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Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.
19 Related JoVE Articles!
New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals
Institutions: Ragon Institute of MGH, MIT, and Harvard, Massachusetts General Hospital.
CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro
. Flow-sorted CD3+
Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.
Infection, Issue 75, Infectious Diseases, Medicine, Immunology, Virology, Cellular Biology, Molecular Biology, Lymphocytes, T-Lymphocytes, Regulatory, HIV, Culture Techniques, flow cytometry, cell culture, Treg expansion, regulatory T cells, CD4+ T cells, Tregs, HIV-1, virus, HIV-1 infection, AIDS, clinical techniques
Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes
Institutions: The University of Florida.
Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-β. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.
Immunology, Issue 79, Cellular Biology, Molecular Biology, Medicine, Infection, Th17 cells, IL-17, Th17 differentiation, T cells, autoimmunity, cell, isolation, culture
Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity
Institutions: Vaccine and Gene Therapy Institute of Florida.
Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30
cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.
Immunology, Issue 82, Cytotoxicity, Effector CD8+ T cells, Tetramers, Target CD4+ T cells, CFSE, Flow cytometry
The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
Institutions: John Curtin School of Medical Research, Australian National University.
Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3
. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro
and in vivo
Immunology, Issue 44, carboxyfluorescein diacetate succinimidyl ester (CFSE), labeling, lymphocytes, proliferation.
Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation
Institutions: Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center, Virginia Commonwealth University- Massey Cancer Center.
It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2
. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo
for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3
. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4
. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo
expanded T cells is determined.
Immunology, Issue 47, Adoptive T cell therapy, Breast Cancer, HER-2/neu, common gamma chain cytokines, Bryostatin 1, Ionomycin
Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens
Institutions: The Ohio State University Medical Center, The Ohio State University Medical Center.
The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro
culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.
We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.
Medicine, Issue 53, Glioblastoma multiforme, glioma, temozolomide, therapeutics, drug design
Isolation of Double Negative αβ T Cells from the Kidney
Institutions: Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine.
There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6
. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7
due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8
in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d α-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.
Immunology, Issue 87, Double Negative (DN) αβ, T cells, CD45+ T cell isolation, renal lymphocytes, non-lymphoid-tissues, T cells purification, Ischemia Reperfusion Injury, Acute Kidney Injury, Tissue Resident Lymphocytes, Lymphoproliferative Disorders, Erythematosus Lupus
Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance
Institutions: Medical College of Wisconsin , Medical College of Wisconsin , Medical College of Wisconsin .
Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro
assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro
suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro
suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic β-cells. The main goal of this piece is to describe an in vitro
human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1
. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro
suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.
Immunology, Issue 53, suppression, regulatory T cells, Tregs, activated T cells, autoimmune disease, Type 1 Diabetes (T1D)
Peptide-based Identification of Functional Motifs and their Binding Partners
Institutions: Morehouse School of Medicine, Institute for Systems Biology, Universiti Sains Malaysia.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
Virology, Issue 76, Biochemistry, Immunology, Infection, Infectious Diseases, Molecular Biology, Medicine, Genetics, Microbiology, Genomics, Proteins, Exosomes, HIV, Peptides, Exocytosis, protein trafficking, secretion, HIV-1, Nef, Secretion Modification Region, SMR, peptide, AIDS, assay
Expansion of Human Peripheral Blood γδ T Cells using Zoledronate
Institutions: University of Tokyo Hospital, MEDINET Co., Ltd.
Human γδ T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1
The majority of γδ T cells in peripheral blood have the Vγ9Vδ2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1
Therefore, γδ T cells are attractive candidate effector cells for cancer immunotherapy. Vγ9Vδ2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2
and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3
In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of γδ T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and γδ T cells activation.3
Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to γδ T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating γδ T cells with aminobisphosphonates. 4
Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5
Monocytes that accumulate IPP become antigen-presenting cells and stimulate Vγ9Vδ2 T cells in the peripheral blood.6
Based on these mechanisms, we developed a technique for large-scale expansion of γδ T cell cultures using zoledronate and interleukin-2 (IL-2).7
Other methods for expansion of γδ T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8
or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9
All of these methods allow ex vivo expansion, resulting in large numbers of γδ T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded γδ T cells display CD27-
effector memory phenotype and thier function can be evaluated by IFN-γ production assay. 7
Immunology, Issue 55, γδ T Cell, zoledronate, PBMC, peripheral blood mononuclear cells
Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques
Institutions: MD Anderson Cancer Center - University of Texas, University of Miami.
The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3
. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5
with cytokine-producing capability6
following non-specific or antigen-specific stimulation 5,7
JoVE Immunology, Issue 38, Immune Response, Cytokine Production, Flow Cytometry, HIV, Rhesus Macaque, T Cells, Intracellular Cytokine Staining, FACS
The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo
Institutions: Australian National University.
The ability to monitor T cell responses in vivo
is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into >250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+
T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+
T cell-mediated killing of FTA target cells and CD4+
T cell-meditated help of FTA B cell target cells in real time in vivo
by flow cytometry. Since >250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.
the cumulative magnitude of the response) and a qualitative (e.g
. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.
Immunology, Issue 88, Investigative Techniques, T cell response, Flow Cytometry, Multiparameter, CTL assay in vivo, carboxyfluorescein succinimidyl ester (CFSE), CellTrace Violet (CTV), Cell Proliferation Dye eFluor 670 (CPD)
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
Institutions: Roswell Park Cancer Institute, University of Pennsylvania , SciGro, Inc., University of Pennsylvania .
Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro
and in vivo
Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
stem and progenitor cell quiescence, proliferation and/or differentiation6-8
antigen-driven membrane transfer9
and/or precursor cell proliferation3,4,10-18
immune regulatory and effector cell function1,18-21
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11
. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18
. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
Failure to achieve bright, uniform, reproducible labeling
. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.
Suboptimal fluorochrome combinations and/or failure to include critical compensation controls
. Tracking dye fluorescence is typically 102
times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.
Failure to obtain a good fit with peak modeling software
. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.
Cellular Biology, Issue 70, Molecular Biology, Cell tracking, PKH26, CFSE, membrane dyes, dye dilution, proliferation modeling, lymphocytes
Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation
Institutions: Vanderbilt University Medical Center, Vanderbilt University Medical Center, Veteran Affairs TVHS.
Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.
Medicine, Issue 83, Glioma, Malignant glioma, primary orthotopic xenograft, isocitrate dehydrogenase
In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection
Institutions: The University of Texas Medical Branch, The University of Texas Medical Branch, The University of Texas Medical Branch.
An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods – an in vitro
T cell priming assay and an intracellular cytokine staining (ICS) – were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+
T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+
T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro
immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.
Immunology, Issue 93, West Nile Virus, Dendritic cells, T cells, cytokine, proliferation, in vitro
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function
Institutions: The Ragon Institute of MGH, MIT and Harvard, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM).
T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro
assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro
experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10Rα and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 °C, 5% CO2
. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.
Immunology, Issue 80, Virus Diseases, Immune System Diseases, HIV, CD4 T cell, CD8 T cell, antigen-presenting cell, Cytokines, immunoregulatory networks, PD-1: IL-10, exhaustion, monocytes
Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells
Institutions: Pennsylvania State University College of Medicine.
The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.
Immunology, Issue 75, Medicine, Cellular Biology, Molecular Biology, Microbiology, Anatomy, Physiology, Biomedical Engineering, Genetics, Surgery, Type 1 diabetes, CD4+ T cells, diabetogenic T cells, T cell transfer, diabetes induction method, diabetes, T cells, isolation, cell sorting, FACS, transgenic mice, animal model


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Eric Carney, 39, was arrested at a Capitol Hill apartment building just after 5 p.m. Tuesday. His infant daughter was also there and was unharmed.
Police say Carney approached his daughter's mother in the 5200 block of 15th Ave. NE just before noon, threatened her and then grabbed her stroller with the daughter inside.
The mother tried to grab the stroller back, but Carney ran off, police said.
Police say Carney has a history of violent crimes, auto theft and animal cruelty and the mother had a no contact order against him.
Seattle police say an Amber Alert was not issued because there was no car associated with the crime.

domain: law


Eaton 158-3737-001 T Series 11.9 cu.in. Hydraulic Motor
158-3737-001 T Series 11.9 cu.in. Hydraulic Motor
Only 1 left in stock
Eaton 158-3737-001 Hydraulic Motor
This is a surplus NOS (new old stock) item and there is a qty. of one available at this discount price. The actual unit available is pictured. If you have a motor with a different design code (the last 3 digits on the end of the part number) it will replace it as an assembly as long as the first seven numbers are the same. Please note that all surplus items may have some imperfections in the paint or small scuffs and scratches, or other signs of light shelf wear Guaranteed to be new, unused, and to operate correctly when proper start up procedures are followed.
It has the following specifications:
- 11.9 cubic inch displacement
- 5/8″ O-Ring ports
- 2 bolt SAE “B” mounting
- 7/8″ Diameter 13 tooth shaft, SAE B
- 2200 psi maximum continuous pressure rating
- 15 GPM maximum continuous and 20 GPM intermittent
- Torque 3905 inch pound Continuous and 4300 inch pound intermittent
- Eaton 158-3737-001
Buy from professionals who have specialized in steering for agricultural and industrial applications for over 25 years. Free technical support, and we service what we sell from our central USA location, and all products are covered with a one year warranty (Click here for more details).


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[One man’s quest to play and talk about every video game released for the Famicom & NES in chronological order][link]. I’ve finally begun watching the video volumes, which run bout 45 minutes long and cover 15 games each. The [Episode Guide][guide] is indispensable.
[link]: http://chrontendo.blogspot.com/ “Chrontendo”
[guide]: http://www.sylpher.com/chrontendo/ “Chrontendo Episode Guide”

domain: game


text:
Dan Loeb's hedge fund firm Third Point is finally out with its third quarter letter to investors. The first half of Loeb's letter focuses on Ben Bernanke and the second half focuses on Third Point's portfolio. The latter portion is what we'll highlight below.
Third Point recently received AR Magazine's award for event driven fund of the year. Third Point now manages $3.6 billion and at the end of the third quarter its Offshore Fund was up 19.2% for the year. The fund now has seen 18.2% annualized returns since inception, an obvious reason to track them. Recall that we've provided commentary and analysis of Loeb's investments in our newsletter. And if you desire to be a successful investor like Loeb, head to his recommended reading list.
Anadarko Petroleum (NYSE:APC)
Turning to Loeb's recent commentary, he touches on his firm's position in Anadarko Petroleum. We originally revealed this position back in August and he purchased debt securities in June and July due to the opportunity presented as a result of the Gulf oil spill.
Of the investment he writes,
Our analysis was correct and in hindsight, investors could have generated similar returns by investing in anything 'Macondo-related (e.g. Transocean (NYSE:RIG), BP (NYSE:BP) and Anadarko equities). However, Anadarko bonds offered similar upside to the aforementioned securities but with effectively zero downside in the event that either our thesis on the severity of the spill was incorrect or there was a material decline in oil and.or natural gas prices, and so we delivered excellent risk-adjusted returns.
In his commentary, Loeb mentions that all oil spill related securities have rallied furiously since the event. He doesn't mention whether or not Third Point still owns these securities and almost makes it sound like he has since exited the position (but that's speculation on our part).
NXP Semiconductor (NASDAQ:NXPI)
Loeb also reveals a new position in his letter as Third Point participated in NXPI's IPO. He notes that,
the company is in the final stages of completing a substantial operational and capital structure restructuring, which is driving free cash flow, rapid deleveraging and attractive new opportunities like a leadership position (>50% market share) in Near Field Communications, a fast emerging mobile payment technology being adopted by Google (NASDAQ:GOOG) Android, Nokia (NYSE:NOK) and Blackberry (RIMM).
The most interesting thing here is that despite the rally in shares, Loeb sees "substantial upside" in this stock.
While not specifically listed in the letter below, we have since seen Third Point's November portfolio update where the fund lists the following as its top positions:
Delphi (multiple securities held)
Chrysler (multiple securities held)
Of note is the fact that Loeb owns physical gold, not the exchange traded fund. Fellow hedge fund manager David Einhorn of Greenlight Capital stores physical gold and John Burbank of Passport Capital also prefers the physical metal.
Embedded below is Third Point's third quarter letter to investors:

domain: economics


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ST. LOUIS—A future where sophisticated geospatial intelligence (GEOINT) is occurring in real-time for operational use could be less than a decade-and-a-half away if there is a sustained investment in technology, people and partnerships, the chief of research for the National Geospatial-Intelligence Agency (NGA) said on Tuesday.
Technology, people and partnerships are the building blocks of the future vision but there also needs to be a focus on three “attributes” that will play primary roles in fulfilling the promise of more advanced GEOINT, Cindy Daniell said during a keynote address here at the GEOINT 2021 Symposium hosted by the U.S. Geospatial Intelligence Foundation.
The first attribute she outlined is that analysts, through their experiences and inputs from data scientists and operators, will remain key drivers in the GEOINT enterprise, making them “indispensable to transparent and informed decision-making.” Moreover, analysts will also help drive efforts in other areas such as “training algorithms, developing novel analytical methods, or identifying more efficient policies and pathways for rapid and secure data dissemination.”
Technology that is enhanced by artificial intelligence (AI), in particular machine learning (ML), that automatically detects anomalous activities, rapidly fuses traditional and non-traditional GEOINT sources for decision makers, and makes sense of large amounts of unstructured data will free up people to focus on challenging task and major decisions, she said.
“AI and ML are the tools for accelerators of GEOINT in the future, letting us make more effective and more efficient use of our workforce,” Daniel said.
Leveraging AI and ML will “enable our decision advantage” and result in “new human-machine teams,” she said.
In her GEOINT vision of the future process, NGA operators speak into computer screens and computer tablets, “creating and manipulating a multitude of real-time GEOINT products at the speed of their voice.”
The final attribute is the need for “adaptive” workflows and workforce amid changes in the threats and technology, she said.
“This means creating a GEOINT enterprise that is sensor agnostic in its ability to capture and assess government or commercial data, has sufficient capacity to process large volumes of dynamic information and is responsive to a variety of user missions, from warfighting to intelligence, from tactical to strategic, or from immediate to foundational,” she said.
The technology landscape in particular has changed dramatically from the explosion of space-based assets due new market entrants and the lower costs to launch satellites, and design and build them. Daniell pointed out that during the Cold War few countries had accesses to space-based sensors and the means to launch them.
So far in 2021, close to 1,400 satellites have been launched, eclipsing the total in 2020, which was the first year more than 1,000 were placed into orbit, Daniell said, citing data from the United Nations.
“As a result, high quality, timely and cross-phenomenology data is now available to a whole new set of consumers,” she said. “On the government side, this has led to an exponential growth in available data, both classified and unclassified, structured and unstructured. As a result, our analysts, whose numbers are not increasing as rapidly, will need help sifting, cataloguing, and prioritizing the data before they can even begin to understand it.”
This challenge is being addressed by evolving strategies, putting more focus on operators, emphasizing the integration of emerging technology, being agile in partnering with industry, particularly with non-traditional players that are as innovative as large contractors but may need help understanding NGA’s needs and working the government contracting process, and investing in people, Daniell said.

domain: news


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Town of Huntington plants 200 trees at Koster Park for Arbor Day
Town of Huntington Supervisor Ed Smyth, Receiver of Taxes Jillian Guthman and Town officials were joined by Covanta and local students to plant 200 donated trees at Koster Park in Huntington Station for Arbor Day on April 29.
“It is so important to maintain our tree canopy; it adds shade, which reduces the need for artificial cooling, and it filters stormwater runoff to stop contaminants from entering our water supply,” said Supervisor Ed Smyth. “Thank you to Covanta and our anonymous donor, as well as the Girl Scouts, Boy Scouts and Huntington High School honor students for your dedication to preserving our green space, this is our largest Arbor Day turnout yet!”
“I’m proud to celebrate Arbor Day with Supervisor Smyth, many of our local Scouts, students and Covanta with the planting of trees at Koster Park, one of the many hidden jewels of Huntington,” said Receiver of Taxes Jillian Guthman.
“We are once again delighted to partner with the Town of Huntington and Supervisor Smyth to plant trees in honor of Arbor Day. Initiatives like this one are so important to preserving Long Island’s natural resources. We are grateful to our municipal leaders for joining with us as great environmental stewards,” said Maureen Early, Senior Community Affairs Specialist for Covanta. Joining the event from Covanta were Asset Manager Dawn Harmon and Shari Kleinman from the Covanta Huntington Facility.
Girl Scouts, Boy Scouts and students from the Huntington High School Honor Society helped plant bare root saplings at the park.
“Members of the Girl Scouts of South Huntington are honored to be a part of the Town of Huntington Arbor Daytree planting at Koster Park. The girls will expand their knowledge about caring for the environment, using resources wisely and making the world a better place,” said Catherine Galli, Coordinator for Girl Scouts Service Unit 12 in South Huntington, who was joined by Troop Leaders Christine Reilly, Bonnie Millman, Kristie Mondo, LeeAnne Ramirez and Wendy Aull with Girl Scouts from Service Unit 12, including Junior Troop 161, Daisy Troop 32 and 74, and Brownie Troop 703 to help plant bare root saplings.
Boy Scouts Troop 34 Scoutmaster Mike Fasciano and Assistant Scoutmaster Dominic Dutton were joined by Boy Scouts from Troop 34 for the tree planting.
“The Cub Scouts are all about having fun with a purpose,” said Scott Wallick, Cubmaster for Pack 406 of South Huntington. “There is a reason for everything our boys and girls do in Cub Scouting, and today’s events will help them ‘think globally’ and ‘act locally’ to preserve and improve our environment. We are grateful to have been a part of the Town of Huntington’s Arbor Day tree planting.” Cub Scouts Pack 406 Assistant Pack Leader Jennifer Rodriguez was joined by Cub Scouts from Pack 406 to plant trees at the event.
Joining the event from the Town’s Department of Planning and Environment to teach the students about the trees and demonstrate how to plant the bare root saplings were Christian Granelli, Elana Israel-Edgar, Dave Kaufman and Vincent Cerniglia.
From the Department of Parks and Recreation, Director Todd Jamison and Deputy Director Walter Edwards joined the event with Town Volunteer Parks Stewards David Lesser and Christopher Zonin.
The ceremony involved the planting of a Caliper Pin Oak donated by Covanta and 200 additional bare root saplings consisting of 25 each of Flowering Dogwood, Highbush Cranberry, Buttonbush, Ninebark, Black Chokeberry, Virginia Rose, Witch Hazel, and Winterberry donated anonymously to the Town of Huntington to commemorate Arbor Day; the Town Board accepted the donations at their April 12 meeting.


text:
Sarah W. Caron from Sarah's Cucina Bella shares a recipe.
SAVE ON THIS RECIPE!
If desired, garnish with a fresh whole strawberry.
Another garnish idea: Place a cookie on the edge of the cup.
© 2013 ®/TM General Mills All Rights Reserved

domain: food


text:
In musical terms, conductor and composer Pierre Boulez is a colossus. His influence since the Second World War can be felt everywhere, but particularly so in the world of modern classical music. He voices his controversial opinions when asked, and also when not asked, and as a result was not loved by everyone.
As a figurehead of avant-garde music, Boulez championed serialist music (in the Fifties), aleatoric music (Sixties, with elements of the music left to chance) and the use of electronic elements in instrumental music (Seventies).
Some of the pieces on this collection are from those periods, but most of this is a bit earlier and not as revolutionary. Pieces that stand out is the Lulu suite by Alban Berg (a pupil of Arnold Schönberg) and a really good performance of the Sacre of Stravinsky, mentioned in the Spotify link. Debussy’s La mer is also there, a real good recording. I would not think Boulez would be good for that piece, but he is.

domain: artistic


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Hiring Dunstan will be one of the easiest and best decisions you will make for your event.”Donna
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“I would highly recommend Dunstan for his music, his professionalism and his manner.”
“Dunstan is incredibly talented and professional… he’s organized, accommodating and prompt in his responses, making planning easy. Dunstan was a pleasure to work with and we very highly recommend him! ”Stefanie


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23rd May 2016
The Chairman of the Asian Catering Federation (ACF), Yawar Khan, is among the first community leaders to condemn the “reckless” Indian restaurant owner jailed for 4 years following the conviction for manslaughter of a customer.
“This callous individual has done enormous damage to the hardworking restaurateurs and their staff, who exercise meticulous customer care and strive to build their businesses,” said Khan who welcomed the court’s decision. “This was a needless tragedy and our thoughts go out to the victim’s family.”
Paul Wilson, 38, suffered severe anaphylactic shock and died in January 2014 after eating a takeaway containing peanuts.
Owner of the IndianGarden in Easingwold, Yorkshire, Mohammed Zaman, 52, was found guilty at Teesside Crown Court.
The court heard he cut corners by using cheaper ingredients containing peanuts. Zaman, denied he was responsible but a jury was told he switched almond powder for a cheaper ground nut mix, which contained peanuts.
Mr Wilson, a bar manager from Helperby, North Yorkshire, specified “no nuts” when he ordered a chicken tikka masala – an instruction which was written on his order and on the lid of his takeaway.
He died three weeks after a different customer with a peanut allergy bought a meal from one of Mr Zaman’s six restaurants and had a reaction requiring hospital treatment.
The restaurateur had a “reckless and cavalier attitude to risk” and “put profit before safety” at all his outlets, the jury was told.
Zaman cut costs by using the cheaper nut powder and by employing untrained, illegal workers.
Sentencing him, Judge Simon Bourne-Arton, said Zaman had remained “in complete and utter denial” and ignored warnings from officials after 17-year-old Ruby Scott suffered a reaction to a curry, three weeks before Mr Wilson’s death.
He said he had thrown away his successful business and property portfolio worth £2m “in pursuit of profit”.
Mr Wilson’s mother, Margaret Wilson, from Sheffield, her son was “meticulous” about dealing with peanuts after a reaction to a chocolate bar at the age of seven.
She said the smell of peanuts, or accidentally drinking from the same glass as someone who was eating them, could trigger a reaction.
Paul Wilson asked for a nut-free takeaway and tried to make himself sick when he realised he hadn’t got what he ordered.
Detective Inspector Shaun Page said Mr Wilson’s death was “totally avoidable” and Zaman’s “lack of remorse” had been striking.
Zaman claimed he left managers to run his restaurants and that included ordering stock and hiring staff. The father-of-four was found guilty of manslaughter by gross negligence and six food safety offences. He was cleared of a charge of perverting the course of justice.
“We have been educating members snout the utmost importance of good hygiene control and the dangers of allergens,” said Khan whose ACF is conduction a round Britain series road shows, which has already been attended by the owners of around 1,000 owners of restaurants and takeaways, adding, “It is horrific that someone has died because a restaurant fails to follow standard procedures.”
A repeated theme during the events from one of BBC TV’s ‘Spicemen’ the renowned chef Cyrus Todiwala, was ‘Don’t Buy Cheap!’
Mohammed Zaman has never been a member of the ACF.


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Thousands of gay and bisexual men convicted of abolished sexual offences are to be posthumously pardoned.
The "hugely important" move will see those convicted for consensual same-sex relationships before laws were changed formally pardoned.
Calls for wider action emerged after Second World War code-breaker Alan Turing was given a posthumous royal pardon in 2013 over a conviction in 1952 for gross indecency with a 19-year-old man.
Turing was chemically castrated and died two years later from cyanide poisoning in an apparent suicide - though there have been suggestions his death was an accident.
Announcing the new plan, Justice Minister Sam Gyimah said the Government would seek to implement the change through an amendment to the Policing and Crime Bill.
Anyone living who has been convicted of the now abolished offences can currently apply through the Home Office to have their names cleared through the disregard process. This removes any mention of an offence from criminal record checks.
In another step, the Government is also announcing that it will introduce a new statutory pardon for the living in cases where offences have been successfully deleted through the disregard process.
Mr Gyimah said: "It is hugely important that we pardon people convicted of historical sexual offences who would be innocent of any crime today.
"Through pardons and the existing disregard process we will meet our manifesto commitment to put right these wrongs."
The Government said it will not support a separate Private Member's Bill on the subject, which is set to be debated on Friday and proposes a blanket pardon for the living without the need to go through the disregard process.
Mr Gyimah said: "A blanket pardon, without the detailed investigations carried out by the Home Office under the disregard process, could see people guilty of an offence which is still a crime today claiming to be pardoned.
"This would cause an extraordinary and unnecessary amount of distress to victims and for this reason the Government cannot support the Private Member's Bill. Our way forward will be both faster and fairer."


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Over at Free Book Friday! This week only!
Also, teens (12-18) please vote for your favorite books before Sept 18! WAKE is a contender along with tons of other cool books. If you are not between the ages of 12 and 18, please find someone who is and pass it on.
P.S. Did you have a good summer?

domain: library


The two most recognisable names from the Bitcoin Satoshi’s Vision (BTCSV) side of the Bitcoin Cash hard fork that occurred last November are adamant that the cryptocurrency agreed upon by the market as the original Bitcoin (BTC) chain is going to plummet in price. In fact, Craig S. Wright and Calvin Ayre are so bearish on the digital asset that they state that the price is going to eventually bottom out at zero.
The primary reason cited for the expected crash is utility – or lack there of it. Wright and Ayre believe that the fact that Bitcoin is working towards scaling with solutions such as Lightning rather than increasing the block size will hinder its adoption as a method of payment.
Craig Wright is perhaps the most controversial man in cryptocurrency. He has previously claimed to be the anonymous inventor of the Bitcoin protocol itself, Satoshi Nakamoto, yet has been unable to provide adequate evidence to support this.
Wright holds the “big block” position towards scaling cryptocurrency. It therefore came as no surprise to see him champion Bitcoin Cash after the hard fork that created it in August 2017. In fact, his faith in the premise that Bitcoin needs enormous blocks to serve the globe as a means of exchange is so strong that his influence caused the Bitcoin Cash chain to split in November 2018.
The chains resulting from this hard fork are known as Bitcoin SV and Bitcoin ABC. After the split, Wright vowed to wage a hash war against other blockchains, starting with Bitcoin ABC (now largely referred to simply as Bitcoin Cash). Since fellow big blocker Calvin Ayre, the owner of the largest Bitcoin Cash mining pool, CoinGeek, supported the Satoshi’s Vision side of the fork, Wright claimed to have the resources to destroy any competing cryptocurrency. This never materialised.
Since then, Wright and Ayre have been vocal opponents of Bitcoin (BTC). For the two, the lack of block space hinders the digital currency’s utility. The latest outburst from either occurred today when Wright arrogantly Tweeted:
In fact, Wright has spent much of today on Twitter. The Australian computer scientist posted close to 30 different Tweets today alone. Most of these were focused on the supposed shortcomings of the BTC chain whilst shilling his own vanity project, Bitcoin SV.
Of course, Wright is not alone in his belief that Bitcoin SV will one day reign supreme and Bitcoin (BTC) will crash to zero. Perhaps the only well-known member of the cryptocurrency community he has not managed to completely alienate, gambling magnate Calvin Ayre, agrees with him.
In late 2018, Ayre spoke to UK news publication The Express about his prediction for the original cryptocurrency:
“The hard fork was a distraction but the downturn is because the SegWit coin that’s erroneously still called ‘bitcoin’ now follows the old-fashioned financial trends. It’s also because people wrongly focus on market cap when it should be about scalability and actual usability. Bitcoin SV is a currency, not an asset just to be held, and has real utility.”
However, what Ayre and Wright completely forget is that it is not for them to decide the future of either digital asset. Sure, they can shill whichever they like. However, adoption of users is what is important. Those users might see that the best way to use a digital asset is to buy things with it or they might prefer to use it as a store of value. The point is that it is up to them.
Ayre and Wright’s opinions of the cryptocurrency market are therefore entirely irrelevant. If the wider market is willing to back a centralised digital coin that offers transactions with less fees then so be it. However, cryptocurrency users might decide that they prefer those validating transaction be less at risk of coercion due to their disparate locations and greater number. This is equally fine. The point is that it is the market that will decide the eventual winners and losers.
Related Reading: BCH Tussle: Bitcoin ABC May Reign Supreme over Craig Wright’s “Satoshi Vision”


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Co-Chairs of the Father's Day 5K on Midday Maryland
Greater Living - GBMC HealthCarehttps:/www.gbmc.org/greater-living
June 11, 2020
The GBMC Father's Day 5K and 1-Mile Fun Walk, which benefits the Neonatal Intensive Care Unit (NICU), has been going on for more than 30 years, but this year it will look a little different. Co-Chairs of the event, Ellen and Chris Dew appeared on Midday Maryland to talk about how the race is going virtual and how people can still help support GBMC's tiniest patients.
Click here to register!


text:
I Like To Bang Posters
I Like To Bang Posters is very impressive and makes a popular wall decoration, this dimension isand the price is $14.98. If you're trying to seek out top recommended I Like To Bang Posters with penny price, this item is the illustrious deals for you.
The color is a bit different than shown on the produce image, a bit more than just monitor differences. All around amazing experience, this one is flawless, without a single wrinkle. The sizeand the colors fit certainly onto any wall and give a rather exciting experience. The glistening colors, crisp details, and just appropriate, the one has a bit of a sheen to it but only so much as to make the picture stand out. I Like To Bang Posters comes in superior condition and the paper it is printed on is useful. The poster picture is simple, yet tasteful, this product has awesome price for the large size. This item is written in a no non-sense and splendid style with various ideas and ways to implement them. This one is of sixpenny, there are no problems and this produce is as decribed. The produce is great heavy ablaze paper and simply quite big. For the price $14.98 I Like To Bang Posters is a admirable poster with electric color.

domain: hobby


My air compressor keeps running - what should I do about it?
It’s incredibly annoying to have tools that don’t work properly but instead of moping around and complaining, you can try to find out what is really wrong in order to fix the problem. When it comes to air compressors the most nagging problem is that some have a tendency to run continuously without stopping. And when your compressor stops working, so does your job!
These tools rely on air pressure to power different tools for different purposes. For users’ safety they are ordinarily designed stop at a certain maximum pressure and restart when air pressure in the tank reaches a set low.
Causes – What Makes It Continue to Run?
Leakages are the most common reason why an air compressor runs nonstop. If there is any pressure lost along the way, the air pressure tool will not reach the set cut-out point which should trigger it to stop.
This quick video shows you how to troubleshoot a leak:
To ensure that your tool is not leaking check that the drain valve is closed and dab the connectors on airlines between the tank and compressor with soapy water. If there are bubbles when you turn on the compressor, you obviously have a leakage problem. In this case, experts recommend disassembling the head to check for worn or damaged parts.
Another problem that would cause an air compressor to run without stopping is dirt and grime gathered in crucial parts of your compressor. Dirt in the air compressor’s filter would mean that the tool is unable to reach the level of pressure that it must in order to stop occasionally during its job.
The problem might also be as a result of faulty extension cords that do not deliver enough power for the device to build enough pressure. If you must use an extension cord to reach far ends with an electric air compressor, avoid choosing a very long one as it might compromise the quality of power.
Sometimes the hose, tube and other fittings could cause the compressor tool to run nonstop. If there are holes, loose fitting or any damages to the tool attached to the air compressor chances are that air pressure will be lost causing it to hum continuously.
Solutions – How to Stop This Nonsense
There are many solutions to continuous humming depending on the reason behind it. It can be easy to fix some problems but others demand the attention of an experienced professional to correct safely. Never disassemble any tool if you cannot return it to its original state as that could expose you to serious injury.
Do not ignore this problem as it could lead up to serious damage of your compressor and the tool attached to it. If you just bought the air compressor and find out that it won’t reach the cut-in point, return it to the vendor who should exchange it or refund your money according to the terms of warranty.
It’s important to set the air compressor tool at the right pressure bar to avoid annoying instances when the tools keeps on starting and stopping. Pressure bar refers to the difference between a tools CFM and PSI rating and when it is low, your work will keep being interrupted. In the long run some of the compressor’s parts will be damaged leading to the more annoying problem of endless whirring.
Gas powered air compressors pose a great risk to users if they run endlessly because of the highly flammable fuel used. If you suspect that there is a problem with such a tool’s pressure, shut it off and wait for it to cool down before troubleshooting.
Always empty the tank to avoid excess contact with gasoline that could lead to serious injury. If it’s an electric device, unplug it from the socket before trying to disassemble in order to avoid risk of electric shock. (read more about gas vs electric air compressors here)
What if it keeps happening?
It is evident that air escaping from the tank is the most common reason why an air compressor will keep running but sometimes it is simply because the tool is too old for the job. Experts believe that repairing a tool twice is one time too many, meaning that if an air compressor constantly poses the same problem you should definitely consider shopping for a replacement.


text: Adult skeletal muscle mass possesses outstanding regeneration capacities. molecularly regulated and exactly how satellite tv cells are essential in diseased and aging muscle. The accurate variety of satellite television cells is normally reduced because of the changing specific niche market during ageing, leading to attenuation of muscles regeneration capability. Additionally, in Duchenne muscular dystrophy (DMD) Rabbit polyclonal to CD48 sufferers, the increased loss of satellite television cell regenerative capability and decreased satellite television cell number due to continuous needs for satellite cells lead to progressive muscle mass weakness with chronic degeneration. Therefore, it is necessary to replenish muscle mass satellite cells continually. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is vital for maintenance of satellite cells like a muscle mass stem cell pool throughout existence. In addition, we discuss tasks in the stem cell market for satellite cell maintenance, as well as related cell treatments for nearing treatment of DMD. mice, in which the gene is definitely inserted into the locus, and thus manifestation of nLacZ recapitulates endogenous mRNA manifestation, revealed that approximately 10% of quiescent satellite cells are LacZ(?), indicating the heterogeneity of Nemorubicin quiescent satellite cells (Kuang et al., 2007). To support this, RT-PCR centered gene expression studies in single satellite cells demonstrated that a portion of Pax7(+) satellite cells communicate Pax3 and/or MyoD (Sacco et al., 2008). Open in a separate window Number 1 Molecular markers for quiescent satellite cells, activated satellite cells, and myocytes. Quiescent satellite cells are triggered by signals from muscle mass injury and start cell division which include symmetric and asymmetric divisions to produce activated satellite cells and self-renewing satellite cell-stem cells. After several round of cell division, activated satellite cells (myogenic precursor cells or myoblasts) exit their cell cycles and give rise to myocytes which fuse each other to form multinucleated myotubes. Markers indicated in each cell types are summarized (blue characters). The characteristics of satellite cells will also be distinguished depending on muscle mass types with the unique variations of gene manifestation and cell behavior and mice (Collins et al., 2005). A single myofiber having a few satellite cells offered rise to a large number of myofibers as well as self-renewed satellite cells. In addition, the number of myofibers generated by tibialis anterior (TA) muscle was significantly less than those formed from EDL or soleus (SOL) muscle (Collins et al., 2005). These observations strongly suggest that satellite cells possess intrinsically different properties depending on the origin of muscle. Sacco et al. conducted the experiments that quiescent satellite cells [CD45(?) CD11b(?) CD31(?) Sca1(?) 7-integrin(+) CD34(+) cells] were isolated Nemorubicin from adult muscle by FACS, and single quiescent satellite cells were transplanted into irradiated muscle. As a result, they found that a single satellite cell has a remarkable ability of proliferation and differentiation, and further revealed that some transplanted satellite cells generated Pax7-expressing satellite cells after engraftment (Sacco et al., 2008). These findings are strong evidence that satellite cell populations are heterogeneous and exhibit high potency of self-renewal gene knockout (KO) mice display reduced significant reduction in satellite cell number, resulting in the failure of muscle growth and neonatal lethality of most KO mice (Seale et al., 2000; Oustanina et al., 2004; Kuang et al., 2006). Following a cardiotoxin-induced skeletal muscle injury, the KO mutant displayed significantly reduced muscle regeneration capacity. These results strongly indicate that is essential for normal skeletal muscle growth and regeneration through the maintenance and rules of muscle tissue satellite television cells (Oustanina et al., 2004; Kuang et al., 2006). Spontaneous conditional double-mutant mice possess proven that Pax7 is essential Nemorubicin for satellite television cell maintenance in juvenile mice, while adult satellite television cells usually do not need either or for muscle tissue regeneration (Lepper et al., 2009). Nevertheless, a more latest publications from many groups proven that constant inactivation of induces cell routine arrest, myogenic differentiation, and impairment of muscle tissue regeneration gene KO ((Zammit et al., 2004). Many Pax7(+)MyoD(+) activated satellite television cells or myoblasts go through Pax7(?)MyoD(+)Myogenin(+) myocyte differentiation, whereas a subset of Pax7(+)MyoD(+) myoblasts down-regulate MyoD expression and come back into Pax7(+)MyoD(?) reserve cells, that are inside a quiescent condition and are regarded as an equal cell human population to quiescent satellite television cells (Yoshida et al., 1998; Zammit et al., 2004). These cells possess the to re-enter the cell routine under growth circumstances and can ultimately bring about differentiating myocytes and self-renewing reserve.

domain: medical


text:
If you like your songs served up with a twist of heartache and a whole lot of Americana soul, Bob Harp is your bartender. The San Franciscan musician is making his way down to Culver City on Saturday to play at the Cinema Bar.
Harp grew up on the East Coast in the Washington, D.C., area, where he worked for an independent record store, among other odd jobs. This gave him the musical resources and inspiration he needed to pick up a guitar.
“That’s where I got my vinyl addiction. That exposed me to a lot of new music, and I heard a lot of things I had never thought to listen to before,” Harp said.
Listening to old jazz, folk and R&B, Harp became rooted in American classics and migrated to California to get serious about his music career.
“Ever since the Gold Rush, that’s where everyone comes for something new,” Harp said. “I just wanted to get a fresh start musically.”
There in San Francisco he met Anton Patzner of Judgement Day, who played violin on both of Harp’s albums, “Good Misery” in 2005 and “Collecting Dust” in 2009, and Patzner sometimes accompanies Harp live. They became good friends and musical allies.
“Bob found me playing on the street in Berkeley. We hit it off, and he asked me to play on his album. On some songs we would just improvise, and on others we already had ideas for a melody, so we’d play that. On some songs we doubled up the violin to make a more epic, orchestral sound,” Patzner said.
Another addition to “Collecting Dust” was Jordan Feinstein of The RituaL, who played keyboards and some bass, as well as helped produce a song on the album. Feinstein notes Harp’s capability to combine the traditional sound with his own.
“Bob plays very authentic American roots music. It’s not very commercial ““ Bob’s is serious music. He’s got all those influences in there, like Bob Dylan and Neil Young, but it still very much sounds like him,” Feinstein said.
Harp himself realizes his ability to elude preconceived notions of genre and set up his own sound within an established structure.
“I just write music I like. It’s hard to say that I fit into any genre. I like things with a little groove and soulful vibes, so a lot of my music is slow and melancholic. It’s a sort of timeless, moody folk music,” Harp said.
Much of Harp’s originality comes from lyrics, which are carefully interwoven into his music to create a tapestry of a story, feeling or scene.
“Some of them are story songs. I’d say heartache is a big part of me, writing about what I’ve lost, both women and other things. I spend a lot of time driving, so there’s definitely a road element to my music, too. Setting is important,” Harp said.
Other people take note of Harp’s gift with words. Patzner said his favorite thing about Harp’s music is his lyrics.
“He’s a writer and a poet. He’s got some interesting things to say. His vocals also have a lot of vulnerability in them that you don’t get to hear in pop music today. I see Bob as a lone cowboy type of guy,” Patzner said.
While he may be the lone cowboy type, Harp has layered his most recent album, “Collecting Dust,” with more instrumentation and variety, with the help of musicians like Patzner and Feinstein. Harp said after four years of experience since his last record, he had more of a vision of what he wanted.
When he is not in the studio, Harp is either listening to, writing or playing music. One of his first shows was with a friend’s band opening for Journey, playing in front of thousands of people. While his concerts since then have been a bit more intimate, he still enjoys playing live. As far as future plans, the road’s wide open.
“It’s hard to say. It’s a tough road, you just gotta stick with it. I’m definitely always writing and playing guitar,” Harp said. “Sometimes I like to change my songs around a little bit when I’m playing them to try and keep things fresh.”
Although Harp is rooted in timeless tradition, he revitalizes his music with new ideas. In addition to developing his sound, Harp is expanding his territory. This is his second show in Los Angeles.
“There’s a big scene down there for music, and there are a lot of outlets for it so that’s why I’d like to make it to L.A. more often. Hopefully this will be the second show of many more to come down there,” Harp said.

domain: entertainment


The Media department is considered one of the first departments of Al- Farahidi University, Media College now. It is one of fourteen departments in this University that offers scientific and practical programs. It has been one of the colleges approved by the Ministry of Higher education and scientific research.
That is why our graduates receive bachelor’s degree in broadcast journalism and television, journalism, and public relations. We have one of the most distinguished professors with professional experience who are able to keep up with the scientific spirit of the age of technical progress in all fields. The Media Collage topped all the Iraqi Universities -Public and Private- in scientific content exam for the years, 2017, 2018, and 2019 receiving a clear 100%.
The Media collage was founded based on the needs and requirements of the local and Arabic media market competencies trained that can keep up with the ever evolving technical, scientific, and artistic fields of communication. We seek to see the evolution of visual mentality of the college in accordance with the certainty, rather than dreams of the vision and that was necessary to allow our students to be able to integrate in the real world flawlessly.
The overall design and implementation of training programs is based on a deliberate and inspired scientific basis of the reality and the need to teach the foundations of media work, which seeks its way to complete the lack of information to address the problems of the media, in order to help future leaders to meet the challenges they face.
We also organize specialized workshops and establish forums for the exchange of opinion and ideas with the rest of the faculties and departments of media nationally and internationally.
I invite you to learn about the department up close and look at all the information through this guide.
Assist. Prof. Dr. Muayed Kasim Jerjis
Dean of College
The College’s vision stems from its belief that it is one of the most important sources that provide media institutions with graduates capable of practicing media work professionally, objectively and morally. Hence, the comprehensive vision should be effective in the graduation of students with scientific and professional competence, enabling them to keep abreast of rapid media developments.
The College aims at graduating qualified media professionals in order to meet the needs of the public and private sectors, and excellence at the local and regional levels in the media field, relying on the principles of professional media work.
The College seeks to provide an educational and training environment capable of increasing the talents of students and preparing them for the requirements of the local and Arab markets in line with the spirit of the times and rapid technological developments.
The college aims to prepare graduates with a high degree of competence in the field of media and other fields, and to qualify them scientifically, technologically and professionally. It also provides students with the knowledge and skills necessary to practice the media work, based on the human heritage and Arab culture, to meet the needs of the labor market and the requirements of competition locally, regionally and internationally.
The College was established with the Department of Media at Al-Farahidi University College in 2013, under the order of Ministry of Higher Education and Scientific Research No. 2678 on 26/5 -2013. The courses are taught according to high-level plans prepared by distinguished universities in Iraq and the world. It balances between the practical and the theoretical aspects, and focuses on materials related to media theories and the arts of media. The College currently has (530) students. The number of faculty staff members are thirteen of different scientific ranks and disciplines.
The College aims to prepare a professional media personality who can communicate with the community and develop it by means of personal communication or through various media, starting with forming a community awareness and ending citizens’ quest to participate in development plans to build a modern society.
After getting a bachelor’s degree, the College hopes to be aware of the ethics of journalism and its seriousness in influencing public opinion. The College also seeks to achieve the following goals:
- Upgrading the scientific level.
- Developing the faculty members.
- Pursuing graduate studies.
- Providing a scientific atmosphere that is equal and fair to all college students.
- Enabling students to acquire the necessary expertise to practice media work.
- Training students through communication and information technologies.
- Helping students get jobs in the job market of media.
- Developing courses in communication.
- Developing practical training systems for students.
- Supporting student’s activities.
- Developing students’ personal skills.
- Achieving justice among college students in the educational process.
- Conducting an annual festival for the college in which the students participate in presentation.
- Developing the radio and television studio according to the highest standards and technologies.
Students are trained in all radio and television arts through the training studio that was established for this purpose.
Students are also trained in the production of journalistic forms, both in writing and directing, as practical training on work systems within press institutions, and the student is evaluated on the basis of his production and not on the basis of the written examination only, and under the supervision of practical training professors.
The college maintains close relations with most of the official and private media institutions in Iraq, with the aim of deepening the scientific field, providing practical training opportunities for students, and strengthening the service of the Iraqi community in general.
The college is linked to many cultural agreements with Arab universities and international organizations, and UNESCO and the International Development Program are among the international organizations that support the activities of the college and specialized workshops, and modernize devices and laboratories.
- United Nations Educational, Scientific and Cultural Organization، (UNESCO).
- Monte Carlo Doualiya، (MCD).
- BBC Arabic News Channel Network.
- Iraqi Media Network (IMN).
The students of the fourth stage (the final stage) are assigned to prepare a practical project, depending on what they have been trained in in the past years.
The student presents a clear plan (Script) for the graduation project, which includes the general idea of the topic, and the foundations and steps of production.
The student is discussed by the academic committee in the college, and after obtaining approval, he is asked to prepare a scenario that includes the details of the work to be submitted to the supervising professor, who in turn facilitates the students ’task, especially in projects that require approval for their completion.
Graduation projects consist of (producing a television program – for radio and television journalism students, issuing a newspaper – for journalism students, and producing a series of television advertisements – for public relations students
- 1. Launching a website and social networking pages for the university and the College of Media, for the benefit of university students in general and college students in particular, to familiarize students with everything that interests them, and to see news, directions and interaction.
- 2. Organizing an annual festival for student creativity in the field of media.
Plans for the Future:
- 1. Issuing a newspaper for the college that students edit, produce and distribute.
- 2. Issuing a college magazine that is part of the training program for students.
- 3. Launching a university-specific radio station, administered by college students, in writing, presentation and direction.
- 4. Concluding cooperation agreements in the field of training with local and international media organizations.
Dr. Mouayed Kasim JarjesDean


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Panda Tip: Length can vary immensely, but probably 300-1500 words or more, depending on the nature of your research.This is one of the most important sections of your research proposal.
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Some sections also include suggested content templates to be filled in, but given the nature of a research proposal as academic, this can unfortunately only be limited.This is the section that requires the most preliminary research: make sure you spend some time in an academic library and using search engines for relevant academic papers before presenting this.You do not need to discuss every work in your area, but you need to present a competent outline, and, especially if this is a proposal for doctoral research, you need to be sure that no-one else has already done the same project.Example Naturally, the scope of this project is limited. It also will help you to know the scale of your work in the preliminary stages of planning, and help you to have realistic expectations of yourself.Panda Tip: The reference list should always begin on a new page.A good way of presenting a literature review coherently is in the form of a narrative, which can either be chronological or thematic.Example There has been a Panda Tip: Length varies, but probably similar length to literature review.This is where you sell your research proposal to the reader.You need to explain, clearly and simply, how your research will complement the field you have just described in your literature review: what you will add, how it fills an existing gap, why the academic world would benefit from your research, etc.50-1000 words depending on the nature of your research. Which ones hold the books and documents you will need? Panda Tip: Probably less than 200 words, unless you have a very detailed plan already in mind. It is surprisingly important, as it shows that you can recognise the limited scale of your work.This is where you explain how and where you plan to carry out your research. Note: this is like a preliminary contents page, but it does not need to be very specific, and can suggest sections rather than chapters at this stage, but the academics reading your proposal will be impressed to know that you have some idea how you may wish to present your work, and that you have some way in mind of translating your research to paper. Every project needs distinct limiting factors in order to be manageable. This section is optional, but may be helpful to show your potential supervisors that you are being realistic and recognise that your project has limits.


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Still working on the rest of it; but,...
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Thank you, sir.
Yeah, I have usually got a more balanced lighting setup; but I deleted a lot of the lights in scene and mostly the blue ones
Oh yeah. lol.
Closing in on finishing the conference room area. Lots of cleanup still needed when it's done; but, gettin there.
..a few more shots from today's progress so far
Thank you. ;)
Trying to get some more done this morning. :)
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thoroughly happy with them; but,...
Yeah, I did those to plan; but, doubt that's what the plan intended. So they're gonna get rounded off lol.
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Separate names with a comma.


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Playtech announced its latest partnership deal with Flutter Entertainment, linking to a live casino studio for the Sky Betting and Gaming brand, Sky Vegas. The new specialized studio will be located at Playtech Live Studios in Riga. On the first launch, Sky Vegas Live Casino will include live blackjack and roulette from Playtech, which allow a limitless number of players to enjoy the content.
Gaming1 is a leading sports betting and gambling provider who won four awards at the Malta iGaming Excellence Awards, which is a record-breaking achievement. The brand awards for: Malta’s Best Gaming Company of the Year, Malta’s Best Industry Achiever, Malta’s Best Omni-channel Provider and Malta’s Best Gaming Mark of Excellence.
Playtech is delighted to announce the roll-out of its online casino in New Jersey via BetMGM’s gaming network for the first time. According to the deal, the supplier will deliver its own leading iGaming casino software to the venue.
Shimon Akad, Playtech COO, said: “We are delighted to launch with leading operator BetMGM in New Jersey.