weeks. I’ve practiced well the last few days and not been waking up with soreness or stiffness.’
‘I feel like when my mind isn’t free of anything that might be frustrating me away from the court, I can’t focus as well as I need to, when my mind’s clear I can go on the court and play, not worry about anything else, I can play much better and think a lot better on the court.’


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Driver, 20, crashes his car on a bridge and crawls out the window then falls 70 FEET to his death
- Chad Lee Melnyk from Floral City, Florida, crashed his car on Sunday evening
- The crash occurred on a bridge over Ocklawaha River near Silver Springs
- Melnyk was pronounced dead on the scene after he fell over the guardrail
A driver who crashed his car on a Florida bridge and then crawled out a window has plunged more than 70 feet to his death, authorities say.
A release from the Florida Highway Patrol says 20-year-old Chad Lee Melnyk struck a concrete railing with his car on Sunday night on the State Road 40 bridge, spanning the Ocklawaha River near Silver Springs.
After the crash, the left side of the vehicle was pinned up against the concrete barrier.
It appears Melnyk crawled out of the driver's side window and then fell over the guardrail onto ground below, the statement said.
Chad Lee Melnyk, 20, fell to his death after he crashed his car into a concrete guardrail on a bridge over Ocklawaha River near Silver Springs, Florida
Authorities say it's unclear why the car had crossed from an eastbound direction into the westbound lane, striking the railing with its left side.
The crash occurred just before 10pm on Sunday after Melnyk's car crossed over onto the other side of the road.
The 20-year-old from Floral City was driving a 2013 Chevy on the bridge.
He was pronounced dead at the scene by a fire rescue official.
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Original Acrylic on Wood Panel
A humpback whale rolls gently through an abstract sea, golden orbs floating through blue paint dribbles.
DetailsOriginal Acrylic on Wood Panel
Heavy-duty D-Rings attached, wired and ready to hang. This is a very heavy piece, so I would recommend using a stud-finder to locate the studs in your wall before you display this piece.

domain: artistic


With the footage rolling 24/7, it's only a matter of time till these cameras pick up some funny footage. We have put together a compilation of 20 hilarious CCTV footage moments caught on tape from the Newsflare archive.
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A mountain lion was seen near Corte Madera School in Portola Valley this morning (March 25), San Mateo County emergency officials reported.
The big cat was seen near the school on Alpine and Corte Madera roads at about 7:15 a.m., according to the county. No evacuations were necessary, officials said.
Anyone who has a close encounter with a mountain lion should not run from the animal, but face it and make a lot of noise, according to wildlife officials.
Mountain lions are typically most active at dawn and dusk.
More information on the predators can be found online at the state Department of Fish and Wildlife website.


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Tweens and teens are invited to learn about the basics of disaster prep in this after-hours zombie-filled event. An EPIC game of tag will conclude the night.Registration Required.
Will the humans be able to hold out or will the zombies rule the night?
Oak Creek Public Library, 8620 S. Howell Ave., (414) 764-4400, www.oakcreeklibrary.org
Your link to the biggest stories in the suburbs delivered Thursday mornings.
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- "Brass at the Border" concert
- Cudahy Board of Education Approves Inquiry into Purchase of South Shore YMCA Building
- Friends of Pulaski Park-Cudahy Planning Park Upgrade
- Cudahy School District Increases Aquatics Programming
- End of an Era
- Oak Creek Library Make and Take Craft Program
- An Open Letter to Governor Walker Regarding "Easy" Teacher Licensure
- Lego Club at St. Francis LIbrary
- 2015 St. Francis Police Department Citizen Academy
- Sharpen your sleuthing skills for New Berlin Rotary's Murder Mystery Feb. 7


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Opera Dining Chair
The Opera Dining Chair by Domitalia is sleek and contemporary with a completely upholstered steel frame. Its curved back creates geometric interest as well as provides a comfortable back support. A real statement, Opera is perfect for the design lover.
Available to order in several different leathers and fabrics - please enquire.


Beaver Men Suffer Seventh Consecutive Loss
On Monday, Feb. 18, the Penn State Beaver men's basketball team traveled to Franciscan University where it suffered its seventh consecutive loss when it was defeated by its host in a nonconference game, 77-75. The loss followed Beaver's 71-68 defeat by host Penn State DuBois on Saturday, Feb.16.
In Saturday's game, Beaver sophomore Zac Fetchin (Aliquippa/Hopewell High School) scored a team high of 17 points. Sophomore guard Tim Kubis (Aliquippa/ Hopewell High School) added 13 points while junior Matt Franetti (Beaver Falls/ Blackhawk High School) chipped in 14 points and eight rebounds. On Monday sophomore Zack Fetchin (Hopewell High School) led Beaver with 24 points and nine rebounds. Other double-digit scorers for the Lions included sophomore Tim Kubis (Hopewell High School) with 16 points, freshman Billy Stoughton (Slippery Rock High School) with 13, and Franitti with 12 points. Kubis also contributed with six assists.
The Beaver Lions, now 6-19 overall and 6-10 in the PSUAC, play their final season game when they host Penn State Greater Allegheny at 7 p.m. Saturday, Feb. 23.
For information about Beaver athletics, contact Andy Kirschner at email@example.com or (724) 773-3826 or visit http://www.br.psu.edu/StudentLife/Athletics/sports.htm.


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Monture TED LAPIDUS TL100702 Tortoise
Lunettes Ted Lapidus / Man
An acetate theme with a « retro » look. The large temples are decorated with an openwork metal plate featuring the Ted Lapidus logo . Two rectangular fronts are available: one low and another higher.


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To view this PAPER PRESENTATION, search for the session title in the Browse by Titlelisting. (See the session title located immediately below ["In Session:"])
In Session: Democratization, Nationalism, and Reconciliation in East Asia: Challenges to Reconciliation Study
1: Challenges in Japan’s Bilateral Relations in the Regional Context
Friday, March 26, 2021
12:00pm – 1:30pm EDT
Middlebury Institute of International Studies, United States
Japan’s multiple conflicts with its East Asian neighbors are rooted in the prewar and wartime history of the regional relations concerned. Ideally, resolution of these conflicts requires a common understanding among the parties about the historical facts, causes, and consequences of those conflicts but, at a minimum, recognition of the need to resolve the conflicts peacefully for the future benefit of all concerned. Unfortunately, the moral, legal, and political implications of the conflicts are exceedingly complex and the national perspectives on them have been mutually irreconcilable and continue to frustrate attempts at reconciliation. Contemporary political dynamics in and between the nations also compound the situation. This paper discusses the main factors behind Japan’s strained relations with China and ROK at four levels of analysis: the individual level (i.e., Prime Minister Shinzo Abe and his nationalist beliefs and revisionist view of modern Japanese history); the domestic level (i.e., the political context characterized by a succession of weak leaderships (until Abe’s premiership) and growing nationalist sentiment among the public and their largely negative attitudes toward China and Korea; the bilateral level (i.e., Japan’s contentious relations with China and Korea); and the regional level (i.e., the changing balance of power relations in East Asia, particularly the rise of China and the uncertain but indispensable military role of the United States, complicated by the deeply integrated economies of East Asian countries). The paper then proposes some principles to guide Japan’s regional relations with a view toward ameliorating the existing tensions.


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Police: Hit-and-run driver kills woman outside Wal-Mart store entrance
FOUNTAIN, Colo. — A woman driving a stolen car hit and killed another woman Monday who was in the crosswalk outside of the entrance to a Wal-Mart store in Fountain.
It happened at the store on South Highway 85/87 at about 11:20 a.m. The victim was 67 years old.
Fountain police said the driver, 26-year-old Rebecca J. Champigny was driving through the store’s parking lot in a stolen 2015 gray Hyundai Sonata when when she hit the woman.
“The driver, after striking the pedestrian, continued across Camden Blvd and collided with a parked car in a parking lot at 200 Plaza Blvd. Drugs and/or alcohol are not believed to be a contributing factor,” a police statement said. “The driver, Champigny, was apprehended, arrested, and charged with multiple charges including vehicular homicide, accident involving death duties, and motor vehicle theft.”
She was being held in the El Paso County Jail.


PATTANI - Fifty-six people were confirmed injured when two bombs went off at Big C Supercenter in Muang district on Tuesday afternoon.
The first explosion occurred about 2.10pm and the second shortly after. Another suspicious object was found at Diana department store in Muang district, Pol Col Preecha Prachumchai, a deputy superintendent at Muang police station said.
The first bomb went off near the food centre inside the Big C building and the second followed shortly after outside the building, after shoppers and staff had left the building in panic, security authorities said.
The second bomb was inside a bag placed in a pickup truck parked outside the building, they said.
At least 56 people were injured by the explosions. All were sent to Pattani hospital. The hospital called for urgent blood donations, especially Group A/B and B.
The pickup used for the bomb outside the hypermarket had Yala registration plates. Authorities were checking on the ownership.
Big C Supercentre in Pattani's Muang district is damaged by two bombs. (Photo by Adbulloh Benjakat)
Video from www.facebook.com/montsakarn.kasamsuk