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Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.
19 Related JoVE Articles!
Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant
Institutions: Baylor College of Medicine.
Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105
/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture – total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106
cells that is reduced by >90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.
Immunology, Issue 51, T cells, immunotherapy, viral infections, nucleofection, plasmids, G-Rex culture device
Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray
Institutions: Akonni Biosystems, Inc..
Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis
(MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.
Immunology, Issue 86, MDR-TB, gel element microarray, closed amplicon, drug resistance, rifampin, isoniazid, streptomycin, ethambutol
Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
Institutions: Washington University School of Medicine, Washington University School of Medicine, Washington University School of Medicine.
As DNA sequencing technology has markedly advanced in recent years2
, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3
. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5
. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8
. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9
and a novel software package1
for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (https://www.ibridgenetwork.org/wustl/splinter). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.
Genetics, Issue 64, Genomics, Cancer Biology, Bioinformatics, Pooled DNA sequencing, SPLINTER, rare genetic variants, genetic screening, phenotype, high throughput, computational analysis, DNA, PCR, primers
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus
Institutions: UT Southwestern Medical Center, UT Southwestern Medical Center.
In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2
. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4
. γHV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo
, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in γHV68 lytic replication both in vivo
and ex vivo
. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during γHV68 acute infection in vivo
. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during γHV68 lytic replication ex vivo
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo
and ex vivo
Recently, we have discovered that γHV68 usurps an innate immune signaling pathway to promote viral lytic replication5
. Specifically, γHV68 de novo infection activates the immune kinase IKKβ and activated IKKβ phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, γHV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.
Immunology, Issue 56, herpesvirus, gamma herpesvirus 68, γHV68, signaling pathways, host-virus interaction, viral lytic replication
Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
Institutions: Tufts University.
Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRα, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4
), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80°C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.
Cellular Biology, issue 10, retrovirus, transfection, 293T cells
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
Perform plating procedures without contaminating media.
Isolate single bacterial colonies by the streak-plating method.
Use pour-plating and spread-plating methods to determine the concentration of bacteria.
Perform soft agar overlays when working with phage.
Transfer bacterial cells from one plate to another using the replica-plating procedure.
Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
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
Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus
Institutions: Baylor College of Medicine , Baylor College of Medicine , University of Texas M.D. Anderson Cancer Center, Baylor College of Medicine , Baylor College of Medicine .
Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4
We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8
However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells.
In an effort to better mimic the in vivo
priming conditions of naïve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9
At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10
At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2.
CB mononuclear cells we are able to generate upwards of 150 x 106
virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11
These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.
Immunology, Issue 63, Cytotoxic T Lymphocytes (CTL), virus, stem cell transplantation, cord blood, naïve T cells, medicine
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
Institutions: China Medical University , University of Arkansas for Medical Sciences , University of Arkansas for Medical Sciences .
A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1
). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1
. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2
). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2
in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105
cells per well typically used in ELISPOT assays1,3
, 1,000 T-cell clone cells in the presence of 1 x 105
autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4
Immunology, Issue 61, Interferon-γ enzyme-linked immunospot assay, T-cell, epitope, human papillomavirus
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
Collection and Extraction of Saliva DNA for Next Generation Sequencing
Institutions: The Research Institute at Nationwide Children's Hospital, The Ohio State University, The Ohio State University.
The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.
Medicine, Issue 90,
DNA collection, saliva, DNA extraction, Next generation sequencing, DNA purification, DNA
Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining
Institutions: CNRS UMR 5534, Université de Lyon 1, LabEX DEVweCAN, CNRS UPR 3296, CNRS UMR 5286.
Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ
in animal models. We describe a DNA-fluorescent in situ
hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 µm tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.
Neuroscience, Issue 83, Life Sciences (General), Virology, Herpes Simplex Virus (HSV), Latency, In situ hybridization, Nuclear organization, Gene expression, Microscopy
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3
. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells
Transformation of Plasmid DNA into E. coli Using the Heat Shock Method
Institutions: University of California, Irvine (UCI).
Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42°C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37°C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.
Issue 6, Basic Protocols, DNA, transformation, plasmid, cloning
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Electroporation of Mycobacteria
Institutions: Barts and the London School of Medicine and Dentistry, Barts and the London School of Medicine and Dentistry.
High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.
Microbiology, Issue 15, Springer Protocols, Mycobacteria, Electroporation, Bacterial Transformation, Transformation Efficiency, Bacteria, Tuberculosis, M. Smegmatis, Springer Protocols

domain: biology


text:
Ellsworth Duncan has an interesting family history. He’s heard all about it, even though he lives across the country from the square where his father grew up.
Then comes the invitation to return to the square to hunt down the last treasure of his great-great-great… grandfather John Matthew Smith. The family is in trouble, and only a child can discover the treasure John Matthew has hidden in a house on the square.
Ellsworth returns to find lots more secrets hidden in the square. The riddle requires the whole family to work together, even the cousins who weren’t talking, Ellsworth’s reclusive grandfather – but will they put aside their differences to rediscover their common roots?
Interesting and mysterious, The Last Treasure by Janet S. Anderson will hold your attention to the end.
Recommended for ages 8 and up.
Side note: The book opens with the ghost of John Matthew haunting the square and Ellsworth & his distant cousin Jessica have strange dreams. John Matthew’s ghost doesn’t factor into the rest of the story, and I’m not bothered by the strange dreams which cause the children to take action – it happens in real life too.
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domain: literature


text:
Contact temperature meter devices including Temperature meters with probes
- Cole-Parmer offers Digital thermometers (digital indicators) to quickly verify temperatures of foods, liquids, baths, air/gas, incubators, refrigerators and freezers.
- Omega’s Handheld Instruments for Temperature Measurement cover a wide range of sizes and types.
- PCE Instruments offers temperature meter products that include handheld Infrared Thermometers in addition to contact temperature sensors.
- Rees Scientific provides automated continuous monitoring to a limitless number of applications from Pharmaceutical to Blood Banking, Laboratory Animal Research to Biotech and Hospitals.

domain: chemistry


text:
IUML president rejects extremism charges
Malappuram: Rejecting charges that the party had links to extremist elements, IUML Kerala unit chief Panakkad Hyderali Shihab Thangal on Saturday the party had been in the forefront of the campaign against terrorism and extremism.
The campaign that extremist elements `are infiltrating into IUML is a conspiracy` hatched by the party`s rivals, he said at a conference of the party`s Malappuram District unit.
Thangal said the party had to "face criticism" for its strong stand against terrorism and extremism, but did not elaborate.
His comments come in the backdrop of the US diplomatic cables leaked by WikiLeaks causing embarassment to the IUML by suggesting divisions in the party over its approach towards extremist outfit National Development Front (NDF).
The whistleblower site revealed that a cable allegedly sent to Washington by the US consulate in Chennai a few years ago quoted IUML leader MK Muneer as having said that party leaders like PK Kunhalikutty "shielded" NDF activists to secure "selfish" interests.
For more news in Malayalam, click www.zeemalayalam.com
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- 1st Test, Day 1 – Sri Lanka vs Australia - As it happened...

domain: news


text:
Back in December 2021, a slate of upcoming European motorcycling events had been canceled due to the latest COVID-19 surge. With both recorded cases and hospitalizations back on the rise in various countries, event organizers saw the writing on the wall and opted to postpone many events until 2023 in hopes that the situation will improve over the coming year. However, COVID Cancellations for European Moto Events still Continue Through 2023 AMERiders explains.
Germany’s Elephant Rally (or Elefantentreffen), IMOT, Augustusburg Winter Meeting, and Supercross Dortmund all announced cancellations for 2022 before the new year had the chance to show its face. Now that it’s the middle of January 2021, how are things looking?
Out of an abundance of caution, additional motorcycle events have announced cancellations for 2022. Motorbike World Lake Constance (Motorradwelt Bodensee), which was originally scheduled to take place in Germany from January 29 through 31, 2022, is canceled. It has instead been rescheduled for January 27 through 29, 2023.
Motorbikes Dortmund (Motorräder Dortmund) was originally planned to take place from March 3 through 6, 2022. Like other events listed here, it has now been canceled for the remainder of the year. Organizers have cautiously booked March 2 through 5, 2023 as the next dates for this motorcycle trade fair to take place.
Motorbike Fair Leipzig (Motorrad Messe Leipzig) has also canceled its annual fair, originally scheduled to take place in February 2022. Organizers say they tentatively plan to hold the next event in February 2023. They have not yet given exact dates at this time, but there’s more than a year for them to make that announcement.
Currently, motorcycle events scheduled toward the beginning of 2022 have mostly been canceled, with plans to resume activities in 2023. However, Intermot organizers still plan to hold Intermot 2022 in Cologne later this year, from October 4 through 9. As there are many months between now and then, the situation could of course change. We’ll be sure to keep you up to date if plans change between now and then.
~And as always…
~Live Free Ride Hard~
COVID Cancellations for European Moto Events Continue Through 2023 AMERiders explains.
Don’t forget to come visit us for all your Motorcycle Apparel needs.
And as always don’t forget to send us your stories, pictures, and events for posting to GALLERY.AMERIDERS @ GMAIL.COM and we will post them for you. The more people that know about your event the better and we are offering free advertising. We would also love to hear about your rides and love to see those bikes so send those stories and pictures.
Like what you just read? Share it on social media ( Facebook, Twitter, Pinterest, Google+, and Instagram) with others and let them get the information and benefit from it as well.


O-ring rubber seal gasket used for concrete pump pipeline. O-Rings are used in sanitary pipeline systems for the Food, Dairy, Beverage, Pharmaceutical and Bio-Tech industries. Used in concrete pump truck, concrete pump pipeline and pipeline connection seal in construction work equipment. Characteristics of Silicone Rubber O-Rings, …
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An O-ring seal is meant to plug the opening between the two connections without extruding through the opening, as seen in Figure 2. An extruded O-ring will hold the seal while a machine is running, but if the machine is turned off for repair or inspection, the O-ring will relax, causing it to crack or flatten out.
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China Factory Produces Black NBR/HNBR/EPDM/Silicone Rubber Product Framework Oil Seal/ Sealing Ring. FOB Price: US $ 1.64 / Piece. Min. Order: 500 Pieces. Material: Nitrile Rubber. Application: Machinery, Industrial Component, Electronic Product. Effect: Wiper Seal. Cross-Section Shape: O-Rings.
EZ-STIK is an a ll weather preformed joint sealant made of Butyl rubber material in flexible rope form and is used to seal all types of concrete structures. It meets and/or exceeds all requirements of AASHTO M-198 and ASTM C 990 section 6.2.1, Butyl rubber sealants. Compare this to many competitive products which only meet ASTM C 990 section 6.1.1.
EZ-STIK is an a ll weather preformed joint sealant made of Butyl rubber material in flexible rope form and is used to seal all types of concrete structures. It meets and/or exceeds all requirements of AASHTO M-198 and ASTM C 990 section 6.2.1, Butyl rubber …
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Our Rubber Ring Joint reinforced concrete pipes are suitable for both irrigation and stormwater use. Available in Class 2, 3 & 4, our Flush Joint pipes are AS4058, ISO 9001:2015. These pipes use a unique rubber ring system to create a strong, lasting seal that you can count on.Civilmart are also able to custom-manufacture Flush Joint pipes to
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Friday night an FBI agent confirmed to CBS 5 a search warrant was being served on the Coolidge home of a man taken into custody that afternoon, suspected of bombing a federal office with an IED 20 miles away.
They made entry just after 9 p.m. after securing the home and making sure it was safe to go inside.
The explosion violently shook the Social Security building on Marshall in downtown Casa Grande, but it didn't injure anyone Friday morning. The blast went off before the office opened for business.
Shortly after that bombing, the Feds descended on a neighborhood in Coolidge.
Residents of the Martin Valley development said Coolidge Police blocked off their neighborhood and kept them out of their homes all day. They were finally let back in to the neighborhood around 7 p.m. Friday.
Neighbors tell CBS 5 and property records confirm the home crawling with investigators, belongs to 47-year-old Abdullatif Aldosary.
"He's pretty quiet. He don't really come out of the house," said neighbor Miguel Pascual.
It was a scene other neighbors tell CBS 5 they had witnessed before. They asked not to be identified.
Court records show Aldosary served prison time in Safford four years ago for aggravated harassment against his then employer Hunter Contracting in Gilbert, and he is currently involved in an assault and disorderly conduct case in Casa Grande.
"I was pretty shocked about it. Around this little town, who knows to expect something like this," said Pascual.
The FBI said they're investigating the explosion in Casa Grande and are "utilizing all necessary resources" including: investigative personnel, the Evidence Response Team, Special Agent Bomb Technicians and the Hazardous Materials Response Team.
The blast was caused by a device that detonated at the rear of the building, according to Agustin Avalos of the city of Casa Grande.
It happened around 8:15 a.m. in a building that houses several other downtown businesses. All of them were evacuated, Avalos said.
"It was very scary. I'm still shaking inside. I never heard anything like that," said Alicia Perez, who works in the building.
No injuries were reported and the type of device had not been determined, Avalos said. But a source close to the investigation says it appears an accelerant and some type of plastic object was used in the blast.
"I don't know what would go through somebody's mind to do that," said Patty Rodriguez who works nearby.
Witnesses inside at the time said the explosion shook the building violently.
"I was afraid to go into the back because it was so close," said Perez.
The FBI is heading up the investigation, but officials from the Bureau of Alcohol, Tobacco, Firearms and Explosives, Homeland Security and Casa Grande Police Department are also investigating, Avalos said.
Aerial news footage over the bomb scene showed extensive damage to a doorway in the back of the building and to the side of an car near the doorway.
Casa Grande is about 50 miles south of Phoenix.
Stay with cbs5az.com and CBS 5 News as this story develops.
Copyright 2012 CBS 5(Meredith Corporation). All rights reserved.
Friday, April 18 2014 8:20 PM EDT2014-04-19 00:20:57 GMT
A Tigard woman found guilty of murder for the death of her 4-year-old son was sentenced to life in prison with the possibility of parole after 25 years. A jury found Jessica Dutro-Boggess, 25, guiltyMore >
A jury found Jessica Dutro-Boggess, 25, guilty on charges of murder, murder by abuse and assault earlier this month.More >
Friday, April 18 2014 10:12 PM EDT2014-04-19 02:12:08 GMT
An investigation into a two Nike employees accused of selling rare Nike shoes for their own financial gain led to a Florida man's arrest this week. A Washington County Sheriff's Office detective's affidavitMore >
An investigation into a Nike employee accused of stealing rare Nike shoes from the company's world headquarters and then selling them for his own financial gain led to the arrest of a Florida man.More >
Wednesday, April 16 2014 10:51 PM EDT2014-04-17 02:51:53 GMT
A Portland police officer was wounded and his K-9 partner is missing following a shootout with a suspect in southwest Portland.The officer survived the shooting and he's in "very good spirits" while recoveringMore >
An officer was shot in the legs and his K-9 partner was killed during a confrontation with a burglary suspect in Portland. More >
Friday, April 18 2014 10:05 PM EDT2014-04-19 02:05:06 GMT
Police said a lengthy multi-agency investigation led to a traffic stop and the discovery of methamphetamine hidden in a car, as well as a meth lab at a Salem home. Police said a 2002 Subaru Impreza drivenMore >
Police said a lengthy multi-agency investigation led to a traffic stop and the discovery of methamphetamine hidden in a car, as well as a meth lab at a Salem home.More >
Friday, April 18 2014 4:42 PM EDT2014-04-18 20:42:29 GMT
An 18-year-old was killed and a boy was critically injured in a car crash in Independence. Police were called to the scene on Stryker Road at around 5 p.m. Thursday. Investigators said a Dodge DurangoMore >
An 18-year-old was killed and a boy was critically injured in a car crash in Independence.More >
Friday, April 18 2014 12:55 PM EDT2014-04-18 16:55:01 GMT
A priest known as Father Isidore who was associated with the St. Anne Orthodox Church in Corvallis has been arrested on child pornography charges. Albany police said Stanley Brittain, aka Father Isidore,More >
Albany police detectives say they found evidence of child porn when they raided a home yesterday morning.More >
Friday, April 18 2014 3:24 PM EDT2014-04-18 19:24:49 GMT
A theft suspect is accused of forging TriMet passes, possibly more than a thousand of them. Transit police served a search warrant at the Central City Concern's Estate Motel, 225 N.W. Couch, on WednesdayMore >
Efren Escarsega Jr., 39, was arrested and charged with multiple counts of first-degree theft, theft by receiving and conspiracy to commit forgery.More >
Saturday, April 19 2014 1:41 AM EDT2014-04-19 05:41:59 GMT
Four people were taken to the hospital after police said a car ran a red light and slammed into the outdoor eating area of a McDonald's restaurant in Longview. It happened a little before 3 p.m. More >
Four people were taken to the hospital after police said a car ran a red light and slammed into the outdoor eating area of a McDonald's restaurant in Longview.More >
Friday, April 18 2014 8:39 PM EDT2014-04-19 00:39:01 GMT
Two teens were arrested Wednesday, accused of trying to steal pot from another person. One of the teens is a well known high school football player. According to court documents, Billy Nelson, 16, andMore >
Two teens were arrested Wednesday, accused of trying to steal pot from another person. One of the teens is a well-known high school football player.More >


text:
Police Charge 2 Women For Assaulting A Transgender Woman At A Bar In North Carolina!
Police in North Carolina say two women sexually assaulted a transgender woman at a Raleigh bar. 38-year-old Amber Harrell and 31-year-old Jessica Fowler are charged with second-degree kidnapping and sexual battery. The woman told Raleigh police she was inside the bar’s bathroom in December when Harrell and Fowler started verbally abusing her, exposed themselves and started attacking her. Posted By Persist

domain: law


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Electromagnetic vibrating feeder is a kind of equipment that implements flow automation.
Bucket elevator is a series of hoppers that are uniformly fixed on endless traction components.
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text:
This fall is all about gorgeous patterns in rich shades of gold, red and more.Make your nails look as luxe as your jewelry by choose a few fall shades and add embellishment for an elegant manicure. There was no shortage of creativity backstage at the nea
This Pretty winter nails art design inspirations 15 image is part from 80 Pretty Winter Nails Art Design Inspirations gallery and article, click read it bellow to see high resolutions quality image and another awesome image ideas.
You've been cleaning your oven wrong your entire life. WaterSpray bottleBaking sodaA ragVinegarA small bowl This is how you do it: The oven: Remove the oven racks Mix a couple spoons of baking soda with some water in the bowl. The goal is.

domain: artistic


Question I want to use some blender functionality to close these holes in mesh objects and remove the vertices. I have alot hence i need to automate the process. Im scripting my way about through the Python API. I cant seem to find any function in Blender I can call to do this. I'm not smart enough to develop a "hole detection"-algorithm myself. Does anyone know how to solve this or could point me in the right direction, then I would be pleased :)
Why? (Background) Because they are small non-important details, that have a lot of triangles in them. I use the model in an industrial 3d application, so they have to be optimized from their original format to run on all kinds of hardware. Sometimes my scenes(unity) have several hundred or even thousands of models displayed at the same time.


text:
Get flash to fully experience Pearltrees
What do you want to watch? The answer used to depend on limits -- what day it was, what time it was, what channels you got. A handy little thing called TV Guide laid it all out.
Academy Awards History (By Decade and Year) A full and complete history and commentary on the Academy Awards from its early beginnings to the present is available, by decade and by individual year.

domain: library


text:
The Rise of Socialism in New Zealand
1890 to 1916
Why Socialism in New Zealand?
The external essay standard AS29134 asks you to explain the significance
of an event to New Zealand.
A major historical force was socialism which demanded equality and
fairness and a redistribution of wealth.
Emerging from the social deprivation of the Industrial Revolution
socialism called for equal treatment for all, for a sharing of wealth and for
a welfare net.
New Zealand had a growing reputation for egalitarianism.
In New Zealand the forces for change were also seeking reform.
The key to this is what were the forces of change and how did each
country deal with these demands for change?
New Zealand in 1890
New Zealand was a British colony.
It was mainly British, the majority being English
although a significant number were now New
All of its systems of Government and Justice
originated in Britain.
In the 1896 census there were only 42,000
Most settlers had migrated to New Zealand to
escape the social and economic restrictions of
the ‘old country’.
This combined with the work ethics of
‘Mateship’ led to a desire for egalitarianism
and social justice.
The Migrants: Push and Pull
Early attempts at migration had sought to
bring a particular type of migrants.
In 1850 the Wakefield schemes targetted
specific groups within Britain.
They had sought a ‘slice’ of British society,
including all social classes.
This did not happen and most of the
migrants were working or middle class.
Many had some level of education.
Many of them wanted better working
conditions than they had left behind.
Most wanted their children to have more
opportunities than they had had.
Samuel Parnell & the 40 hour week.
On the voyage form GB Parnell, a builder was asked to
build a shop. The employer offered the same conditions
as applied in Britain. (Start at 6am, a 12 hour and a 6
'There are,' he argued, 'twenty-four hours per day given
us; eight of these should be for work, eight for sleep,
and the remaining eight for recreation and in which for
men to do what little things they want for themselves. I
am ready to start to-morrow morning at eight o'clock,
but it must be on these terms or none at all.‘
'You know Mr. Parnell,' Hunter persisted, 'that in
London the bell rang at six o'clock, and if a man was not
there ready to turn to he lost a quarter of a day.' 'We're
not in London', replied Parnell.
Other employers tried to impose the traditional long
hours, but Parnell met incoming ships, talked to the
workmen and enlisted their support.
The eight hour working day thus became established in
the Wellington settlement and around the country.
Since the 1890’s labour day was celebrated as a holiday.
Mateship in the Bush Frontier
Most early migrants wanted land.
In GB only the wealthy owned land.
In NZ owning land was seen as the path to
This meant purchasing and breaking in land for
farming or working in the ‘Bush’.
The work was dangerous and male orientated.
In the “Bush frontier’ men often lived and
worked in ‘gangs’. Some stayed together for
Neighbours often assisted each other in large
The frontier developed a particular type of
male-centric society with values based upon
hard work, honesty and the equal treatment of
Dishonesty or shirking were anathema to a good
The Long Depression 1878-1892
In the 1860’s NZ had prospered.
Cheap land and high farm prices saw many
farmers borrow heavily on future earnings.
In the 1870’s wool prices fell, gold began to
become scarcer and the rabbit plague
destroyed many farms in the South.
The Bank of Glasgow had lent heavily to both
NZ public and private investors.
In 1878 the Bank of Glasgow collapsed. Other
banks also suffered.
Its international investments were recalled
creating a worldwide depression.
The Government was heavily in debt which
did not encourage business leaders.
In 1882 the Australian market for NZ wheat
Economic downturn led to Political instability.
The downward spiral (Recession become a
for wool fall. This signals
a general decline in
As suppliers incomes
fall they also cut costs
employing fewer workers
and cutting back on spending
Public & Private Retrenchments
continue. Business confidence is low
so little is invested in new ventures –
people prefer to keep their money.
fall, they cut costs
by employing fewer
labourers and cutting
back on spending.
Lenders (banks) demand
loans be repaid.
Reduced tax revenue
forces the Government
to reduce spending,
especially in employment
and supplies. (Retrenchment)
Negative growth in 3
consecutive quarters is an
Conservative Government, Conservative policies.
Governments tend to reflect the type of people
who elect them.
While the franchise was limited to the wealthy
the Government and its policies favoured them.
Since most of the voters were farmers of
manufacturers, laws tended to support them.
From 1860 to 1891 the Government was
conservative in its policies and its legislation.
Politics however began to change, the franchise
was expanded and by the 1890’s more
politicians owed their support to urban workers
and later to the female vote.
After the 1890 Maritime Strike, Labour Unions
began to look towards Parliament to create
The Liberals were the first to benefit from this
The Franchise(s) and Electoral Equality
1840 New Zealand was administered by the Governor
and a chosen cabinet.
1852 – First Election: Voting for Males aged 21 who own
or rent land (£50 Rural £10 Urban). Maori
disenfranchised through communal ownership.
1860 – Franchise extended to (Gold)Miners.
1867 - 4 Maori Seats established. A ‘short term’
1879 - Franchise broadened – land ownership
requirement abolished. All males over 21 may vote.
1889 – Representation Act establishes a country quota.
(Smaller constituencies). Favoured farmers.
– Ends Plural voting.
1893 – Women receive the Vote.
– Effective Universal Suffrage.
– Women would not receive the vote in the US and GB
until after WWI.
Trade Unions: defending workers
The idea of Unions was exported
Unions began in the 1850’s, but they
were few and far between.
Governments were openly hostile to
From 1876 the Trades Council in
Auckland was able to co-ordinate
Union activities in areas of common
Most laws actually favoured the
employer, until the 1878 Trade
Union Act they had no legal standing.
The Long depression made it harder
for workers as wages and conditions
were driven down.
The ‘Sin of Cheapness’
As the number of factories grew so did
demand for labour and despite lower
rates paid to women, many saw more
opportunities in this work than in
In 1889 Dunedin Reverend Waddell
preached his sermon on the ‘Sin of
Cheapness’. (Think ‘The Warehouse’)
Hours were long and pay was usually
low. (Piece)Work was often taken
home to fulfill quotas.
Few factories complied to any
standards and were very dangerous to
work in. (No Social Welfare)
It was amongst women that the first
Unions began to flourish.
The 1890 Maritime Strike
The Wharves and Shipping were the
lifeblood of New Zealand’s trade.
By 1890 export of meat, wool and dairy
products were becoming our largest export
The Unions wanted to improve their wages
They chose to support a strike in Australia
by closing our wharves as well.
There was little public support and the
Government took the upper hand.
Scabs were employed to break the strike.
A strike of several weeks was broken and
the Unions were left severely weakened.
After losing the 1890 Maritime Strike, Unions chose to
support the Liberals against the Conservatives.
The Liberal’s wanted a strong central Government.
Government should treat everyone equally.
To care for the Disadvantaged.
Where Monopolies existed they should be able to
They wanted to encourage people to stay in the
country (rural areas) by breaking up the Big Estates.
The previous Conservative Governments had
supported Wealth and Privilege over Democracy.
They wanted Labour Reform and created a Bureau of
Industry (the Labour Department)
A “Social Laboratory”
Under the Liberal Government 1893-8 New
Zealand earned a reputation as a “Social
Laws passed during this period seemed aimed at
creating a “better world”.
Equality was created through Universal Suffrage.
Workers conditions were protected in factories
and shops (Acts).
Collective bargaining was allowed and created a
settled Industrial climate.
Social Welfare began with the Pension.
Many felt New Zealand was leading the world in
Social, Economic and Political equality.
This became a point of pride for many.
A progressive NZ had created a better world for
the working man.
The Liberals: their Acts of Parliament
1891 Land and Income Tax Assessment Act: taxed land especially absentee
1892 Land for Settlements Act: Set aside £50K to buy large estates.
1892 Lease in Perpetuity Act: Allowed existing Leasers a 999 year lease on their
1892 Compulsory Purchase: Allowed government to buy land
1893 Local Option Act: Allowed Local voters to decide on Prohibition in their
1893 Advances to Settlers Act: Allowed farmers to lend cash to improve their
1893 Government Electoral Bill: gave Women the Vote
1894 Bank of New Zealand Guarantee Act: Gave the Government power to
appoint its President and Auditor in return for a Guarantee that saved it.
1894 Factory Act: Required factories be registered and inspected, setting minimum
1894 Shop and Shop Assistants Act: Regulated work and conditions in shops.
1894 Industrial Arbitration and Conciliation Act: Set up to settle industrial
disputes, its decisions were binding on both parties.
1898 Old Age Pension: Paid a pension to those of ‘good’ character.
Political Equality = Social Change: Suffrage
The campaign began in the late 1880’s when attempts
at Temperance failed.
The WCTU led by Kate Sheppard sought to effect social
change through the franchise.
Alcohol was their target. At first temperance later
By denying women the vote, it was claimed the
Government had classed them with juveniles, lunatics
The franchise department of the WCTU took the three
major petitions to Parliament in 1891 to 1892.
The petitions were presented by Sir John Hall,
supported by Alfred Saunders, John Ballance and
Many of middle class men were teetotalers.
In 1893 despite interference by Seddon Suffrage was
Sir John Hall
Labour: Arbitration & Conciliation
The rise of Unions and Collective Bargaining
had led to considerable Industrial unrest in
This had eventually led to the 1890
Losing this confrontation led the Unions to
seek political support in the Liberals.
The 1891 election saw the most urban
electorates swing across to the Liberals.
William Pember Reeves sought to stop
unnecessary confrontation when he
introduced the 1894 Industrial Arbitration
and Conciliation Act.
Now Unions and Employers were forced to
accept decisions by a tribunal when
negotiation broke down.
Social Welfare: Pensions
In Victorian societies the view was held that the poor
were poor because they did not work hard enough.
One aspect of every frontier society was the large
number of men. (Miners, Bush Workers etc)
With no immediate or wider family to call on they
relied upon charity to care for them as they grew old.
By the 1890’s the number of “Indigents” threatened to
overwhelm the local charitable organisations.
The Liberals encouraged by Seddon, introduced a
It excluded those of “poor character” and the Chinese.
Maori were excluded because they lacked the birth
In 1898 NZ was the first nation to introduce such a
Defying the Arbitration Act.
From 1900 Unions began to feel that the Act was being
used against them.
In 1906 the Auckland Tram Drivers went out on the
first illegal strike.
Wellington Slaughtermen soon followed and won a pay
In 1908 the Blackball Miners forced the Mine Co. to
double their lunch break to 30 minutes after a 3 month
“Right Minded New Zealanders were pitted against
Socialist Union Agitators”.
The Miners Federation became the Federation of
By 1910 Watersiders, Shearers and Tramwaymen had
also affiliated. (joined)
The Federation of Labours journal was called the
The Maoriland Worker
• It contained traces of
the new Unionism along
with a sprinkling of
• It regularly condemned
the effects of
The Socialist Threat
By the 1890’s more and more countries
found themselves under threat from the
evils of Socialism and Unionisation.
Socialist wanted better conditions, better
pay through a fair distribution of wealth.
The US based organisation International
Workers of the World (Wobblies) led
The 1905 Revolution in Russia was driven
by these sentiments and almost toppled
the Tsars Government.
World wide unions were coming under
attack from Employers, the Media and
Governments as enemies of the
Union members were harassed and
attacked, beaten and jailed.
Who has benefited from the ‘product of labour’?
Liberals v. Reform and Unions
The Liberals under Joseph Ward, won again in 1908 but
were only a shadow of their former selves.
Ward relied on Union support and was unwilling to
confront the Unions.
In the next 4 years Unions made more demands that were
resented by the Employers.
By deregistering from the Arbitration Act they could freely
In 1912 the Reform Party under Massey defeated the
Liberals with support from both social Conservatives and
Immediately the Government took a harder line against
The 1912 Waihi Strike
The Arbitration Act no longer seemed to work for the
Tribunal Judgements lacked consistency and decisions
often favoured the Employers.
When a dispute over wages failed to reach a resolution the
Employer attempted to bypass the existing Union by
creating a new Union.
Under the Act any settlement with new Union would
become binding on all workers on the site.
The Unions called a strike.
The Employers brought in Scabs to work.
In a final incident an attack on the Union Hall led the
wounding of a constable and the death of a Unionist.
The Strikers &Union supporters were driven from the
“Socialism, Syndicalism and Anarchy”
Internationsally unions were seen as
Socialists – enemies society.
The United Federation of Labour (UFL)
was created in July 1913 at the Unity
400 Delegates represented 60,000
The Employers Federation responded
by created a “defence’ fund to “oppose
extreme agitation, syndicalisers and
Massey’s government made it clear
they would support any assault on
The Great Strike of 1913.
By 1900 refrigeration had created a
national system based around the
farming of sheep & cattle, their
processing, transportation and
export to overseas markets.
Impediments (Strikes) to this system
threatened the entire economy.
Unions controlled both
transportation by rail and sea.
When trouble began on the
Wellington wharves, Massey was
determined to crush the challenges
to good order. (and profit).
Town vs Country
About 1912 New Zealand became an urbanised
Traditional (Rural) society felt threatened.
Unions (Syndicalists) were seen as a symbol of
Massey hoped to use the Farmers Union against
the Militant Unionists.
Initially rural volunteers showed little interest in
Later they called upon 25,000 strong Territorial
Thousands of “Mounted Specials”, and hundreds
of foot answered this call.
Many of the farmers felt they had a duty to
defend the nation from the socialists.
Strikers grew to hate the “Specials” and almost
all violence was aimed at them.
Crushing the Unions: Bring on the Cossacks
The Strike quickly grew from a minor dispute
between the shipwrights and the Union
Months of negotiation proved fruitless.
The Shipwrights then affiliated (joined) with the
Wellington Watersiders .
Returning from a stop work meeting in October
the shipwrights were locked out.
Scabs were employed. The strike then escalated.
Soon wharves all around the country were
The Employers were determined to crush the
United Federation of Labour.
Massey wanted to use his Rural supporters. To
force the issue.
Mounted Police at Mercer Street prepare to charge
War in Wellington
“Wellington had become an armed camp. At
night the sinister sound of massed hoof beats
could be heard ...... as hundreds of “specials”
wended their way back to Buckle Street.”
The streets of Auckland, Wellington and
Christchurch would echo to the sounds of riot
Fighting included clubs, stones, bottles and
eventually rifles and pistols.
Gunfire was exchanged across Taranaki Street.
Soldiers manned machine guns outside
Strike leaders did not encourage attacks on the
Police but the Cossacks.
The Unions Defeated
Initially the Unions held the upper hand.
But by December 1913 the Unions were
Scabs were quartered on the wharves, or on
ships making them difficult to reach.
The wharves were defended by lines of
Massey’s ruthlessness and the ever present
Specials wore them down.
Union leaders were jailed and imprisoned as
were some strikers.
In Wellington they were forced out of the
Opera House to the Olympia Skating Rink.
Unable to stop the loading of ships and
losing public support they were forced to
call off the strike action.
Why did it fail?
The strike failed because most businesses were small
and their working relationships more personal.
Workers and Employers often worked together.
Most workers were not as a group unhappy enough
to support a total strike.
The Unions lacked the organisation and ability to
wage a long campaign.
Unions lacked the political support in Parliament.
Mainstream Media were almost completely against
Finally when scabs were employed on board the
ships, (Freyberg = stokers etc.) the strike was
What was the outcome of these strikes?
The “Red Feds” were for a short time
excluded from many sites but began to
work their way back into the Unions.
It left behind an entrenched class based
political system. (Melanie Nolan)
It reinforced the growing urban/rural split
especially in political support.
The Reform Party was now the established
party of rural NZ and social Conservatives.
With the Liberals in decline the Unions
realised they needed a new political ally.
The Social Democratic Party (SDP) held
out some hope, and it would eventually
become the Labour Party in 1916.
Many of the union leaders from
1908/12/13 would go on to become
important political figures. (Holland,
Socialism and WWI
Socialists were against the War. (or any
They could not see any reason why workers
should become involved.
The war seemed to be an argument
between Governments about a trivial
matter. (The assassination of Franz Josef)
Increased Imperial rivalries reinforced by
expanding armouries appeared to be for
the benefit of large companies, not workers
Increasing Nationalism also seemed
designed to drag workers into believing in
war as a good thing.
Many Union leaders, including Holland,
Semple and Fraser were imprisoned
because of their opposition to the war.
Many men refused to serve in the war,
they were ‘conscientious objectors’.
Some objected on religious grounds
(Jehovahs Witnesses or Christians)
Many refused because of their Political
beliefs. (Mainly Socialists)
Many socialists believed the war was a
It was fought to keep the rich, rich and
was fought by the poor. (Workers).
While there were volunteers this was
only a small issue.
After 2 years fewer were willing to go to
Until 1916 this was not an issue, but
Conscription made this illegal.
Why was the strike significant?
It was a demonstration of the growing demand for
equality in both New Zealand and around the world.
It was an example of major changes in New Zealand’s
NZ had just become an Urbanised society and with it came
a shift in emphasis.
Urban workers demanded more rights and better wages.
Such Socialist or “Communist” ideas threatened the
It was an example of the older established order fighting
back. (Country Vs Town)
It showed the power of the Government and its
willingness to attack the Union movement.
The Government used the law/changed the law and was
prepared to employ the armed forces (Navy & Army) to
enforce its will.
Their Significance to People
The 1913 Strike was an opportunity for the “Red Feds” to confront the
Government and regain the standing lost at Waihi the previous year.
For the Reform Party it was a chance to reinforce their dominance over the
Unions and to stop the march of Socialism in the Cities and return to more
traditional values and standing.
There were few in the country who were not effected.
– Thousands of Mounted Specials descended onto the cities determined to
enforce their values onto the weak and immoral city, leaving farms untended
– For thousands of striking workers it was weeks of wages lost and families that
went hungry, or accepted charity.
While the strike lasted barely 6 weeks its effects were felt for decades.
– Red Feds were jailed.
– Many were unable to obtain work in their normal trades.
– Unions left the Liberals and moved towards the SDP and later Labour.
– Union Leaders would eventually lead the Labour Party to victory in 1935.
Perspective Assessment: Group Work
Identify the groups that would be best to interview
– Get the widest range of views.
Identify 4-5 people who could be interviewed.
For each person identify:
– The group or trade they might be a member of.
– Their age....?
– Their family situation....?
– Their level of education....?
– What their view of the strike might be....?
– Why would they hold this view.....? (See above)
– How might the strike have effected them...?
– What roles/actions they might have taken in the strike...?
– Why might they have done this......?
– How significant might they see the strike in their personal lives or for the
Eliminate the weakest candidate(s) .... KEEP THREE!!!!

domain: history


text:
Cyberpunk 2077 is right around the corner, and CD Projekt Red is giving you a great reason to pick the game up on Steam. Pre-order the upcoming title and you’ll receive a short story written by Story Lead, Tomasz Marchewka, along with a few other digital gifts.
Marchewka’s short story is titled 2AM – She Calls. It will give you a closer look at “the hostile streets of Night City as seen by Frank, an ex-NCPD officer working the night shift as a Combat Cab driver.” Marchewka clearly knows how to deliver a powerful story – as he worked on The Witcher 3 – and it’ll be interesting to see how his latest short story plays out. At the very least it’ll offer another perspective on the world of Night City and its crime-ridden populace.
You’ll also get a set of avatars, posters, and a wallpaper for your Steam pre-order.
Steam isn’t the only outlet getting in on the pre-order action. Picking up Cyberpunk 2077 from CD Projekt Red’s own GOG will net you exclusive Cyberpunk 2077 posters, a digital comic (Big City Dreams), and an additional set of wallpapers and avatars.
If you’re picking up Cyberpunk 2077 on Xbox or PlayStation, your pre-order options are a bit spartan. All pre-orders come with the original score, Cyberpunk 2020 sourcebook, an art booklet, digital wallpapers, and the Cyberpunk 2077: Your Voice digital comic, but console players don’t have many exclusive bonuses. Your best option might be Best Buy, as pre-ordering from the electronics giant will net you a free steelbook. If you’re a collector, that’s probably more than enough to make it worth your time.
Cyberpunk 2077 is set to release on December 10 after getting hit with numerous delays throughout the year. Preparing a game of this scale for PC, next-gen, and current-gen platforms has been difficult for CD Projekt Red, but launching in the middle of pandemic no doubt played a factor as well. Thankfully, it seems that the end is near, and the game will be in our hands in just a few weeks.
READ NEXT: Just Cause 4, Rocket Arena, And Worms Rumble Are Your Next PS Plus Games
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Jon Bitner is an Associate Editor for TheGamer. His passion for gaming started with his first console (Sega Genesis) and he hasn’t stopped playing since. His favorite titles include The Legend of Zelda: Ocarina of Time, Team Fortress 2, Rainbow Six Siege, Pokémon Sword & Shield, Old School Runescape, Skyrim, and Breath of the Wild. He can usually be found playing the latest RPG, FPS, or some obscure mobile game. Before working as Associate News Editor, Jon earned a Biology degree and worked in the Biotechnology sector — experiences that taught him how to put words together and make sentences. When not playing or writing about the gaming industry, he enjoys sleeping, eating, and staring at birds.
Source: Read Full Article

domain: game


Multifunctional catalyst for de-NOx processes: The selective reduction of NOx by methane
A multifunctional catalyst has been applied for the reduction of NOx by methane in excess of oxygen. The catalyst includes a heteropolyacid (HPW) and a noble metal (Pt) deposited on a ceria–zirconia mixed oxide. The presence of hydrogen is necessary for reducing NOx into N2 with methane. During several alternate cycles of NOx storage (2 min) and reduction (1 min), the average NOx reduction percentage into nitrogen was of ca. 60% with a constant storage efficiency of ca.70%. A coherent picture of the catalytic process emerged from two separated experimental tests: water gas shift and steam methane reforming reactions.
Journal: Catalysis Communications - Volume 8, Issue 3, March 2007, Pages 400–404