text:
A module for light sheet or single plane illumination microscopy (SPIM) is described which is easily adapted to an inverted wide-field microscope and optimized for 3-dimensional cell cultures, e.g., multi-cellular tumor spheroids (MCTS). The SPIM excitation module shapes and deflects the light such that the sample is illuminated by a light sheet perpendicular to the detection path of the microscope. The system is characterized by use of a rectangular capillary for holding (and in an advanced version also by a micro-capillary approach for rotating) the samples, by synchronous adjustment of the illuminating light sheet and the objective lens used for fluorescence detection as well as by adaptation of a microfluidic system for application of fluorescent dyes, pharmaceutical agents or drugs in small quantities. A protocol for working with this system is given, and some technical details are reported. Representative results include (1) measurements of the uptake of a cytostatic drug (doxorubicin) and its partial conversion to a degradation product, (2) redox measurements by use of a genetically encoded glutathione sensor upon addition of an oxidizing agent, and (3) initiation and labeling of cell necrosis upon inhibition of the mitochondrial respiratory chain. Differences and advantages of the present SPIM module in comparison with existing systems are discussed.
24 Related JoVE Articles!
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
Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry
Institutions: Henry Ford Hospital.
Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro
and in vivo
models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro
, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.
Medicine, Issue 83, Primary Cell Culture, animal models, Nervous System Diseases, Neoplasms, glioblastoma, neurosphere, surgical specimens, long-term self-renewal
Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress
Institutions: CEA DSV iRCM SCSR, INSERM, U967, Université Paris Diderot, Sorbonne Paris Cité, Université Paris Sud, UMR 967.
Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.
Neuroscience, Issue 87, EdU, BrdU, in utero irradiation, neural stem and progenitor cells, cell cycle, embryonic cortex, immunostaining, cell cycle checkpoints, apoptosis, genotoxic stress, embronic mouse brain
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
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
Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion
Institutions: University of Dundee, A*Star, Singapore.
3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.
Biomedical Engineering, Issue 85, tumor microenvironment, stromal fibroblasts, extracellular matrix, tissue engineering, dermal equivalent, collagen, native matrix
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma
Institutions: Advanced Cell Diagnostics, Inc..
The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ
hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ
analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.
Medicine, Issue 85, RNAscope, Head and Neck Squamous Cell Carcinoma (HNSCC), Oropharyngeal Squamous Cell Carcinoma (OPSCC), Human Papillomavirus (HPV), E6/ E7 mRNA, in situ hybridization, tumor
High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli
Institutions: Aix-Marseille Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
Escherichia coli (E. coli)
is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli
cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.
Bioengineering, Issue 89, E. coli, expression, recombinant, high throughput (HTP), purification, auto-induction, immobilized metal affinity chromatography (IMAC), tobacco etch virus protease (TEV) cleavage, disulfide bond isomerase C (DsbC) fusion, disulfide bonds, animal venom proteins/peptides
Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
Institutions: National Institutes of Health, National Institutes of Health.
Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.
Stem Cell Biology, Issue 89, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
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
Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Institutions: California Institute of Technology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, University Of Washington Medical Center, Johns Hopkins University School of Medicine.
The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for C
omputer tomography-guided f
radiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.
Neuroscience, Issue 81, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells
Institutions: Columbia University, Columbia University.
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the Fo
-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
Bioengineering, Issue 77, Microbiology, Cellular Biology, Molecular Biology, Biochemistry, life sciences, roGFP, redox-sensitive green fluorescent protein, GO-ATeam, ATP, FRET, ROS, mitochondria, biosensors, GFP, ImageJ, microscopy, confocal microscopy, cell, imaging
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
Institutions: University of Birmingham.
Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo
transplantation. In vivo
transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.
Cellular Biology, Issue 43, Skeletal muscle stem cell, embryonic tissue culture, apoptosis, growth factor, proliferation, myoblast, myogenesis, satellite cell, skeletal muscle differentiation, muscular dystrophy
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
Mouse Bladder Wall Injection
Institutions: Stanford University School of Medicine.
Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.
Medicine, Issue 53, stem cell, bladder cancer, intramural injection, bladder wall injection, bladder
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
Institutions: Pennsylvania State College of Medicine, Pennsylvania State College of Medicine, University of California Los Angeles, School of Medicine.
Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro
Developmental Biology, Issue 56, CD133, liver stem cell, oval cell, liver cancer stem cell, stem cell, cell isolation, non-parenchymal fraction of liver, flow cytometry
Production and Detection of Reactive Oxygen Species (ROS) in Cancers
Institutions: Baylor College of Medicine.
Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2
(hydrogen peroxide), NO (nitric oxide), O2-
(oxide anion), peroxynitrite (ONOO-
), hydrochlorous acid (HOCl), and hydroxyl radical (OH-
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1
, autoimmune diseases 2
, etc…) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4
. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6
, migration 7
etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3
. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8
. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10
. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.
Medicine, Issue 57, reactive oxygen species (ROS), stress, ischemia, cancer, chemotherapy, immune response
Planarian Immobilization, Partial Irradiation, and Tissue Transplantation
Institutions: University of Utah School of Medicine, UCSB, Howard Hughes Medical Institute, Stowers Institute for Medical Research.
The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2
. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3
. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4
, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6
and partial irradiation7
have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea
. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8
. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.
Developmental Biology, Issue 66, Neuroscience, Molecular Biology, Medicine, transplantation, partial irradiation, rescue, immobilization, planaria, flatworm, stem cell, regeneration
Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection
Institutions: DFG - Research Center and Cluster of Excellence for Regenerative Therapies Dresden, Germany.
Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1
. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2
. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4
. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo
. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero
electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8
. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9
. Both approaches, whose basic principles were already described by other video protocols 10-14
, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e.
by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.
Stem Cell Biology, Issue 68, Neuroscience, Developmental Biology, Neural stem cells (NSC), brain development, adult neurogenesis, cyclin-dependent kinase 4 (cdk4), cyclin D1, in utero electroporation, viral stereotaxic injection
Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK
Institutions: Saint Louis University.
Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.
Infection, Issue 70, Immunology, Virology, Medicine, Infectious Diseases, Ophthalmology, Herpes, herpetic stromal keratitis, HSK, keratitis, pathogenesis, clinical evaluation, virus, eye, mouse, animal model
A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics
Institutions: Hospital for Sick Children, Toronto Medical Discovery Tower, Princess Margaret Hospital, Toronto Western Hospital.
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.
Cancer Biology, Issue 76, Medicine, Biomedical Engineering, Cellular Biology, Molecular Biology, Genetics, Neuroscience, Neurobiology, Biophysics, Anatomy, Physiology, Surgery, Intracranial Window, In vivo imaging, Stereotactic radiation, Bone Marrow Derived Cells, confocal microscopy, two-photon microscopy, drug-cell interactions, drug kinetics, brain, imaging, tumors, animal model
Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma
Institutions: Mary Babb Randolph Cancer Center, West Virginia University, West Virginia University , West Virginia University .
Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo
bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu
mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo
application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.
Medicine, Issue 53, Invasion, mouse model, two-photon microscopy, tongue, orthotopic, head and neck cancer
Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes
Institutions: University of California, Irvine (UCI).
In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.
Cellular Biology, Issue 5, mosquito, malaria, dengue fever, genetics, infectious disease, Translational Research

domain: health


Indium (In - 156.1oC)
Ingots: 1 kilo, 500g
Bismuth, Tin, Indium, Cadmium, Lead, Antimony, Zinc, Aluminium,
Copper, Nickel, Molybdenum, Tungsten, Rheinum, Tantalum.
Indium is a very soft, silvery-white metal with a brilliant lustre, primarily produced as a by-product of zinc and lead smelting.
Indium is used to coat the bearings of high speed motors since it allows for the even distribution of lubricating oil. Indium is used to dope germanium to make transistors. It is also used to make other electrical components such as rectifiers, thermistors and photoconductors, and has a use in the production of flat screen TV's and computer displays. Also used to produce low melting alloys such as our Lens alloy117 and 136, Indium is very good at withstanding low temperature and this makes it useful for gaskets and seals in cryogenics.
Melting Point 156.17 oC
Boiling Point 2000 oC
Thermal Conductivity @ 20oC 0.0204cal/(s.cm. oC)
Specific Heat @ 20oC 0.057cal/g
Latent Heat Of Fusion 6.8k-cal/g-atom
Brinell Hardness 0.9
Nearly 15% of Indium consumption goes in the manufacturing of electrical components.
Indium has a very low melting point and the ability to conduct electricity, it is used in the making of solders and alloys.
Indium also finds extensive use in coatings of glasses, aircraft windows, building windows, doors, refrigerators and ovens.
Indium tin oxide is popular in low pressure sodium lamps.
Indium is used as a solder alloy owing to its ability to fill the difference of the coefficient of thermal expansion (CTE) of two different metals that cause breakage between metal joints due to contraction and expansion.
This material is available in the following forms
Ingots: 1 kilo & 500g


text:
Name: XXX Sex: Male
National: Han origin: Tangshan City, Hebei Province
Academic qualifications (degree): Undergraduate Political outlook: Party members
Profession: Industrial Design
Phone: XXX-XXXXXXXX phone: 13XXXXXXXXX
Contact Address: Hebei Province Yanshan University West Campus Box 1140 Zip: 066004
Email Address: firstname.lastname@example.org QQ No.: XXXXXX
Graduate institutions: Hebei Yanshan University 2004.9 - 2008.7 Design Industrial Design
(1) hand-painted to highlight the capacity, performance and results at fast mapping, and there are a number of individual works
(2) master: Alias studio tools Rhino 3D max Vray auto CAD CorelDraw Illustrator PhotoshopCS2 such as two-dimensional mapping of three-dimensional graphics and design software.
1. University on several occasions during the school organization to Beijing, Shanghai, Hangzhou, Guangzhou, Shenzhen and other cities study design was to good blue map, wave, Haier, Shanghai pan-Asian design interns to visit companies and enterprises.
2. University during the Head of the Department of Industrial Design School Design and more to the project together, the "Fortis" socket design company, "Fortis," the company's exhibition space design, the design of Apple's keyboard.
3. Their single man-made "beautiful self-made" cosmetics store sales for the design, checkout and future design, has been completed renovated four stores, all designed by me. And so on.
(Note: The above is absolutely true practical experience, can provide the works and the original files.)
I have good knowledge of art and artistic quality, hand-painted to highlight the capacity, good performance and the effect of rapid mapping, according to the company's need for creative design production, master a wide range of computer software, able to efficiently complete the work. Practical ability, and good at self-learning. The design of active thinking, I am a strong sense of responsibility, character, cheerful, active thinking, highly creative and easy to communicate, have good sense of team.
Reward and performance:
2004 - 2005 spring semester to obtain a second-class scholarship
2004 - 2005 In the fall semester professional scholarship prize fight
2005 - 2006 was third in the fall semester scholarship
2005 - 2006 was named the outstanding member of
2005 - 2006 was named the hospital-level students Miyoshi
Was named colonel 2007-2008 Miyoshi students
2007-2008 Guangzhou Auto Cup Design Competition Excellence Award
2007-2008 has been incorporated into the design of New Horizons World Chinese Directory
Cheerful, modest, self-discipline, self-confidence
Another: the most important thing is the ability, I believe that your company will feel that I am a suitable candidate for this position! Look forward to working with your interviews!

domain: artistic


text:
In the wake of the tragic death of Trayvon Martin, associations throughout the country are now reassessing their involvement in neighborhood watch programs in their communities. My comments to a reporter with the Associated Press on the matter were published in a recent article that appeared in news outlets nationwide (click here to read the report), and it now appears likely that Martin’s parents will be filing a wrongful death lawsuit against the community association.
In reaction to this and other news reports about the legal implications of the actions of neighborhood watch volunteers within community associations, the Community Associations Institute (CAI) recently issued a press release with helpful guidelines and recommendations for community associations that wish to implement watch programs manned by volunteer residents in their communities. The press release, which can be accessed by clicking here, stipulates that associations should work with their local police department to implement these programs, create a process for recruiting responsible volunteers who will follow all of the written procedures for the security measures, and continuously reinforce these procedures and the do-not-engage rules with the volunteers. Our firm is very active with the South Florida CAI chapters, and we applaud the organization for issuing this press release to help associations gain a better understanding of the proper procedures for implementing neighborhood watch programs in their communities.
Security has traditionally been one of the most important considerations that associations feel compelled to address, but budget constraints limit their ability to hire professional security guards for on-site monitoring and protection for their residents. In response, many associations resort to creating neighborhood watch programs, which are typically comprised of volunteer owners who agree to keep a watchful eye for suspicious activity.
The Trayvon Martin case illustrates the concerns for associations that organize their own watch programs. In light of this tragic case, many associations are likely to avoid partaking in the organization and implementation of these programs, because doing so could result in significant liability for the association. Notwithstanding these concerns, if associations feel compelled to participate in the organization of a watch program and endorsing it in their community, they should do so with the utmost precautions detailed in the CAI release. These include organizing workshops with their local police department to establish procedures and training for the individuals who volunteer to participate in order to help ensure that they limit their involvement to watching and listening for suspicious activity and contacting the police when necessary, rather than taking on active duties to follow and engage individuals who are suspected of being involved in criminal activity. It is also important for the associations to stress in their written procedures that these individuals are not allowed to conduct armed patrols in the community.
In addition, the associations should consult with their insurance carriers and agents to determine whether they are covered for liabilities that may be caused by the actions of the watch volunteers, who should be vetted by the association with a criminal records background check. If the association learns of any questionable conduct or history of criminal activity by the volunteer, they should take immediate steps to disallow any involvement in the watch program by the individual.
There are many reasons why associations should avoid formally creating these watch groups and leave it up to the individual owners to band together to develop their own efforts outside of the auspices of the association. However, for associations that cannot or will not distance themselves from the formation of the watch groups, they should follow these and other guidelines, including those suggested by CAI, and consult with their own attorneys in order to limit their potential liability to the greatest possible extent.

domain: sociology


text:
Thomson Holidays, which is suffering diminished profits and disappointing millennium sales, launched its range of no frills holidays called "Just". The budget brand offers customers the "real essentials" of a flight and hotel room. "Unnecessary extras" such as food on the flight, transfers from airport to resort, and tour guides must be paid for on top of the package.
"It's a reduced holiday package, but not an inferior one," said Shaun Powell, deputy managing director of Thomson. "It's for people who only want the bare essentials - just a flight, just a room, just a beach."
The concept was inspired by the success of easyJet and British Airway's carrier, Go, which offer basic flights without food or in-flight entertainment. But Jeremy Skidmore, editor of Travel Weekly, said yesterday he doubted the concept would work for holidays. "You're going to end up paying extra for just about everything except flight and hotel room," he said.
"You may not realise it, but you get an awful lot for your money when you buy a package holiday. If people think they are getting an all-in package holiday with `Just' they are sorely mistaken. This is not a package holiday as we know it."
Mr Skidmore emphasised that flight supplements mean what may appear like a pounds 100 holiday could end up costing double. "If you fly from airports in Scotland or Northern Ireland on a Friday, Saturday or Sunday between 5am and 7pm you have to pay a pounds 80 supplement. That's a sizeable increase on what looks like a very cheap holiday."
Should the "Just" holidays prove successful, he said they were still not a solution to Thomson's present problems. The company has issued two profit warnings in two months following a dire half-year performance which led the City to revise its year-end profit forecast from pounds 110m to pounds 85m.
Keith Betton, head of corporate affairs at the Association of British Travel Agents , said the new brand signalled a return to the price wars of last decade.
"One of the problems in the 1980s was that there were very cheap holidays, but they were not backed up with quality," he said.
The millennium is not proving to be the goldmine that travel companies had hoped. "Demand for short-haul destinations has not lived up to expectation," Mr Betton said. "All the travel companies are trying to build in a bit of extra profit, but in reality the demand does not really justify it."
After an initial rush for Thomson's millennium packages, which were launched 14 months ago, interest died down. "The millennium holidays sold exceptionally well initially," he said. "Then there was so much hype that the vast majority of people thought there was nothing left. We need to get people to realise that they can still go."
t Most adults in Britain are still undecided as to whether they will do something special to mark the dawn of the new millennium, according to a survey published yesterday.
Only one in 10 of the 1,000 adults polled by NOP said they intended to visit either a pub, club, restaurant, show, or take a holiday.Reuse content

domain: economics


Sooner Great Dane was originally established in 1974 as TIMPTE of Oklahoma. In 1988 the dealership was changed to Sooner Great Dane. The company is one of the largest trailer parts inventories in the southwest. It offers a complete line of parts for Great Dane, Pines, Mac, American, Circle-R, CPS and Morgan. Sooner Great Dane is also a distributor for Maxon, Todco and Midland. Its products include rolling tarps, liquid containers and cover systems. Sooner Great Dane has the knowledge and ability to customize equipment for specific uses, such as roll offs, end dumps and belly dumps. Sooner Great Dane is headquartered in Oklahoma City.


To determine if your Cadillac has Magnetic Ride Control, check for the presence of a button or switch labeled “MRC” or “Magnetic Ride.” Additionally, you can consult your vehicle’s owner’s manual for information about the specific features and options of your Cadillac model.
Magnetic Ride Control is an advanced suspension system that adjusts the damping characteristics of your Cadillac’s shocks in real-time, providing a smoother and more comfortable ride. If you’re unsure, it’s always best to contact your local Cadillac dealership for assistance or schedule a service appointment to have your vehicle inspected by a professional technician.
Understanding Magnetic Ride Control
Magnetic Ride Control is a suspension technology developed by Cadillac that uses magnetic shock absorbers to improve the driving experience. Unlike traditional suspension systems that rely on mechanical valves to control damping, Magnetic Ride Control utilizes magnetorheological fluid and electromagnets to adjust damping in real-time. This allows for a more responsive and adaptive suspension system that can quickly and continuously adjust to changing road conditions.
One of the key benefits of Magnetic Ride Control is its ability to provide a smooth and comfortable ride while also improving handling and performance. The system can automatically adjust damping based on factors such as vehicle speed, road conditions, and driver inputs, resulting in a more controlled and enjoyable driving experience. Additionally, Magnetic Ride Control can also help reduce body roll and improve stability during cornering, further enhancing the overall handling characteristics of the vehicle.
The evolution of suspension systems has been a continuous process aimed at improving ride comfort and handling performance. From conventional suspension systems to electronically controlled systems and now to Magnetic Ride Control, advancements in suspension technology have allowed for greater control and adaptability. Magnetic Ride Control represents the latest evolution in suspension technology, providing a smoother and more refined driving experience for Cadillac owners.
Identifying Magnetic Ride Control In Cadillacs
When trying to determine whether your Cadillac has Magnetic Ride Control, there are a few visual clues you can look for. Firstly, check for the presence of a button labeled “Tour” or “Sport” on the center console or dashboard. This button is typically associated with Magnetic Ride Control and allows you to switch between different driving modes.
Additionally, you can locate Magnetic Ride Control features by reviewing the vehicle specifications. Look for phrases such as “Magnetic Ride Control suspension” or similar terminology. These specifications can usually be found in the owner’s manual or on the Cadillac website.
If you’re still unsure, you can confirm the presence of Magnetic Ride Control by verifying with the vehicle identification number (VIN) and dealership resources. Provide the VIN to your local Cadillac dealership and they can provide you with detailed information about your vehicle’s features, including whether it is equipped with Magnetic Ride Control.
Models And Years With Magnetic Ride Control
Here is a year-wise breakdown of Cadillac models with standard Magnetic Ride Control:
|Escalade EXT, XLR-V
|CTS-V, Escalade, Escalade EXT, XLR-V
|CTS Coupe, CTS-V, DTS, Escalade, Escalade EXT, SRX, XLR-V
|CTS Coupe, CTS-V, Escalade, Escalade EXT, SRX, XLR-V
|CTS Coupe, CTS Sedan, CTS-V Coupe, CTS-V Wagon, Escalade, Escalade ESV, SRX, XTS, XLR-V
Additionally, some special editions of Cadillac models have unique Magnetic Ride Control setups. These include limited editions like the Cadillac CTS-V Black Diamond Edition and the Cadillac CTS Crystal Red Special Edition.
Measuring Performance Impact
Cadillac’s Magnetic Ride Control is a cutting-edge technology that enhances the performance of your vehicle. It uses magnetorheological fluid in the shock absorbers to adjust the suspension in real-time, providing a smooth and comfortable ride.
When it comes to measuring the impact of this technology on your Cadillac’s performance, it’s important to consider ride quality. One way to do this is by comparing the ride with and without Magnetic Ride Control. Without this feature, your vehicle may feel stiffer and less responsive, especially over uneven road surfaces. With Magnetic Ride Control, the suspension adapts to the road conditions, offering improved handling and comfort.
Owners’ testimonials highlight the advantages of Magnetic Ride Control. Many customers appreciate how it enhances their driving experience, providing a more connected feel to the road. They mention the improved stability during cornering and the ability to switch between sporty and comfortable modes based on their preferences. Overall, Magnetic Ride Control adds a level of sophistication to your Cadillac, ensuring an enjoyable driving experience.
Upkeep Of Magnetic Ride Control Systems
If you own a Cadillac with Magnetic Ride Control, it’s essential to know how to determine if your vehicle is equipped with this advanced suspension system. One way to identify this feature is to look for a button or switch labeled “MRC” on the dashboard. Magnetic Ride Control systems use magnetic fluid and electromagnets to adjust the suspension in real-time, providing a smooth and controlled ride.
Maintaining your Magnetic Ride Control system is crucial for optimal performance. Regular maintenance tips for Magnetic Ride Control include inspecting the suspension components for any signs of wear, ensuring the electromagnetic coils are clean and free of debris, and checking the magnetic fluid levels periodically. If you encounter any issues with your Magnetic Ride Control, it’s important to troubleshoot the problem promptly. Common issues may include error messages, leaks, or harsh ride quality. Upgrading your current suspension to Magnetic Ride Control is also a possibility if you wish to experience the benefits of this advanced technology.
Enhancing Your Cadillac With Magnetic Ride
If you are wondering whether your Cadillac has Magnetic Ride Control, there are a few ways to tell. Firstly, check your owner’s manual or contact your dealership with your vehicle identification number (VIN) to determine if Magnetic Ride Control was included in your Cadillac’s original specifications. Another way to check is to look for the Magnetic Ride Control button on your dashboard, which allows you to switch between different driving modes.
If your Cadillac doesn’t have Magnetic Ride Control but you want to upgrade, there are aftermarket options available. However, it’s important to consider the costs involved. Retrofitting Magnetic Ride Control can be expensive, as it requires specialized parts and installation. Additionally, finding a certified installer is crucial to ensure the proper installation and functionality of the system. Research different installers, read customer reviews, and ask for recommendations from other Cadillac owners to find a reliable and qualified professional.
By following the steps discussed you can easily identify if your Cadillac has Magnetic Ride Control. From checking the owner’s manual to looking for specific indicators, such as a button or badge, you can be confident in determining whether your vehicle is equipped with this advanced suspension system.
By understanding the benefits and characteristics of Magnetic Ride Control, you can enhance your Cadillac’s performance and luxury experience.


text:
Bees are simply amazing. Some fun facts
- To create one pound of honey, bees have to visit 2 MILLION flowers to generate enough nectar and pollen.
- In her lifetime (which is about 4 months), a honey bee will generate 1/12 teaspoon of honey.
- One hive can generate 3-5 gallons of honey each year.
- When harvesting, we only take their excess and leave them more than enough to live on through the winter.
- The honey bee's wings stroke incredibly fast, about 200 beats per second, thus making their famous, distinctive buzz. A honey bee can fly for up to six miles, and as fast as 15 miles per hour.
- A hive of bees will fly 90,000 miles, the equivalent of three orbits around the earth to collect 1 kg of honey.
- It takes one ounce of honey to fuel a bee's flight around the world.
- The queen bee lays up to 2500 eggs per day. The queen bee has control over whether she lays male or female eggs.

domain: biology


text:
EA continues to milk the Ultima franchse, as they've announced Ultima Online 7th Anniversary Edition, bundling everything from the previous Ultima Online games. Here's the pr:
Electronic Arts (Nasdaq:ERTS), the world's leading interactive entertainment software company today announced the Ultima Online(TM) 7th Anniversary Edition, commemorating the game's historic seven-year run. The special version of the longest-running online game is currently in development and scheduled for release this summer.
With Ultima Online 7th Anniversary Edition, players will be able to enjoy one of the most immersive online worlds because in addition to the original Ultima Online game, the following five expansion packs will be included: Utlima Online(TM) The Second Age(TM), Ultima Online Renaissance(TM), Ultima Online(TM): Third Dawn(TM), Ultima Online(TM): Lord Blackthorn's Revenge and Ultima Online(TM): Age of Shadows(TM). Players will also receive one of seven rare and powerful anniversary gifts, each offering an exclusive benefit to the lucky recipient, and a collector's edition of the Ultima Online Fan Guide, which includes exclusive art from the history of Ultima Online and interviews with the development team and community leaders.
As an added bonus, included in the Ultima Online 7th Anniversary Edition package will be a full version of the classic Ultima(TM) IX: Ascension, the last game in the Ultima series that was hailed as one of the greatest fantasy adventures of all time and was the catalyst for Ultima Online.
"We are very proud and excited about UO's upcoming seventh birthday, a record in the online gaming world," said Anthony Castoro, producer of UO. "To celebrate, we want to give our players the total UO experience plus a few unexpected surprises."

domain: hobby


text:
you are viewing
LINTON CELTIC TRIOS
Bring the sounds of the Celtic culture to life at your event by booking a Celtic Trio to perform live: weddings, birthday parties, festivals, and more. Choose from our database of talented singers and musicians here on GigMasters.
Please note these Celtic Trios
will also travel to Strasburg, Hazelton, Cannon Ball, Braddock, Westfield, Hague, Kintyre, Fort Yates, Moffit, Napoleon, Solen, Zeeland, Pollock, Herreid, Wishek, Wakpala, Sterling, Lincoln, Driscoll, Menoken, Lehr, Steele, Dawson, Saint Anthony, Mc Laughlin, Mound City, Bismarck, Ashley, Eureka, Fredonia

domain: entertainment


text:
Skip to comments.Seven Democratic candidates for governor, none pro-life
Posted on 02/27/2014 3:30:52 PM PST by franky8
The seven Democratic candidates for Pennsylvania governor were asked during a recent joint appearance if they were pro-life or pro-choice. Not a single one said, pro-life. Hear their responses for yourself in this brief video clip.
One of the seven, front-runner Allyson Schwartz, was the former executive director of a Planned Parenthood abortion facility in Philadelphia for 13 years, until it finally went bankrupt.
Since the video was made, an eighth Democratic candidate has entered the race: former Pennsylvania Auditor General Jack Wagner.
I doubt any of the democrat gubernatorial candidates nationwide are anywhere near pro life.
Higher level Democrats are not allowed to be pro-life. It is all a shell game designed to fool the more conservative states.
Then again, governors have very little impact on the issue of life ..
Anti-life, anti-liberty, anti-property, anti-pursuit of happiness.
Life, liberty and the pursuit and destruction of totalitarians.
Laboratories of democracy are the stomping grounds of socialists ALERT!
They influence it in that they influence the culture. Every Pro-Abortion slob out there is best countered by one pro-life candidate.
True enough. I certainly wouldn’t trust my progressive republican governor with more power on the issue.
Granted he’s signed bills that have made abortions harder through regulation and licensing but I don’t think it has anything to do with a pro life sentiment.
Even though they have been losing population for decades, PA people are still on board for “population control” and “sexual freedom”.
We should have known when they denied Bob Casey senior.
The “esteemed” Gov of N Y is a classic example.
captain obvious is not surprised.
Was that the 1972 Democratic convention or the 1976 convention? One of those years they came out in favor of abortion and the gay agenda. Pat Buchanan made a lot of noise about it but the Dems skated right through it. The Dem voters are either asleep or just waiting for their handouts. I am glad that the Republicans at least held their ground then and have for a long time but now they seem to want to be like the Democrats.
I don’t disagree with that - but it’s around the margins only.
I don’t disagree with that - but it’s around the margins only.
The day Tom Ridge (pro-abortion Republican) became governor of Pennsylvania, inspections of abortion mills ceased. Result: Kermit Gosnell. And other horrors.
Don’t worry. In about 40 years, from their prison cells, the Catholic bishops will issue a warning that they are thinking about considering setting up a committee to ponder the question of whether the Democrat party might be an organization that Catholics possibly should not join.
AS WE HAVE THOUGHT
OBAMA ADMINISTRATION ‘DOING THE WORK OF SATAN’
A top executive for Americas leading pro-life organization, the American Life League, says the Obama administration is doing the work of Satan through its abortion and abortifacient mandates.
The conclusion comes from Jim Sedlak, vice president of the pro-life group that faithfully follows Catholic teaching and opposes not only abortion but other death-related agendas such as euthanasia.
Sedlaks comments came only a day after the Washington Post quoted Exorcist author William Peter Blatty calling a graphic abortion procedure demonic.
At virtually the same time, Obamas Health and Human Services Secretary Kathleen Sebelius was waffling in testimony before Congress on whether health care insurance policies would be available under Obamacare that allow religious believers to opt out of funding abortion.
We have always described the work of Planned Parenthood as Satanic, Sedlak told WND on the issue of abortion and Blattys reference.
If you look around the country, if Satan were to establish an organization today, what organization would be most like that one, he said. The answer is Planned Parenthood, which does the work of Satan with its attacks on our young people with sexual sin.
It also promotes the total devaluation of human life, he said.
We frequently refer to those that are radical pro-abortion as doing the work of Satan, he said.
Yes, there really is a devil. Discover the arguments in Spiritual Warfare,
In this regard, certainly the [Obama] administration is doing the work of Satan.
Blattys comment came during a conversation about his petition to the pope to instruct Georgetown University to follow the teachings of its founder, the Roman Catholic church, or lose its Catholic identity.
According to the Washington Post, Blatty said the last straw for him regarding Georgetown was its invitation to Sebelius, who was radically pro-abortion even when she was the governor of Kansas, to speak at commencement.
Sebelius has a record of supporting abortion rights, and abortion is the issue that really sets Blattys nerves on fire, the newspaper said. He describes, his voice trembling, a particular abortion procedure in graphic detail. He pauses. His voice is nearly a whisper. Thats demonic.
A counsel to Blatty explained to WND that the graphic abortion procedure was partial birth abortion, often done by Sebelius friends in the abortion industry. In the process, the live babys body is delivered, but before the head emerges, the skull is crushed and the brains vacuumed out.
Sebelius, in testimony before Congress on the catastrophic failure of the Healthcare.gov website, responded to questions about the mandate that consumers pay for abortions as part of their health care.
There also is a requirement for abortifacients, a topic that already has been the subject of dozens of lawsuits against the Obama administration. In large numbers of cases, judges have ruled that requirements by the Obama administration that company owners violate their religious faith and pay for abortifacients cannot be enforced before a final judicial determination on the constitutionality of the demand.
Sebelius was asked during the congressional hearing Wednesday whether offers for insurance to consumers would be clearly identified and so people can understand if theyre going to buy a policy that has abortion coverage or not because right now you cannot make that determination.
Sebelius said, I will check and make sure that that is clearly identifiable.
But Sedlak said the Obamacare system was set up so that people must sign up and agree to purchase insurance before they are told whether abortion funding is included in the premiums they pay.
He pointed out there is no question about abortion funding. He said in a court case in Pennsylvania, Department of Justice attorneys said if Catholic Charities objected to paying for contraceptives or sterilizations, the insurance company would have to provide those services and seek reimbursement from the government.
That, he said, puts taxpayers on the hook for abortion funding.
We get phone calls in the office increasingly from people asking how do I know if my insurance policy funds abortions. I dont want to sign up for any police like that, he said.
Right now theres no way to tell, Sedlak said.
He said, however, it aligns with Obamas long-term, radical dedication to abortion.
He voted against, while he was a state senator, bills to help babies born alive, Sedlak noted. Hes radically pro-abortion.
Another pro-life organization, Operation Rescue, also raised concerns about Sebelius claims not to know about abortion payments.
We know Sebelius to be a radical abortion activist who, as Kansas governor, invested the better part of her two terms covering up criminal abortions and blocking legislation that would have supplied oversight to abortionists who were running amok over the health and safety of women, wrote Operation Rescues Cheryl Sullenger.
As governor, Sebelius covered up for filthy and unsafe abortion clinic conditions when she vetoed common sense clinic safety laws in 2003 and 2005. In her 2003 veto statement, Sebelius proudly declared that Kansas abortion clinics met the highest standards for medical care in the country. Meanwhile, abortion clinics in Kansas City and Wichita were operating in blood-smeared facilities infested with roaches and other vermin while George Tillers late-term abortion clinic in Wichita was sending one woman after another to the emergency room and one to the morgue.
Operation Rescue has documented Sebelius holding a secret soiree for late-term abortionist Tiller in the governors mansion while Tiller was under criminal investigation by the attorney generals office.
Later, she appeared at a birthday party thrown in her honor by Planned Parenthood where jubilant abortion supporters danced the conga line in celebration of Sebelius policies that aided their cause, Operation Rescue reported.
We know the truth, even if Sebelius wont admit to it. Obamacare has carefully obscured numerous ways to fund abortion through premiums in some cases and through a surcharge in others. That surreptitious funding represents billions of dollars of taxpayer funded bailout money to an abortion industry that has been imploding in recent years. Just this year alone 46 abortion clinics have closed and abortion numbers continue to fall.
Lila Rose of the activist Live Action organization also spoke to WND on the issue.
Live Action is known for its undercover video stings of abortion facilities, which have been caught violating laws regarding covering up and facilitating the sexual exploitation of minors, providing medical misinformation, using manipulative coercion and facilitating race- and sex-based abortion.
Rose told WND that Obamacare was written for the benefit of the abortion industry.
The architects of Obamacare have a clear, extreme pro-abortion goal, including abortion coverage in many different ways. Some are public Others, I fear, we will discover as the massive government takeover of health care continues, she said.
Already required are payments for abortifacients, she said. Another funding mechanism for abortionists will be the relationships with the community health partners, a position for which Planned Parenthood already is lined up, she said.
Obamas plans represent the greatest expansion of the abortion industry since Roe v. Wade, she said. Theres a whole world out there of other avenues where people can people can receive money.
She commended Blattys comment.
If you believe that evil exists in the world what could be more evil than the dismemberment of an unborn child, a child when theyre the most vulnerable and defenseless, she told WND.
She said for American culture to be free from this evil, the nation needs to confront it.
Voices like Blattys are refreshing, calling out evil for what it is.
The Obama administration is threatening a veto of legislation that would permanently ban federal funds for abortion. In a statement on Monday, the administration said the measure would intrude on women’s reproductive rights and prohibit the ability of individuals to spend their own money on insurance coverage for abortion under President Barack Obama’s health care law
There are NO Pro-Life Democrats...NONE!
Sodomites are swarming across our country like fire ants.
Repent as commanded, America. Time is short.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.

domain: politics


With our quick chiler systems, we offer customers the opportunity for process optimisation in the cold stores. Our customised rapid-cooling solutions ensure that carcasses can pass through the cooling process faster and can be hung up in cold storage sooner. At DGS, we offer two different rapid cooling solutions:
These quick chiller systems freeze the product using sprays of water and cold air. An entirely natural process that is food safe and ensures that carcasses are quickly brought to a low temperature.
The DGS Quickchillers work on cooling elements. The difference is that these quick chillers do not use water sprays and air supply, which makes this solution ideal for smaller quick chilling rooms.
In addition to this acceleration, quick chilling offers even more advantages. Rapid cooling with the DGS solutions limits bacterial growth. Moreover, you lose less weight to dehydration, so you keep more meat after the cold store.
We are happy to discuss the possibilities with you.
Complete solutions for the cooling process
After the clean slaughter line, it is up to the abattoir to realise the highest possible yield per carcass. With the use of various innovative techniques, the maximum profit per carcass can be guaranteed. Stemming from our passion for process optimisation, we create and develop complete solutions for our customers in no time. Contact us to enquire about the possibilities.
- Customer-specific solutions
- Innovative blast chilling
- Various quick-chilling techniques
Service by DGS
Maintaining your machines and systems is crucial for a streamlined process. That is why we provide not only tailor-made systems, but also the proper service required. At DGS, we ensure that your systems are maintained properly and efficiently. Does a component need to be replaced or has an unexpected malfunction occurred? Then our specialists have the knowledge and skills to get your operations up and running again as soon as possible. In addition, our experts also offer remote assistance and will gladly draw up a maintenance plan together with you to avoid unforseen maintenance costs.
- Maintenance and repairs
- On-site and remote service


Preview Justin Bieber And David Guetta’s New Collaboration, “2U”June 8, 2017
Song of the Summer contender.
Listen to 13 magical seconds from David Guetta and Justin Bieber’s new tune.
“2U” doesn’t drop until Friday, but appropriately Victoria’s Secret model, Sara Sampaio, is sharing a teaser of it beforehand.
From what we can make of it, Bieber is rolling into a massive chorus before Guetta flings in a huge drop.
I know it’s only a brief glimpse at the track, but it sounds like the duo has a Song of the Summer contender on their hands!


Students at a private high school in Sydney’s west have won the backing of the NSW...
Stoush over a school uniform policy has been settled – for now.
A boy reported missing from Mountain Creek has been located
Half of Sunshine Coast public schools require girls wear skirts
School girls must be able to choose to wear pants or shorts
“Proper arch support is required.”
Distraught students from two high schools are too scared to go back
"I’m not going to buy another pair of shoes.”
Salesman admits he had sex with stepdaughter


text:
This rugged cotton canvas wallet from Browning combines Mossy Oak Break-Up Infinity camo with distressed brown top grain leather. The Browning Men's Canvas Camo Bi-Fold Wallet has 4 card pockets, 1 clear ID window, 2 side pockets, and a currency pocket for bills, giving you all the room you could possibly need in a wallet.
- Rugged cotton canvas fabric in Mossy Oak Break-Up Infinity camo
- Distressed brown top grain leather interior and trim
- Woven Buckmark logo tab
- Fully lined with 300 denier polyester fabric in Mossy Oak Break-Up Infinity camo
- 4 card pockets, 1 clear ID window, 2 side pockets, 1 currency pocket
- Closed size: 3 1/2 x 4 1/2 inches

domain: design


text:
If you aren’t familiar with the term Brony, it’s the word used to describe those that are super fans of My Little Pony. While it may seem like an odd obsession, it has become hugely popular over the last few years, thanks in a large part to 4Chan. To give you a better understanding of who Bronies are, here are 21 things you didn’t know about them. If you want to learn more, there is an annual My Little Pony Census filled with stats and info on the subculture.
1. The demographic of Bronies is predominantly, but not exclusively, male. Approximately 4 out of every 5 Bronies are guys.
2. While many think the main demographic is teenagers, the average age of a Brony is 21 years old.
3. Over half of all Bronies are from the United States, with no other country even making up 6% of the Brony population.
4. Interestingly enough, the percentage of Bronies from the US dropped 6% from 2013 to 2014, which means the phenomenon is spreading far beyond North America.
5. 1 out of every 4 Bronies report that they’ve been in a serious relationship in the last year.
6. The largest percentage of Bronies have never been married, while less than one percent of the group has formerly been married and now divorced. Married individuals only make up around 5% of the group.
7. The continent with the least amount of Bronies is Africa, which doesn’t have a single country with more than one Brony in it.
8. The United Kingdom and Russia make up the biggest population of Bronies in Europe with Germany also beginning to grow into a Brony hub.
9. South America is one of the fastest growing Brony areas, as Chile, Brazil, and Argentina have become incredibly active with fans over the past few years.
10. In the United States, Utah has the most Bronies per capita with 9 out of every 100,000 people being a Brony.
11. California has the largest US Brony population of any other state with 1238 on record.
12. The median household income of Bronies for 2014 is $61, 578.
13. Approximately 3 out of every 4 Bronies still live at home with their parents, which makes sense considering the average age is 21, an age when most people are in college or about to being college.
14. The largest race of Bronies is caucasian, but the fastest growing group is Hispanic, which grew from 5% to 13% from 2013 to 2014.
15. The vast majority of Bronies have siblings, with only 16% of them reporting as only children.
16. According to their 2014 Census, there are even two Bronies in Antarctica.
17. The most isolated Brony in the world is the lone Brony in Fiji, nearly 2,880 miles from fellow Bronies in Australia.
18. Only about 1/3 of Bronies have divorced parents, with an even smaller percentage having a parent that has passed away.
19. Nearly one in ten Bronies are active members of the military or were part of the military service in the past.
20. 65% of Bronies report that they are heterosexual with no interest in the same gender with less than 2% saying they were exclusively homsexual.
21. The most popular way to watch My Little Pony? You guessed it, good old YouTube.


text:
US President Joseph Biden has vehemently condemned the “hateful” assassination of his Haitian counterpart, Jovenel Moise, expressing concern over the country’s political crisis and demanding further information about the attack.
“We condemn this heinous attack and send sincere wishes of recovery to the First Lady of Haiti. The United States offers its condolences to the people of Haiti and is ready to provide assistance as we continue our efforts to strengthen security in Haiti. “, said Joseph Biden, according to Reuters.
“We need more information, what has happened is very worrying for the situation in Haitii “, underlined the president of the USA.
Haiti’s ambassador to the United States, Bocchit Edmond, has called for security assistance from Washington, including for the training of Haitian police officers.
U.S. Republican Sen. Marco Rubio has called on the Joseph Biden administration to bring the assassins of the Haitian president to justice. “We cannot allow this cowardly and evil attack to create additional difficulties for Haitian citizens and create more instability,” said Marco Rubio.
Four Democratic members of the House of Representatives – Val Demings, Yvette Clarke, Andy Levin and Ayanna Pressley – have called for an independent investigation into the assassination of the Haitian president.
For the most important news of the day, broadcast in real time and presented equidistantly, LIKE our Facebook page!
Follows Mediafax on Instagram to see spectacular images and stories from around the world!
The content of the website www.mediafax.ro is intended exclusively for your personal information and use. It is prohibited republishing the content of this site in the absence of an agreement from MEDIAFAX. To obtain this agreement, please contact us at [email protected]
Source: Mediafax by www.mediafax.ro.
*The article has been translated based on the content of Mediafax by www.mediafax.ro. If there is any problem regarding the content, copyright, please leave a report below the article. We will try to process as quickly as possible to protect the rights of the author. Thank you very much!
*We just want readers to access information more quickly and easily with other multilingual content, instead of information only available in a certain language.
*We always respect the copyright of the content of the author and always include the original link of the source article.If the author disagrees, just leave the report below the article, the article will be edited or deleted at the request of the author. Thanks very much! Best regards!

domain: news


text:
Unless you are very, very lucky, you likely have contended with a body issue at some point in time. Whether no amount of running could slim your thighs or you have tried diets and remained at an unhealthy weight, sometimes winning the battle doesn”t always come in the form of diet and exercise. This is true for weight-loss surgery approaches, ranging from liposuction to a tummy tuck to life-saving bariatric surgeries.
If you’ve tried diet and exercise regimens, but can’t seem to lift or lose an area of your abdomen, thighs, buttocks, upper arms or even your neck, liposuction can be an option. This outpatient procedure involves removing bulges or pockets of fat utilizing small incisions and inserting special tools.
Liposuction isn’t for everyone — you must be in good health and have enough skin elasticity so your skin can snap back when the fat portion is removed. If you follow a diet and exercise routine, but just need a little help when it comes to trouble spots, liposuction may be for you.
You don’t have problem areas — you have a problem area: Your stomach. If it never snapped back after giving birth or you’ve experienced sagging skin after a recent weight loss, a tummy tuck can help. Also known as an abdominoplasty, a tummy tuck helps to remove sagging skin or tighten areas where your abdominal muscles may have become weakened or separated, particularly following childbirth.
Much like liposuction, a tummy tuck requires you to be in generally good health and at a stable weight. If your weight goes up and down as much as the stock market, the procedure may not be for you because it won’t offer lasting results.
Bariatric Surgery: Hope for a New Life
An estimated 34 percent of Americans are classified as obese, according to the National Center for Health Statistics1. This number equals more than 72 million Americans who are plagued by obesity and the chronic health conditions, such as diabetes, hypertension and heart disease that can often accompany obesity.
If you have tried every diet and failed, are morbidly obese and concerned for your health and appearance, bariatric surgery can offer you hope — if you are ready to change your lifestyle and your body. Bariatric or weight-loss surgery encompasses a variety of approaches that can help you lose dramatic amounts of weight. This includes the Roux-en-Y or gastric bypass surgery that involves altering the size and shape of your stomach to create a smaller stomach pouch. Because you become full with less food, you are able to lose significant amounts of weight in a short time.
Another option is gastric banding or LAP-BAND surgery that involves putting a band around your stomach, which creates a smaller, but less-permanent stomach alteration than a Roux-en-Y procedure does. Each surgical type has its pros and cons, and there are variations on each procedure that may be a good fit for your unique health conditions.
Whatever your weight-loss procedure choice, learning more about these options could not only change your life, it also could save it.

domain: medical