text:
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
23 Related JoVE Articles!
Monitoring Actin Disassembly with Time-lapse Microscopy
Institutions: Harvard Medical School.
Cellular Biology, Issue 1, cytoskeleton, actin, timelapse, filament, chamber
Aip1p Dynamics Are Altered by the R256H Mutation in Actin
Institutions: University of Iowa, University of Iowa.
Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro
studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo
using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 ± 0.30 μm/sec in R256H cells compared to 1.60 ± 0.42 μm/sec in wild type cells, p < 0.005).
Developmental Biology, Issue 89, green fluorescent protein, actin, Aip1p, total internal fluorescence microscopy, yeast, cloning
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
Institutions: Imperial College London, Institut Pasteur, Unité Macrophages et Développement de l'Immunité.
is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri
by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro
using tissue culture cells and in vivo
using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.
Infection, Issue 91, ATG8/LC3, autophagy, cytoskeleton, HeLa cells, p62, septin, Shigella, zebrafish
Assessing the Secretory Capacity of Pancreatic Acinar Cells
Institutions: Weizmann Institute of Science.
Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo
culture will represent the in vivo
nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.
Cellular Biology, Issue 90, Cell biology, Exocrine secretion, Pancreatic acinar cells, Primary culture, Exocytosis, Actin, Lifeact
Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
Institutions: University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, EZ BioResearch LLC.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g.,
in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Neuroscience, Issue 95, AP2, genotyping, glial feeder layer, mouse tail, neuronal culture, nucleic-acid extraction, PCR, tattoo, torsinA
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays
Institutions: The University of Melbourne, The University of Melbourne.
Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro
myelination assay, an established model for studying CNS myelination in vitro
. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro
myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.
Developmental Biology, Issue 95, lentivirus, cocultures, oligodendrocyte, myelination, oligodendrocyte precursor cells, dorsal root ganglion neurons
Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis
Institutions: RWTH Aachen University, RWTH Aachen University, RWTH Aachen University.
In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g.
, partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.
Medicine, Issue 96, bile duct ligation, cholestasis, bile obstruction, hepatic fibrosis, inflammation, extracellular matrix, jaundice, mouse
Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets
Institutions: The University of Queensland.
Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1
. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2
. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb
(db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.
Medicine, Issue 99, Pancreatic islets, Leprdb, db/db, isolation, liberase, collagenase, 2-photon imaging, exocytosis, insulin, beta cell, diabetes
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level
Institutions: University of Michigan-Dearborn, University of Illinois at Chicago, University of Illinois at Chicago.
Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP
channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo
Bioengineering, Issue 81, Islets of Langerhans, Microfluidics, Microfluidic Analytical Techniques, Microfluidic Analytical Techniques, oxygen, islet, hypoxia, intermittent hypoxia
A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Waterloo.
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H
]-thymidine incorporation, protein abundance, and mRNA expression.
Physiology, Issue 88, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation
Institutions: F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD.
A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1
. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro
and in vivo 2
. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.
Cellular Biology, Issue 32, Mammospheres, HC11, lactogenic differentiation, mammary
A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device
Institutions: University of Illinois, Chicago, University of Illinois, Chicago.
A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 μm diameter and 150 μm depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 μm in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.
Cellular Biology, Issue 35, Microfluidics, Islet perifusion, glucose ramp, imaging, perifusion, beta cells, insulin secretion
Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells
Institutions: Bio-Rad Laboratories.
The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression. siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks. These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology. Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages. To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis. The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa). The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells. We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.
Cellular Biology, Issue 38, Cell Counting, Gene Silencing, siRNA, Namalwa Cells, IL4, Gene Expression, Electroporation, Real Time PCR
Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry
Institutions: Baylor College of Medicine.
Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1
. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.
Cellular Biology, Issue 45, Glucose, FACS, Plasma Membrane, Insulin Receptor, myoblast, myocyte, adipocyte
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Tracking Hypoxic Signaling within Encapsulated Cell Aggregates
Institutions: University of South Carolina, University of South Carolina.
In Diabetes mellitus type 1, autoimmune destruction of the pancreatic β-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2
. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3
, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5
which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8
. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7
. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9
, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10
. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen11-13
. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14
. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15
. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15
. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15
. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1α for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.
Bioengineering, Issue 58, Cell encapsulation, PEG, cell aggregation, hypoxia, insulin secretion, fluorescent imaging
Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach
Institutions: University of Pittsburgh, University of Pittsburgh.
Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro
in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1
. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro
setting. Pancreatic development is known to occur in specific stages 2
, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3
in combination with several growth factors 4-7
. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro
by addition of cyclopamine 8
. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro
maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9
. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.
Stem Cell Biology, Issue 61, Human embryonic stem cells, Endothelial cells, Pancreatic differentiation, Co-culture
Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Institutions: Indiana University School of Medicine, Indiana University School of Medicine.
Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3
. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6
. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15
. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1
). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17
Medicine, Issue 64, Physiology, beta cell, gene expression, islet, diabetes, insulin secretion, proliferation, adenovirus, rat
Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer
Institutions: Baylor College of Medicine , Baylor College of Medicine , Baylor College of Medicine .
Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo
use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1
. They lack chronic toxicity because they lack viral coding sequences2-5
and retain only Ad cis
elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.
Medicine, Issue 68, Genetics, Physiology, Gene therapy, Neurogenin3, Betacellulin, helper-dependent adenoviral vectors, Type 1 diabetes, islet neogenesis
Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots
Institutions: The Ohio State University, The First Affiliated Hospital of Soochow University, The Ohio State University.
Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.
Medicine, Issue 100, encapsulation, microcapsules, thermogenesis, obesity, adipocytes

domain: medical


Coemar ParLite Led ACLJune 16, 2020
Coemar Panorama LedJune 16, 2020
Coemar ParLite FC
ParLite FC is the evolution of the classic ParLite Led with a RGBW Full Color chip LED plus White 3.000 K for a perfect color mixing and a pre-set CCT levels from 2.700 to 10.000 K with color rendering index greater than 90 (CRI > 90).
Ideal for the broadcast market due to the removal of the fan and flicker-free DMX dimming from 600 to 19.000 Hz.
Available in an IP 65 rated version with PowerCon In/Out for easy connection as well.


BET Cast Gabrielle Dennis as Whitney Houston in Bobby Brown Miniseries – Details Here!
Whitney Houston was an integral part of Bobby Brown’s life, so the person who plays her in telling the Bobby Brown story is actually a second lead to Bobby’s character. That second lead in this situation is just as important as the first lead in order to convey the story as the stars lived it.
BET after looking at several potentials has selected Gabrielle Dennis to play the legend Whitney Houston in their upcoming two-part miniseries that’s scheduled to premiere September 2018. The series will be based on the couple’s turbulent relationship which produced successes, struggles and failures during their marriage from 1992 to 2007.
The Bobby Brown Story which is currently filming in Atlanta, is executive produced by Jesse Collins (The New Edition Story) for JCE Films, a division of Jesse Collins Entertainment; written by Abdul Williams (Lottery Ticket, The New Edition Story) and directed by Spike Lee protégé Kiel Adrian Scott.
You may be interested
Reem’o C.U.T.S. by Reem’o Meerak (@reemoapproved)Relisha - December 24, 2019
Nonetheless you don't want to miss out the latest from the #RugerSprayyyyyer Click HERE, and let's see what's UP!!!!!
“Queen and Slim’s” director Melina Matsoukas discusses the Soundtrack at the Spotify x Motown movie Premiere in AtlantaEditorial Dept - November 24, 2019
Emmy winning writer Lena Waithe and director Melina Matsoukas premiered the much anticipated "Queen and Slim" romantic thriller in Atlanta at the…
Ahmir “Questlove” Thompson & Tariq “Black Thought” Trotter Present: ‘Hip Hop: The Songs That Shook America’Editorial Dept - October 14, 2019
From Executive Producers Ahmir “Questlove” Thompson, Tariq “Black Thought” Trotter, Shawn Gee, Alex Gibney and directors Erik Parker and One9, HipHop: The Songs That Shook America, premiered last night, October…
You must log in to post a comment.


text:
Ramsey County Judge Kathleen Gearin reacted coolly this morning to a move from the two Republican legislative transportation chairs and the state’s contractors association to let as much as $100 million flow to road projects around the state despite the government shutdown.
“It really sounds like you want me to become an activist judge,” Gearin told the Republicans, Sen. Joe Gimse and Rep. Mike Beard, as they made their case in St. Paul Wednesday morning. “It sounds to me like you’re asking [Special Master Kathleen Blatz] and I to be super-activist judges.”
Gimse and Beard appeared before Gearin in an attempt to secure funding for 98 road construction projects around the state that they said were already underway. The commonly cited figure for the cost of such order is around $100 million, although Beard said it’s possible the number could end up being closer to the $30 million that was approved for projects in 2005.
On behalf of the attorney general’s office, Solicitor General Alan Gilbert said they would defer to Gearin’s original ruling and oppose the petition. The Dayton administration filed a brief (available here) opposing it explicitly.
In her original ruling June 29, Gearin explicitly said road construction that is not a matter of public safety or necessary maintenance — such as repairing a bridge that might be in danger of collapse — do not meet the standard of a “critical, core” function of government, and therefore must be stopped as long as the shutdown, and impasse at the Capitol, continues.
Beard, for his part, argued that the constitution requires the state to maintain a “trunk highway system,” and that leaving the projects in limbo was in fact potentially damaging to public safety. Allowing the projects to go forward, he continued, would also be in line with Gearin’s order that approves state spending on certain programs to preserve state property.
Despite Beard’s arguments, though, Gearin seemed undeterred. “I’m just having trouble changing my order,” she said flatly. “At what point do we need to have potholes filled on the highway? I mean, I don’t know.”
In looking to limit the petition even further — likely in the hopes of gaining Gearin’s favor in light of the reluctant stance she’s taken throughout the shutdown’s court proceedings — Beard suggested that perhaps only certain partially completed projects that met some more strict public safety standard could be allowed to continue. The executive branch and MnDOT, he suggested, could compile a list that either Gearin or Blatz could consider for approval.
But Gearin wasn’t interested in such a compromise. “[Blatz] didn’t sign up to be commissioner of transportation,” she replied. “I can assure you that’s not going to happen.”
Gearin took the issue under advisement, but warned that it may take some time to issue a ruling given the weight of the issues before her and the time it takes to consider each matter fully.

domain: politics


A Maryland State Police agent may have used an undercover alias to infiltrate a leftist Mount Vernon cooperative in January 2005 - two months before police say their secret monitoring of death-penalty activists began - according to documents released yesterday by the American Civil Liberties Union of Maryland.
Red Emma's, a cooperatively run bookstore and coffee shop, is one of 32 organizations that filed records requests yesterday with state and local law enforcement agencies, wanting to know if they have been under surveillance. The requests were coordinated by the ACLU of Maryland as part of its broadening investigation into spying activity by state police.
Police officials have acknowledged surveillance of death penalty activists in 2005 and 2006, saying the efforts were legal. They said they have cooperated with a separate review panel commissioned by Gov. Martin O'Malley after revelations of the spying this summer sparked concern in Annapolis and Washington. A report on the official probe, led by former Attorney General Stephen H. Sachs, is scheduled to be released today.
But David Rocah, a staff attorney with the ACLU of Maryland, says new records indicating police may have spied on Red Emma's in January 2005 belie statements made by Col. Terrence B. Sheridan, state police superintendent, that his agency's surveillance activity began in March of that year and ended 14 months later.
Police spokesman Gregory Shipley said he was unaware of any police interest in Red Emma's, a bohemian redoubt offering radical literature and vegan food. Shipley said that the agency would comply with the ACLU's public records requests to the extent required by the law.
Rocah said the suspected Red Emma's infiltration was discovered because state officials failed to redact, in documents made public this year, the alias used by a police agent while she spied in 2005 on the Committee to Save Vernon Evans, a death row inmate.
"They were just incompetent," Rocah said. "They meant to redact it, and they goofed."
In the police document, an agent using the name "Lucy Shoup" is described as having covertly attended a meeting at the American Friends Service Hall in North Baltimore. A member of the Red Emma's cooperative who scoured the released police documents checked that name against an e-mail list maintained by the cooperative and surmised that "Lucy Shoup" could be the same person as an "Ann Shoup" on the list, said Mike McGuire, another Red Emma's member.
What Red Emma's found: On Jan. 4, 2005, "Ann Shoup" sent a message to the cooperative expressing interest in an upcoming lecture by Bernardine Dohrn, a juvenile justice activist, ex-fugitive and former member of the Weather Underground, the violent, late 1960s radical group.
"Hi! I would love to come to the event on Feb 6th," wrote "Ann."
In a July 2008 article in The Progressive magazine, Baltimore activist Max Obuszewski - one of the anti-death-penalty spying targets - indicates that "Lucy Shoup" also sent him a message notifying him that her new e-mail address was firstname.lastname@example.org.
McGuire said after discovering the connection, Red Emma's contacted the ACLU of Maryland and asked for their help. "We're appalled by this," he said. "We want to find out the extent of their investigation. ... This is a clear indication that police were being used politically."
The artfully disheveled store, in an English basement on St. Paul Street, features left-leaning zines, free Internet and "Zapatista" coffee. The book section does not have a copy of the infamous Anarchist Cookbook, which has instructions for making explosives, though Red Emma's customers can purchase an actual cookbook by the same name. Independent presidential candidate Ralph Nader is scheduled to speak this evening. With the exception of Red Emma's, the other entities that filed records requests yesterday are traditional activist groups, ranging from anti-abortion to anti-war protesters. Among them are the Humane League of Baltimore, the Maryland Coalition Against State Executions, Christian Peace Witness for Iraq and the Algebra Project.
Rocah said the ACLU was coordinating the mass public records request in order to understand the extent of the police's spying activity. "It will be interesting to see whether ... these groups were spied on as well, or whether they weren't spied on, in which case the [Maryland State Police] has some explaining to do about how they choose which groups to spy on."
Sheridan, the police superintendent, has said that he was "troubled" by the previously acknowledged surveillance but that police monitored death penalty activists out of concern that protests around two planned executions in 2005 might get violent.
According to records obtained by the ACLU, police agents secretly joined the Baltimore Pledge of Resistance, a peace group; the Baltimore Coalition Against the Death Penalty; and the Committee to Save Vernon Evans.
Rocah said it was necessary to file individual records-requests for each of the additional 32 groups because when "you're seeking a document that is about you ... you have a greater right of access than general public does." Under Maryland's public information law, the state police have 30 days to respond to the requests.
Though ACLU officials said they had no information that police spying continued after 2006, several activists who are now ACLU clients said revelations of police surveillance on political activists could have a chilling effect on free speech in Maryland.
"If people are spied on, they'll become intimidated," said Jack Ames of Defend Life, a Baltimore-based anti-abortion group.
"We have a constitutional right to oppose government policy," said Jean Athey of Peace Action Montgomery, an anti-war group. "Illegal government spying is a very serious threat to democracy."
Also yesterday, ACLU of Maryland lobbyist Cindy Boersma said she has been talking to lawmakers about proposed legislation that would "prohibit spying and the compilation of ... intelligence dossiers on Marylanders."
The ACLU bills, drafts of which are currently being circulated among members of the General Assembly, would prohibit law enforcement officials from investigating "lawful First Amendment activities" or keeping records of people's political and social beliefs, Boersma said.
She said the ACLU was waiting to see Sachs' report before approaching Gov. Martin O'Malley for his support. O'Malley administration officials had considered postponing today's release of the report because of the recent Medevac crash that killed two state troopers, but Sheridan preferred to dispense with the matter as soon as possible, Sachs said.


text:
ABC of Medical Law is a Wiley-Blackwell source of information is developed by MedHand Mobile Libraries. Improve your performance with relevant, valid material which is accessed quickly and with minimal effort in the palm of your hand using MedHand’s patented technology.
An understanding of medical law is increasingly important for all health professionals and this new ABC provides a basic introduction to the legal issues faced in health care that is accessible to anyone without any legal knowledge. The ABC of Medical Law provides ideal guidance to the practicing physician - covering just what you need to know without becoming embroiled in complicated legal discussion.
The ABC of Medical Law has up-to-date coverage of the legal issues to be found in daily medical practice, including confidentiality, research, consent, negligence, organ donation and human rights, as well as more contentious issues such as tissue retention and withholding/withdrawing treatment. Well illustrated and presented in a user-friendly format, chapters include summaries and cases to help clarify the points made.
Written by practising clinicians with expertise in medical law and a medical barrister, the ABC of Medical Law will help keep a practice within the constraints of the law and is ideal for GPs, junior doctors and medical students, and anyone wanting to understand the broad basics of medical law.
Authors: Lorraine Corfield, Ingrid Granne, William Latimer-Sayer
MedHand Mobile Libraries offers a SUBSCRIPTION FREE application without edition upgrade. MedHand is the exclusive partner of Oxford University Press, publishing the latest editions of their Medical Handbooks series.
MedHand applications improve your performance with relevant, valid material that is accessed quickly at the point of care!
Offering the most trusted and well recognized medical guidelines provided by excellent publishers such as McGraw-Hill, RPS, PDR Network LLC, Wiley-Blackwell and more.


text:
My son is moving to LR. He lives in Madison, AL now which is just outside of Huntsville, AL. He considered living in West LR but now he's thinking about living outside of LR and commuting to work. Can you offer any suggestions for living in Benton or Bryant. What are the best areas to look for a home in the $250 thousand range with good elementary schools. He has 3 young children age 2 to 6. Thanks for your very kind help.
Moving To Little Rock........Where To Live...........
Recent ActivityView all United States activity »
- 1 Trip Report to Pacific Northwest: August 2014
- 2 Sacramento to State College, PA in late November
- 3 Angels Crest and Rim of the World Scenic Drives: Mid-November 2014
- 4 Three nights in Chicago -- a report
- 5 New York Movie Tours & TV Show Tours on your Mobile
- 6 Hotel in Newton outside Boston - need transportation and related advice
- 7 I am turning 60 and boy do I need help with my trip!
- 8 Relocation: Where Do We Fit In
- 9 What do you do when your VRBO rental is a nightmare in Lake Geneva?
- 10 Thinking about moving from PA to Florida (Dr. Phillips area of Orlando)
- 11 Saturday arrival Seattle airport to Lake Crescent
- 12 USA and Canada in 2 months
- 13 Grand Canyon , Zion and Bryce Itinerary suggestions
- 14 Drive from Grand Canyon to Prescott next week
- 15 Houston Weekend Planning Help Needed, please
- 16 Special Occasion/Fancy Dress shops in South Florida
- 17 Looking for a travel loop in Arizona (Las Vegas to Tucson & back)
- 18 Is the New York subway safe?
- 19 Skiing New Mexico
- 20 Hotel near the Miami airport
- 21 Girls Weekend Trip to Sarasota/Tampa Florida Area
- 22 Where to from Tampa?
- 23 kuai and maui top 10 to see list
- 24 DC in Dec-Restaurant ideas near Hilton Mark Alexandria
- 25 Richmond to Oklahoma City Slowly: what not to miss?

domain: geography


text:
Essex Leopards look to make changes
PUBLISHED: 11:30 07 November 2016
Coach Ogunjimi considering options
Levett Essex Leopards look set to ring the changes before Friday’s clash with Reading Rockets at the Brentwood Centre (8pm).
After their National Cup exit the previous weekend, the Big Cats produced a better performance on Saturday despite going down 83-70 at league leaders Derby Trailblazers.
But their fans can expect to see new players in their line-up on Friday and coach Steve Ogunjimi said: “The season is far from a write-off but clearly things have to change.
“When we put this team together in the summer we had high hopes that we could win things, but for whatever reason it hasn’t worked.
“It’s disappointing and it’s hard to say goodbye to people but we still have a lot to play for and if we’re going to make changes, it makes sense that we do it now.
“I know the team went 1-4 in the season they won the treble, so clearly it can be done. We just need to move sooner rather than later if we’re going to have a successful season.”
A good start at Derby on Saturday proved insufficient as the Big Cats slipped to their fourth defeat of the Division One season.
Ryan Lascelles gave the home side the lead, before scores from Mike Martin and Dave Ajumobi put Leopards 4-2 ahead, and Ladi Brown sunk a trey off AJ Roberts’ assist to put the visitors 9-2 up.
A triple from Dougie Bennett, followed by a Martin lay-up, saw Leopards’ lead peak at 16-5, but only Martin scored for the visitors in the second five minutes of the period as they went into the first break tied at 21-21.
Jamie Hayes had arguably his best game for the Big Cats and opened the second period with a trey and kept his side within a point at 33-32 with four minutes left, before Derby closed out the half strongly to go into the locker room leading 38-33.
The third period proved to be the killer for the visitors as Derby opened with a 9-0 run, and although Martin stopped the rot they trailed by as many as 19 before another Hayes three-pointer cut the deficit to 64-46 at the final break.
Martin opened the fourth period scoring but, although the veteran big man got the deficit down to eight with 5:34 on the clock, the home side held on to maintain their perfect start to the season.
Martin top scored for the visitors, adding eight rebounds to his 18 points, with Brown finishing on 11 points and both Bennett and Hayes adding 10.
The Big Cats will face a Reading side who are also looking to bounce back after a weekend in the north that saw them lose against Team Northumbria and Bradford Dragons.
Despite another summer of heavy recruitment, including the signing of two Americans and several Spanish players, the Rockets are currently 1-3 in league play, while a loss against Worthing in the National Trophy saw them finish second in the group stage.
They have reached the National Cup quarter-final with wins as Cardiff and Hemel, and travel to Kent Crusaders in the last eight.
The Rockets are the Big Cats oldest rivals with the sides having met nearly 50 times in 13 seasons.
There have been plenty of thrillers between the teams, with Leopards’ National Cup final success in December 2005 probably the most notable, and last season was no exception.
A buzzer-beating three from Dave Ajumobi took Rockets into overtime in October’s National Trophy meeting at the Brentwood Centre, and his side went on to win in the extra period.
But both league games went Leopards’ way as they finished strongly to win by four points in the final minute of the game.
The play-off encounter at Rivermead looked set to go the same way, before a dominant fourth period saw the hosts run out 20-point winners.

domain: sports


The Rutgers Scarlet Knights‘ uniform combinations this season have all been pretty slick and their helmet contribution for “Heroes Day” (Nov. 10) is no exception, but they won’t be the only school donning red, white and blue on the side of their lids. The New Mexico Lobos unveiled their own helmets paying tribute to the members of the United States’ armed forces, both past and present.
Despite suffering a loss at the hands of the Kent State Golden Flashes, Rutgers still has a chance for an impressive season. They battle the Army Black Knights this weekend before finishing the regular season with road games against the Cincinnati Bearcats and Pittsburgh Panthers with a clash against the No. 9 Louisville Cardinals to cap off the slate.
Louisville and Rutgers are currently undefeated in Big East play. If both teams remain undefeated, that contest will determine the Big East’s BCS representative.
New Mexico hasn’t had such a banner year. The Lobos boast the No. 5 rushing attack in the country, piling up over 302 yards per game, but have won only four games. Wins against the Wyoming Cowboys, Nevada Wolfpack and Colorado State Rams are musts if New Mexico will even be considered, let alone eligible, for a bowl bid.
As far as “patriotic” helmets go, Rutgers and New Mexico got it right. You can actually tell there’s an individual school paying tribute and the flag’s colors against a solid shell are always the way to go. When it has a certain USFL quality to it, that’s never a bad thing, either. These two get the Seal of Approval®.
Brandon Cavanaugh is a college football columnist for Rant Sports and member of the Football Writers Association of America. Feel free to follow him on Twitter and join in on the conversation.


text:
Hey guys, have you ever heard of the appendix? Many people don’t know much about it, but it actually serves an important function in the body. The appendix is a small pouch attached to the large intestine and its main function is to store good bacteria for the gut. However, sometimes it can get inflamed and cause a condition called appendicitis, which requires surgery to remove the inflamed organ.
What about the Large Intestine?
Speaking of the large intestine, did you know that it is also known as the colon or the bowel? It is a crucial part of the digestive system and plays a major role in absorbing water and electrolytes from the remaining food matter after nutrients have been absorbed in the small intestine. The large intestine also helps to eliminate waste products from the body in the form of feces.
Now, if you experience any abdominal pain, fever or vomiting, do not ignore them. It could be a sign of a serious condition called diverticulitis, which occurs when small pouches in the lining of the intestine become inflamed. This condition can be prevented by maintaining a high fibre diet and staying hydrated regularly.
The Importance of a Healthy Gut
Your gut health is closely related to your overall health and wellbeing. If you want to maintain a healthy gut, it is important to eat a balanced diet rich in nutrients, probiotics, and fiber. It is also essential to stay hydrated, exercise regularly, and get enough sleep. Probiotics, in particular, help promote the growth of good bacteria in the gut and can be found in fermented foods like yogurt and kimchi.
So, guys, let’s take care of our gut health and avoid any digestive problems. Remember, prevention is always better than cure.


text: Social Security Taxes Estimate how much of your Social Security benefit may be considered taxable. You are viewing this website at a small screen resolution which doesn't support calculators. Click here to view this calculator. Have A Question About This Topic? Name Email Address Question Thank you! Oops! Related Content What Is a 1031 Exchange? A look at 1031 Exchanges, a real estate investment strategy that may allow you to defer your capital gains taxes. Put It in a Letter A letter of instruction provides additional and more personal information regarding your estate. The Cost of Procrastination Procrastination can be costly. When you get a late start, it may be difficult to make up for lost time.

domain: economics


text:
Trade union members at Perth College UHI and 19 other colleges across Scotland look set to walk out in protest after members voted overwhelmingly to strike in support of fair pay.
Union Unison said the dispute is the result of college bosses awarding lecturing staff a £450 flat rate rise, while some college support staff were only offered a flat rate of £230.
College support staff such as those working in administration, admissions, funding, catering, cleaning and security, as well as advisors, classroom assistants and technicians, are calling for fair pay and parity with lecturing colleagues.
Unison will take part in national negotiations later this week. But if bosses fail to deliver a fair deal, then strikes look unavoidable.
Chris Greenshields, chair of Unison’s further education committee, said: “Our demand is simple and fair. Pay college support staff the same amount that you gave to our teaching colleagues. We work for the same colleges, help deliver the same courses, support the same students and deserve the same cost of living increase.”
Shirley Sephton, vice-chair of Unison’s further education committee, said: “The colleges should use a small portion of the extra £8.1 million given to the sector this year to address operational pressures to resolve this dispute before things get worse.
“Recent surveys of our membership have shown real low morale in support staff. We have been through a difficult merger process and we can’t allow a two-tier system to develop.
“Unless we get a fair and equitable pay award it is a further kick in the teeth to overworked and underpaid college support staff.
“High student dropout rates recently reported will not be helped by alienating key support staff.”
John Gallacher, Unison’s Scottish organiser, said: “Striking is a last resort, but we will support our members in every way possible to achieve the same fair and reasonable pay settlement as already paid out to teaching colleagues.”

domain: finance


text:
Red X Podcast (hosted by Mayor Lance Olive and Nicole Bernard) exists to raise awareness of human trafficking in North America and empower everyday people to work with their local municipalities to create a safe place for citizens, a hostile environment for criminals, and a second chance for survivors.
I Am Jane Doe is a documentary highlighting the story of four young trafficking victims and their legal battle against backpage.com. You can find it on free on Netflix.
Presentation to HEAL from the Greater New Orleans Task Force (Leanne McCallum). Rethinking Representation: Framing Human Trafficking for Health Professionals. Also dilenates prior legislation.
Unbridled is a family film about a young girl's recovery from the trauma of being sex trafficked by her step-father and the use of equine therapy in her restoration.
The True Cost is a documentary (free on Netflix) that sheds light on the issue of labor trafficking that supports the multi-billion dollar fashion industry.
Renting Lacy is an excellent introduction to how children become sex trafficking victims and the realities of their exploitation.
The Locust Effect by International Justice Mission's Gary Haugen explains the plight of global fight against slavery.
Written by Shield's Nicole Bernard, A War Against the Cowbird helps explain the process of trafficking and is perfect for sci-fi and young adult dystopia lovers.
The Body Keeps the Score explains the complexities of healing in complex trauma.
Girls Like Us is a memoir of a woman who was sex trafficked as a child, found restoration and then founded GEMS a nationally known restoration program in NY.
The Slave Across the Street is a true account of a young girl who was a part of the sex trade as a teenager.
Suite 802, #107 10030 Green Level Church Road Cary NC 27519
Copyright © 2021 Shield North Carolina - All Rights Reserved.
We are asking 200 people to commit to donating $20 a month to help us fight for those who are vulnerable to trafficking and those who have been exploited.
Your contribution makes all the difference!

domain: sociology


BEACH RIOT | Leah Sports Bra - USA
The Leah Top is Beach Riot's classic sports bra with scoop neckline both in the front and back. This top is featured in navy with stars on the top and red and white stripped support band under the bust. Pair this bra with the matching bike short in stars and stripes to complete the look.
- Poly/Spandex blend
- Hand wash cold; lay flat to dry
- Made in the USA


text: AYOOO! I really hope you guys get that I'm a K-Pop fan by now. So, odotter is my saving grace when it comes to covering K-Pop songs. THESE USTS ARE SO GREAT! So, this is my favourite BTS song. I actually made Noke sing my bias's part because Suga is life. Credits UST: odotter Original: BTS Parts and Voicebanks used Jimin: Mario Fuwa EXTRA by @nanaro Jungkook: Noto Hana by me Suga: Kiseki Noke V3 by me Rapmon: Nintone Reicheru by @Rei-Chan J-Hope: Zuki Fuji by @kamonohashi V: Minami Mi by @LilyoftheValley Jin: Q.P. by @Animé Fan You can download my cover from the Soundcloud link.

domain: artistic


text:
Best bets for the horse racing at Kempton Saturday 7th April 2018
I am having a go in two of the ITV televised races from Kempton Park on Saturday and have jumped on nice and early as I like the look of the BPG odds.
They go over six furlongs for the Betfred 'supports Jack Berry House' Handicap at 2.20. Some decent types line-up but most require a career best to win on current ratings. Tropics hasn’t won of this sort of mark since 2015, whilst Boundsy, Mazzini and Raven's Lady are all on higher marks than they have ever won off. One that doesn’t have to improve in theory is Swift Approval the chestnut gelding back down to a rating of 92. Ideally drawn in one I would expect the six-year-old to ping out and make this a real test; stays seven furlongs. The eye shields are on to help with concentration and I have taken a chance at the 10-1.
Smart Call is the star attraction in the Listed Betfred "Home Of Goals Galore" Snowdrop Fillies' Stakes at 3.15. Now trained by Sir Michael Stoute she was, of course, a multiple Group One winner in her native South Africa and though she perhaps didn’t reach the heights hoped for in 2017. She has not been seen in competitive action since finishing seventh behind Highland Reel in the G.1 Hong Kong Vase in December but the six-year-old acquitted herself well enough in races such as G.2 Middleton Stakes at York and G.2 Duke of Cambridge at Royal Ascot to suggest talent remains, was asked quite a bit in G.1’s later in the season, and she should prove too classy for this line-up. Bigger targets will be aimed at later in the summer and I have the 5-2 about her for this. Shenanigans is feared most.

domain: hobby


text:
Rwanda Wildlife Safaris Tour – Places for Rwanda Safaris Tour
Rwanda is a beautiful country located in East Africa and is known for its diverse wildlife and stunning natural landscapes. One of the popular activities for visitors to Rwanda is embarking on wildlife safaris to explore its national parks and see the incredible wildlife that inhabits them, including the critically endangered mountain gorillas.
Rwanda Wildlife Safaris Tour Combined with Gorilla Trekking Holidays
Places for Rwanda Wildlife Safaris Tour – Iconic Rwanda National Parks
Located in eastern Rwanda, Akagera National Park offers a more traditional African wildlife safaris tour experience. The park is home to a variety of wildlife, including elephants, lions, buffalos, giraffes, zebras, and numerous bird species. Game drives and boat safaris along Lake Ihema are popular activities here.
While not primarily a wildlife park, Nyungwe Forest is a haven for primate lovers. The park is home to thirteen different primate species, including chimpanzees, colobus monkeys, and the endangered golden monkeys. Guided walks and treks are available to explore the lush rainforest.
When to Go for Rwanda Wildlife Safaris Tour?
The best time to go for a wildlife safaris tour in Rwanda depends on various factors such as weather, wildlife activity, and personal preferences. Rwanda has a mild climate throughout the year due to its elevation, but there are some considerations to keep in mind.
Ultimately, the choice of when to go depends on your preferences and priorities. If you prioritize dry and easier-to-navigate conditions, the dry season is recommended. However, if you prefer lush landscapes, lower tourist numbers, and don’t mind the occasional rain, the wet season can offer its own unique charm.
It’s advisable to check with Tubale Safaris Ltd for up-to-date information on weather conditions and wildlife sightings to help you plan your ideal time to visit Rwanda for a wildlife safaris tour.
Dry Season (June to September, December to February)
The dry season is generally considered the best time for wildlife safaris in Rwanda. During this period, the weather is relatively drier with fewer chances of rainfall, making it easier to navigate through the parks and spot wildlife. The dry season also coincides with the peak tourist season, so it’s advisable to book your accommodations and permits well in advance.
Wet Season (March to May, October to November)
The wet season in Rwanda is characterized by heavier rainfall, especially in the form of afternoon showers. While the wet season brings lush greenery and fewer tourists, it can make trekking and game drives more challenging. However, the landscapes are beautiful and vibrant during this time, and the prices for accommodations and permits may be lower.
It’s important to note that Rwanda’s wildlife, including the mountain gorillas, can be seen throughout the year. However, it’s worth considering that some trails and paths in the national parks can become muddy and slippery during the wet season, which might affect accessibility.

domain: travel


“Have you heard about Openbay?”
“Have you met Rob from Openbay yet?”
“You have to check out this new startup, it’s called Openbay”
This seems to be what we’ve been hearing A LOT lately.
Openbay is a car care startup founded by Rob Infantino that makes the murky world of car maintenance a whole lot easier to navigate. The company's platform, which allows users to scan the area and search for the best deals for car care service, is one of those marketplace apps that seems like such an obvious problem, it is almost shocking no one has tried to attack the sector before.
Infantino came up with the idea after experiencing something very commonplace, sticker shock at a car dealership's repair costs.
"The genesis for the company started out of some frustration that I had with a dealership here in Boston," Infantino said. "I brought my car in for a simple wheel alignment, and I ended up sitting in the waiting room with this ten to twelve page document and a bill of around $4,000 for the car. I told them they were crazy."
"I went home and looked on the web for an alternative, I wanted to see if I could submit my service to multiple shops and get a response. That's when I realized there was nothing like that on the web; basically, I'd have to call every shop around, talk about my service, get the costs, make and appointment, etc."
"That was the 'Ah Ha' moment for me," Infantino said. "A service like this needs to be created and built."
So Infantino followed some Lean Startup methodology. He connected with both consumers and service providers to see what their pain points were to see how he could build something that could help both parties.
"I got great responses, which helped me move forward building a web-based service," he said.
Openbay went through a long trial/beta period before officially launching in October.
As Infantino said, the launch "exceeded our expectations across the board."
Since the day of launch, they have been rapidly expanding the number of customers and providers they work with. As far as Infantino knows, they are in over forty states right now, "maybe even fifty," he told me. When they launched, they weren't sure how long it would take to make an impact outside of Massachusetts.
The company is growing at a light-speed pace.
Based out of Intrepid Labs, Openbay got some help building the mobile app from Mark Kasdorf's Intrepid Pursuits.
So why all the buzz around Openbay?
For one, the are the only Boston startup from Boston to get funding from both Google Ventures and Andreessen Horowitz. Second, from what I hear, they are one of the rare companies that VC's and angels are hunting down and trying to get a piece of, and not vice versa.
Everyone I speak to, in all corners of the Boston tech ecosystem, have nothing but great things to say about Openbay. As the year unfolds, it wouldn't be shocking if Openbay is one of the companies that tops the list of biggest movers and shakers next December.


House of Blues Dallas
The House of Blues, what more is to say about this ICONIC American institution? Opened in Dallas in 2007 the brand and building has always been home to the best and wildest parties in America! It continues to celebrate and showcase art and music in the best way possible, by throwing outrageous parties and inviting the hottest performers! Much more than Blues, this venue is host to rock, jazz, rhythm, and just about any kind of soulful music you can think of, this home to live music, original folk art, and delta-inspired cuisine has a dedicated fan base and is looking for you to join in on the fun! Book private parties, such as New Years Eve or birthday parties, or snag a few tickets and bring your friends out for an amazing night of cold drinks, delicious food, and live music sure to keep you coming back!
The House of Blues guarantees the bring some of the best live music, making the bar/club a premier destination for music enthusiasts. Steal the dance floor, celebrate an occasion or special event, mingle over drinks at the bar...the options here are endless!
Featured Events Nearby
Upcoming Events @ House of Blues Dallas
Australia's Thunder From Down Under Music Hall 7:00pm Doors | 8:30pm Show Tickets- - $20.00 Reserved 1 - $27.50 Reserved 2 - $27.50 Cabaret 2 - $35.00 Cabaret 1 [more]
Voodoo Stage in Crossroads at House of Blues Dallas Every Sunday, live on the Voodoo Stage in Crossroads at House of Blues Dallas is Lip Sync Showdown! You think you've got what it takes? Come show your stuff and lip sync to your favorite songs! This event is free and open to the public. [more]


ABIS Consulting Group is a Houston, TX based consulting firm specializing in business process automation through technology and software solutions.
Adjutant, our flagship enterprise software solution, enables our clients to do more with less and gives them the insight to make informed business decisions.
Our suite of integrated and automated software solutions allows you to work on your business, not in your business. The old saying, "Time is Money" has never been more true than today. We work hard every day to refine our products and make them as user-friendly and intuitive as possible.
Please do not hesitate to contact us with any questions. We are eager to learn about your business and help in any way we can.
The Adjutant accounting solution delivers advanced AR, AP, general ledger and financial reporting capabilities designed to meet the needs of today's proactive, growth-oriented businesses. The accounting solution is completely integrated with all modules and is the key to maximizing the efficiency and accuracy of your financial data.
The benefits of CRM and SFA are clear: by streamlining processes and providing sales, marketing, and service personnel with better, more complete customer information, they allow organizations to build more profitable customer relationships and decrease operating costs. Our highly customizable web application is both powerful and affordable.
Our suite of BPM software modules combine to create a solution that is second to none. It includes Project Management, Task Management, Resource Scheduling, Automated Alerts, Workflow Automation, Approval Routing, and much more. Contact us today to find out how you can automate your entire company.


Buy a plasma CNC cutting machine from China
January 28, 2015 12:59am CST
Recently,CNC cutting machine is used widely all over the world.Lot’s of people input it to their factory.And other people still have a weak awarness of the cutting machine.Because they are not ususlly live with the machine.In this article, CNC cutting machine manufacture will tell you the detial imformation of the CNC cutting machine. Air plasma cutting machine is a new type of thermal cutting equipment, it works as a working gas is compressed to high temperature and high speed plasma arc as heat source, will be cut metal partial melting, and at the same time with a high-speed air would have been melted metal blown off, forming a narrow kerf. The air plasma CNC cutting machine device can be used for stainless steel, aluminum, copper, cast iron, steel and other metal material cutting, not only fast cutting speed, narrow kerf, cut flat, small heat-affected zone, the low degree of deformation of the workpiece, simple operation, and has significant energy savings. The device is suitable for all kinds of machinery, manufacture of metal structures, installation and maintenance, as in, cut a thin sheet, openings, Patching, and other cutting beveling. Despite the plasma CNC cutting machine,there are flame CNC cutting machine and laser cutting machine using in the market.The future of domestic high-end CNC cutting machine only solid direction towards the progressive development of the above, in order to increase market share, in order to promote the CNC industry toward good, stable direction, and ultimately to achieve the numerical power.


I’m trying to work out my 10-50 Jeffsy after it wasn’t properly setup. I have the gears running through the full range, but the 42 continues to be a problem. It jumps and skips, no matter how I adjust the B screw in 1/4 turn increments.
Any ideas? Is this a cable tension issue?
Here are two tricks for the Shimano freehub body removal, that is stuck.
When the rear hub is laced into the wheel, it is easier adn all you need to have is a wall and maybe some extension for your hex key:
When you have the rear hub removed from the wheel, it gets a bit more difficult, but the brake cable (or shifting cable) and two slabs will do...


text:
President Trump continues to remain defiant and lashing out at Special Counsel Jack Smith, the DOJ, and President Joe Biden over the legal battles he is currently fighting, maintaining that the cases and investigations are politically motivated and meant to hurt him, not effect justice in America.
Particularly, he did so on Truth Social after the Supreme Court rejected a request from Special Counsel Jack Smith to immediately hear a claim from President Trump that he is protected from prosecution in the election case by presidential immunity.
Declaring something of a victory and commenting on what he sees as the ridiculousness of the case, Trump wrote, “The Supreme Court has unanimously rejected Deranged Jack Smith’s “SUPER SPEEDY” lawsuit against me. This sick puppies team of Lowlifes and Radical Left Thugs could have brought this “case” 3 years ago, and it would be long over by now. No, they waited until right in the middle of my very successful campaign for President.”
Continuing, he wrote that he thinks the case is politically motivated and meant to sink his poll numbers, writing, “If I were polling poorly, like everyone else against me is (including Crooked Joe Biden!), or, for some reason, I decided not to run, they never would have even brought this ridiculous and disgraceful lawsuit. It is Election Interference like never before seen in our Country.”
He then added, “In a certain way, it is real “insurrection,” especially when adding the millions of people, from places unknown, that the worst President in the history of the United States is allowing to pour into our Country, totally unchecked and unvetted. This insane Open Border’s decision will prove to be a mistake for the ages, but I will fix it. MAGA!!!”
In a separate but similar post on the matter and SCOTUS ruling, Trump wrote, “The Supreme Court has unanimously rejected Deranged Jack Smith’s desperate attempt to short circuit our Great Constitution. Crooked Joe Biden and his henchmen waited three years to bring this sham case, and now they have tried and failed to rush this Witch Hunt through the courts.”
Continuing in that post, he argued that he deserves immunity, writing, “Of course I am entitled to Presidential Immunity. I was President, it was my right and duty to investigate, and speak on, the rigged and stolen 2020 Presidential Election. Looking forward to the very important arguments on Presidential Immunity in front of the DC Circuit Court of Appeals. Make America Great Again!”
However, a conservative attorney on CNN recently argued that the SCOTUS decision was not a major victory for Trump, saying, “I think it’s not a big deal because I don’t think it’s going to affect the schedule that much, and I think it actually shows the weakness of Donald Trump’s immunity claim. And I say it as somebody, as Norm Eisen said to me in the green room, I’m the only person in the universe who ever won, wrote a brief that won, a Supreme Court immunity case… that was against the president — Bill Clinton, Paula Jones.”
In a different post, Trump alleged that President Biden has pressured the DOJ into investigating him, arguing thathe tactic was reminiscent of those used by tin pot dictators in banana republics. He wrote, “Crooked Joe Biden has pushed DOJ to prosecute his Political Opponent. I told you so! He can’t fight for himself because he is defenseless and inept, so he is using his Justice Department to do the campaigning for him. Banana Republic. FIGHT FAIR AND SQUARE JOE. DROP THE FAKE POLITICAL INDICTMENTS. A VERY DANGEROUS PRECEDENT IS BEING SET!!!”

domain: news


text:
Highland League Cove Rangers' record Scottish Cup run ended with a 3-1 defeat to Paul Hartley's Championship Falkirk in Inverurie.
Police have warned football fans attending a North-east match today against anti-social behaviour.
“It’s the biggest game in Cove’s history.”
Cove Rangers skipper Eric Watson knows his playing days are numbered.
Cove Rangers manager John Sheran says facing Livingston or Falkirk would be a “big test” if they make it through to the Scottish Cup fifth round.
Hamilton striker Louis Longridge has left the club on a permanent basis to join Falkirk.
Jimmy Nicholl could not contain his excitement after sealing a return to Ibrox as Rangers assistant manager.
PFA Scotland chief executive Fraser Wishart has condemned the Falkirk fans who targeted Dean Shiels' disability at East End Park.
Falkirk have apologised and vowed to take action against the culprits after some of their fans threw fake eyes towards Dunfermline midfielder Dean Shiels during last night’s Ladbrokes Championship derby.
Dunfermline manager Allan Johnston hailed Dean Shiels' discipline after Falkirk fans threw fake eyes towards the midfielder.
Falkirk have announced plans to scrap their academy from the end of the year as they focus on gaining promotion back to the Ladbrokes Premiership.
Falkirk's Joe McKee has been handed a four-match ban after being found guilty of taunting Dunfermline's Dean Shiels over his missing eye.
Dundee United midfielder Scott Fraser has thrown the gauntlet down to title rivals St Mirren and challenged them to bounce back like his side did against Dunfermline.
A new steel leopard sculpture by the world-renowned artist behind the Kelpies could put Aberdeen on the map, according to a senior councillor.
This memory match appeared in Wednesday's Evening Express Aberdonian supplement:
Highland League side Formartine United are set to face Premiership club Partick Thistle in the Scottish Cup fourth round.
Falkirk midfielder Craig Sibbald today became the latest to be linked with a move to the Dons.
Aberdeen goalkeeper Danny Rogers has joined Scottish Championship side Falkirk on loan for the 2016/2017 season.
Police are appealing for help to trace a young girl who is believed to be from the North-east.
Falkirk manager Peter Houston has revealed he wants to bring Aberdeen's Danny Rogers back to the club.
Keeper Danny Rogers is determined to push for the No1 spot at Pittodrie next season after signing a new contract.
ALISTAIR Mather will be another one flying the flag for Aberdeen at tomorrow's Scottish Cup final.
INVERNESS Caley Thistle left-back Graeme Shinnie's career will come full circle when he joins home city club Aberdeen next month.


text:
Np/nd (ca.1943): Self-Published. Octavo. Item #025298
98 leaves, typed carbon copy on one side only. Nell Goodale Price was a prolific author of children's plays, poems and stories published in the late 1930's through the 1940's in such magazines as Jack and Jill, Child Life, Wee Wisdom, the Instructor, and Junior Magazine. This includes as she notes in the section 12B The Two Mysterious Marvel Workers (10 pages) which she notes was her first juvenile which was not sold. Ref: John MacKay Shaw, Childhood in Poetry (Gale, 1967). As a freelance writer she had first published two western short stories in the early 1930's but the majority of her writing was aimed at the elementary or juvenile readers. Her children's play "Mother Bunny's Magic Spectacles" was presented not only in the 1940's but in the 1960's in primary schools in the Southwest. Bound in 1/4 beige cloth over maroon cloth lettered in gilt, spine lettering gilt. Near fine.