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Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.
18 Related JoVE Articles!
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
A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants
Institutions: Boston College.
The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii
causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5
. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+
, a signal that is also critical for host cell invasion6-8
. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9
. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12
. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma
we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13
. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma
, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18
. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 °C), but fail to proliferate at the restrictive temperature (40 °C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13
. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11
. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19
. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20
. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.
Immunology, Issue 60, Genetics, Toxoplasma gondii, chemical mutagenesis, egress, genetic screen
Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions
Institutions: University of Wisconsin.
is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii
disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2
starvation, using medium with high a pH, or the addition of interferon gamma (IFNγ). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus
agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2
and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNγ and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii
to bradyzoites using a high pH growth medium with low CO2
and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNγ and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.
Microbiology, Issue 42, bone marrow-derived macrophages, fluorescence microscopy, parasitology, Toxoplasma gondii, bradyzoite development, cell culture, cyst wall
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
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections
Institutions: Universidad de la República, Trinity College Dublin.
Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung’s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages.
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae
inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs’ immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.
Medicine, Issue 90, Sublingual immunotherapy, Pneumonia, Streptococcus pneumoniae, Lungs, Flagellin, TLR5, NLRC4
Intralymphatic Immunotherapy and Vaccination in Mice
Institutions: University Hospital Zurich.
Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.
intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.
Immunology, Issue 84, Vaccination, Immunization, intralymphatic immunotherapy, Lymph node injection, vaccines, adjuvants, surgery, anesthesia
Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Institutions: University of California, Davis, University of California, Davis, Merck KGaA, Darmstadt, Germany.
A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-γ)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.
Medicine, Issue 80, Urinary Bladder, Animals, Genetically Modified, Cancer Vaccines, Immunotherapy, Animal Experimentation, Models, Neoplasms Bladder Cancer, C57BL/6 Mouse, MUC1, Immunotherapy, Preclinical Model
Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species
Infectious prion (PrPRes
) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1
. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3
as well as from environmental sources4-6
. Scavengers and carnivores have potential to translocate PrPRes
material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes
material through the digestive system of American crows (Corvus brachyrhynchos
), a common North American scavenger7
We describe procedures used to document passage of PrPRes
material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.
Infection, Issue 81, American crows, feces, mouse model, prion detection, PrPRes, scrapie, TSE transmission
Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Institutions: University of Colorado Anschutz Medical Campus, University Hospital of Essen.
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo
using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo
pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo
using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo
bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro
and in vivo
and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Medicine, Issue 79, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells
Institutions: University of Michigan, University of Michigan, University of Michigan.
We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh
CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.
tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFNγ and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.
Cancer Biology, Issue 83, Cancer stem cell (CSC), Dendritic cells (DC), Vaccine, Cancer immunotherapy, antitumor immunity, aldehyde dehydrogenase
A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.
Institutions: Office of Research and Development, US Environmental Protection Agency, Shaw Environmental & Infrastructure, US Environmental Protection Agency.
species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12
. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium
oocysts and Giardia
cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium
oocysts and Giardia
cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium
(oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14
. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium
cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11
. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium
oocysts recoveries when directly compared with the Envirochek HV filters4
. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium
from the magnetic beads in some matrices9,13
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.
Immunology, Issue 65, Infection, Microbiology, Medicine, Cryptospordium, hollow-fiber ultrafiltration, EPA Method 1623, heat dissociation, Giardia
Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Institutions: National Institutes of Health, Xiamen University.
Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3
. The malaria parasite Plasmodium yoelii
is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9
. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum
. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii
that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10
Genetic crosses in P. yoelii
and other rodent malaria parasites are conducted by infecting mice Mus musculus
with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11
. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.
Infectious Disease, Issue 47, Genetic cross, genetic mapping, malaria, rodent
Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination
Institutions: Johns Hopkins University.
Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.
Cellular Biology, Issue 5, mosquito, malaria, genetics, injection, RNAi, Plasmodium, TIssue Culture, Cell Culture, Insect
Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient
Institutions: Dynamac, Inc., University of Cincinnati, McMicken College of Arts and Science, Agricultural Research Service, U.S. Department of Agriculture, US Environmental Protection Agency.
is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii
cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii
cysts in meat1, 2
. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5
. To date, research on understanding the prevalence of T. gondii
oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6
. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii
oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii
oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7
Jove Infectious Diseases, Microbiology, Issue 33, Toxoplasma gondii, cesium chloride, oocysts, discontinuous gradient, apicomplexan
Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit
Institutions: University of California, Irvine (UCI).
Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.
Basic Protocols, Issue 11, genotyping, PCR, DNA extraction, Mice


text:
This beautiful bracelet is made of Pink Moonstone, glass beads, goldtone rondelles and a Hamsa charm.
The Pink Moonstone is associated with the inner goddess, a symbol of fertility and sensuality. The life-giving feminine figure is an empowering visualization that gives us the gift of increased intuition, the perfect prescription for cleansing the mind of negative emotions holding you back. With the Moonstone by your side you can channel its healing powers and clear the mind and center the Spirit.
This bracelet is handmade and Reiki Infused.

domain: artistic


Show transcribed image text A 6/4GHz earth station satellite receiving system is shown in Figure Q1.c. The carrier from the desired satellite S1 is [EIRP]1 = 32dBW and from the interfering one S2 is [EIRP]2 = 30dB. The ground station receiving antenna gain in the desired direction [Gar]1 = 40dB and the antenna gain towards the interfering satellite [Gar]2 = 28dB. The polarization discrimination YD = 4dB, and the downlink space loss [SPLD] = 196dB. The remaining parameters of the receiving system are shown on the block diagram. Knowing that the speed of light c=3x10 8 m/sec. the reference temperature To = 290K. and the Boltzmann's constant k= 1.38x10 -23 J/K calculate: [C/I]D The equivalent noise temperature increase (? TE) The total system equivalent noise temperature (Tts) [G/T] [Ps4] [Pn4] [SNR]i [SNR]0.


text:
The ultimate shitposting bot.
This bot is meant for fun commands rather than serious ones.
Slav Bot also has unexpected responses to certain words or messages (This can be turned on/off using the respond command on Slav Bot).
The bot is in development so expect more commands in the future.
You can use the feedback command to report bugs, give improvements or request new commands.
Join our support server for any questions or feedback.
Visit Slav Bot’s website for the documentation.
Support Slav Bot on Patreon
You can support the development on Patreon. More info on the Patreon page
You can use our custom Steam UI by downloading it from steam customizer. Simply pick either the dark or light theme and download the skin file. Make sure to have the Steam Customizer tool downloaded in order to use the file. After applying it, you can show how superior you are to your friends.

domain: game


No radiator, Netatmo thermostat
Comfort in every room with Netatmo thermostatic head
It only works with the Netatmo thermostat
It can be programmed individually for each room separately
Open window detection
Individual manual adjustment
2 year warranty
It saves even more energy
Complete your heating system with the intelligent Thermostatic head from Netatmo and individually control each radiator in the house. Thanks to the programming available for each room, it heats only what you need and saves energy.
Open window detection:
When the Netatmo thermostatic head detects an open window, turn off the heating in that room to prevent waste of gas.
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When needed, the temperature can be raised manually in a certain room for a period of 3 hours.
Power supply: 2 AA batteries with a lifespan of 2 years.
Connectivity: Compatible with WIFI 802.11 b / g / n (2.4GHz); Wireless connection between thermostat and relay: high beam radio waves
Programming: you can create as many programs as you want for each thermostat or you can adjust each thermostat manually
Compatibility: compatible with the vast majority of thermostatic radiator valve manufacturers
Design: transparent plexiglass cylinder resistant to UV rays with 4 interchangeable colors
1 Netatmo intelligent thermostat head
6 radiator adapters to be compatible with most manufacturers of thermostatic radiator valves
Adhesive tape in 4 colors 2 AA batteries


text:
A 230 acre sandstone plateau formation with cliffs, rock shelters and forest located in Hancock Co., Kentucky
The Cliffs are open on Labor Day, September 6th. Please join us for a lunch and unveiling a plaque honoring Mr. Jerry Harris for his family's donation of the cliffs to the public.
Used as a a rendezvous and hideout by Confederate guerrillas during the Civil War.
Hike the Forrest
View at the top.
Located in Hancock County. Ky, just 4 miles east of Hawesville.
Total 230 acres of beautiful forests, cliffs and large rock shelters.
Over 5 miles of hiking trails.
The area is free to the public. It is maintained solely by volunteers and paid for by donations.. We receive no government funds. You can help us in this effort with your tax deductible donation to: Hancock Co. Heritage Commission, Inc., a 501 (c) (3) non-profit organization.
Additional Days for 2021:
Spring Break IN - March 15th to 19th
Spring Break KY - April 5th to 9th
Memorial Day - Monday , May 31st
Independence Day - Monday, July 5th
Labor Day - Monday, September 6th
Fall Break KY - October 4th to 8th
Fall Break IN - October 11th to 22nd
Veterans Day - Thursday, November 11th
Thanksgiving - Thursday, November 25th
Hancock County Fiscal Court
Kentucky Heritage Land Conservation Fund
Hancock Co. Heritage Commission, Inc.
715 Williamette Road, Hawesville, Kentucky 42348, United States

domain: geography


text:
Discussion Guide ignores ‘imperilled’ coastal Douglas-fir forest, plays down clearcutting impacts
Public can now weigh in on future of Municipal Forest Reserve in on-line survey
North Cowichan has released a Discussion Guide designed to better inform citizens participating in round two of the public engagement on future management of the 5,000-hectare Municipal Forest Reserve.
While the document, prepared by Lees & Associates, provides some useful information on potential management scenarios, it is also noteworthy for what it does not contain.
Readers will see several photos of pretty viewscapes and folks on forest walks, but not one image of a clearcut.
In fact, in the entire 18-page document, there is only one reference to clearcutting, based on the results of the first round of public consultation: “Many community members expressed concern about harvesting practices, particularly the impact of clearcutting. Some respondents feel that cut blocks negatively impact views on the mountains, recreational experiences, and the ecological health of the forest.”
The guide also fails to mention a warning from the UBC Partnership Group that due to a legacy of cutting the backcountry, future logging will be increasingly visible to the public. In other words, the potential for denuded viewscapes for decades to come.
There’s also no mention of a report — deep-sixed by the Municipality — that estimates 141 species at risk. Habitat loss including from logging and development are largely to blame.
Perhaps most incredible of all is the absence of any reference to our coastal Douglas-fir forest — a forest type which UBC describes as “imperilled” and which the BC Forests Ministry and other governments, including Cowichan Valley Regional District, are seeking to preserve.
Release of the Discussion Guide today coincides with launch of an on-line survey asking the public to weigh in on four management scenarios presented by UBC on future management of the forest reserve.
You have until Dec. 31 to take the survey: https://bit.ly/3HaWVJV
Status Quo is based on logging 43.7 hectares per year which compares with 20.3 hectares under Reduced Harvest.
Active Conservation would allow the annual harvest of 3.9 hectares — not clearcutting, but for purposes such as restoring/enhancing ecosystem conditions that promote biodiversity — while Passive Conservation would have no logging.
UBC did a 30-year revenue projection for the various scenarios.
While logging produces more revenue early on, cash from the sale of carbon credits ultimately prevails — and by a wide margin.
Under the projection, Passive Conservation generates a net revenue of $39.6 million, Active Conservation $35.1 million, Reduced Harvest $30.7 million and Status Quo $31.3 million.
The Municipality is hosting in-person information sessions on Nov. 30, 3:30 p.m. and 6 p.m. at the Maple Bay fire hall.
Sign up for on-line sessions, December 6 and 12, 6–8 p.m.
To view the Discussion Guide, visit https://bit.ly/3gBwlOX .
(Photo: municipal clearcut atop Mount Prevost)
Subscribe free to sixmountains.ca for ongoing coverage of the forest consultation. More than 24,000 unique visitors.


text:
Home Affairs Minister Malusi Gigaba will finally account to the Portfolio Committee on Home Affairs, after it agreed to hold a full inquiry into the process that led to members of the Gupta family being granted early naturalisation.
The committee met on Tuesday to discuss a series of emails tabled by Democratic Alliance MP Mohammed Hoosen.
Hoosen said there were more Gupta family members who had received citizenship beyond the initial five that the department first disclosed to the committee.
He proposed that it was, in the public interest, time to put the issue to bed once and for all and set a date when they could call the department and Gigaba to Parliament.
"It will give the public at large the comfort that we have dealt with it and we then decide how we take the matter forward," he proposed.
"All of us in the committee have a responsibility to hold the officials accountable for that."
What they wanted to know was whether or not there was any impropriety in granting the family early citizenship and, if so, who granted it, he said.
"Only the minister can answer that," he added.
ANC MPs support proposal
The ANC caucus seemed to finally have reached firm consensus on the issue. ANC MP Maesela Kekana declared it time to deal with the issue decisively.
"Having read the documents, I'm convinced there is a case to answer. Without a waste of time, this matter is of public interest. We are here as public representatives to work for the people who put us here," he said.
"We are not here as praise singers, we are here to make sure we play our role as members of Parliament."
ANC MP Dineo Raphuti agreed with her colleague, but not before "raising her concerns" about the whistleblower who made the information public.
"Did this whistleblower go through someone's emails? Is it for personal gain? We need to look at... here is someone who got ahold of emails," she mused.
But she supported the inquiry, she eventually said.
Timelines and documents
Economic Freedom Fighters MP Hlengiwe Mkhaliphi said the inquiry was long overdue, as previous battles to hold had it led to a reshuffle of chairpersons in the committee.
"We must also remember - some of the Guptas - they are fugitives."
ANC MP Patrick Chauke said the committee needed to prepare and put the facts together.
He also had one last warning to those who would appear before them.
"Anyone who has lied before Parliament, there are sanctions. There are very clear sanctions," he said.
The committee will start setting up timelines for the inquiry and requesting documents from the department and minister.
Parliament goes on recess over Easter, and will sit again on April 16.
In the end, the decision was unanimous.

domain: law


text:
Research by graduate student James Radich has revealed a previously unexpected sensitivity to UV light of reduced graphene oxide (RGO) on TiO2 nanoparticles in aqueous suspensions. Hydroxyl radicals, produced in the surrounding water, further oxidize the RGO film and prolonged exposure to UV irradiation fully mineralizes the organic material. The work not only calls into question several proposed applications of RGO in photocatalysis and fuel cells, for example, but also suggests potential strategies for remediation of related carbon contaminants in the environment. (Chem. Mater. 2014, DOI:10.1021/cm5026552).
The work was highlighted in C&EN ‘news of the week’ on August 4th.

domain: chemistry


Azerbaijan, Baku, November 19/ Trend , corr I. Alizade/ Nasib Nasibli, member of MP group of opposition Musavat Party in the parliament says the legislative body should hold special hearings about the situation of Azerbaijanis living in Georgia.
"The Azerbaijani parliament needs to a great extent to hold hearings on the situation of Azerbaijanis living in Georgia as the life condition of our compatriots are not good," member of the MUsavar MP group said to journalists on November 19.
According to unofficial reports, nearly 500,000 Azerbaijanis live in Georgia. They are mostly populated in Kvemo-Kartli (Borchali) province.
Nasibli said he has several times initiated to discuss condition and problems of Azerbaijanis living in Georgia in the parliament. However, his proposal failed.
"At least International Relations and Interparliamentary Ties Commission of the parliament can hold such discussions," MP added.
The pressures on Azerbaijanis living in Georgia still continues. A month ago, Georgians were appointed director and deputy director of a secondary school in Qochulu village, which is totally comprised of Azerbaijanis. The Azerbaijanis in this village protested against it, Nasibli said.
"The number of Azerbaijanis living in Georgia is dwindling rapidly. There is no any progress in poor life conditions of the community there. It means that our compatriots living there are facing serous danger. Two years ago all school directors were planned to be replaced by Georgians. But Azerbaijanis prevented it by protest. They want to carry out this plan again. All these developments show that pressure on Azerbaijanis has become part of Georgia's state policy. Because a school is state-owned and it is government officials who put pressure on Azerbaijanis," Nasibli said.
MP noted that a delegation of Azerbaijani parliament should visit Georgia in order to study life conditions of our compatriots on a local level. Parliaments across the world hold hearings on various problems. But Azerbaijani parliament does not have such experience. Such hearings can be held in a closed form as well, he said.
Georgia's Ambassador to Azerbaijan Nikolaz Natbiladze rejects pressures on Azerbaijan. "These are all issues discussed by the media. There is no fact about pressure on Azerbaijanis living in Georgia".
The correspondent can be contacted at : email@example.com


text: Also at: http://wordsandsinew.blogspot.com/2012/08/someone-like-you-by-sarah-dessen.html"Read her once and fall in love." So says the blurb in the back of this book. But honestly, I don't think truer words have ever been spoken. Dessen is like, the YA writer of all YA writers, and if you read her novels and see all that she's accomplished and been recognized for you just pretty much can't refute that. Someone pointed out once that though she uses the same devices in her novels, she makes them work. You don't question it. You don't roll your eyes and say, "Here we go again." You just read and you go, "That was your typical Sarah Dessen novel, and damn, it was good."Reading this novel is a lot like eating comfort food. You take the first bite and this sense of relief just floods over you. There's something entirely soothing about Scarlett and Halley's friendship in that way that they're completely willing to be there for each other, will sacrifice whatever to be there for one another no matter what the situation entails. As you read, there's no question about it how authentic and pure this friendship is -- and it's very comforting to read because you feel that close bond and tightness. It's lot like seeing a happy child laugh -- you laugh along with them because the joy is infectious. Seeing the loyalty and love between Scarlett and Halley, you can't help but feel that too and it makes you feel better. This is the kind of novel I'll read if I just don't feel good because like comfort food, I know it'll make me feel better.Halley, the main character, changes so much throughout the course of this novel. And it's not the atypical POINT A to POINT B kind of way. She goes from Point A to Point B to Point C back to Point A. She becomes this slew of various people, changing and learning and figuring out what kind of person she is. The thing that never wavers is her loyalty and love but she sheds things like her naivety, her innocence and gains things like a backbone and independence. When Halley finally stood up for herself, oh man, I wanted to cheer because it is a very defining moment for her. I remember just rereading that portion because to have a character change that much and to see such a defining moment, it's something worth reading over again.Everyone from Scarlett to Macum to even Micheal were are just so fleshed out and real. These characters are all real. And I don't mean real like I could imagine them wandering around town somewhere, I mean real as in they feel so real I could imagine what their reactions would be or what they would say to things outside the novel. They are just so insanely defined, but then again Sarah Dessen is known for her ability to over and over again create a cast of characters so well-developed that that's what makes you fall in love with her novels. Not only that, she is able to take real life feelings and situations and make them so simple that you feel she talking right to you. All the stuff tackled in here -- friendship, love, parents, identity -- it's so very well written which is where another reason as to why this novel is so comforting comes in.I'll be honest: I didn't completely understand the depth of the title, Someone Like You. I just thought, "What does that really have to do with the book?" Then I read one single line and it just all fell in place for me: "Not people like us, Halley. Not people like us." Throughout the course of novel, statements like that would appear. Like, people like Halley don't have people like Macum Faulkner chasing after them. People like Halley and Scarlett don't have sex without saying "I love you" first. They make it sound like if you're labeled a certain way, there are certain guidelines you have to follow and everyone knows who you are and the things you do because of them, which is bullshit. Just because you are a certain way, that doesn't restrict you from doing other things or finding love with people different than who you are. People change and become different people over time; they step outside of that "someone like me" perception and just become "me", doing whatever the hell they want. It really reminds me of a segment in the show, How I Met Your Mother. There are these dopplegangers of the gang around New York who are completely different from who they are. So Ted is talking to Robin, who has just gone through a break-up because she chose love over her career and it back-fired on her, and tells her, "We've all been searching for the five doppelgangers, right? Well eventually, over time, we all become our own doppelgangers. These completely different people who just happen to look like us. Five years ago? That girl was pretty great. But doppelganger Robin? She's amazing." This correlates really well with the novel. The exact same thing happens to Halley; she becomes doppelgangers of her herself as she learns how to be her own person outside. She is no longer Mom's-best-friend-Halley or Macum Faulkner's-Girlfriend-Halley. She just becomes Halley in the end.(Side note: If you don't How I Met Your Mother, I would highly recommend you do. In fact, you aren't allowed back if you don't. The show is so insightful, funny, amazing, and also has Neil Patrick Harris. You can't argue against Neil Patrick Harris. He is Neil Patrick Harris.)Someone Like You covers a lot of things from friendship to loss to love to especially identity. It has characters who breathe, live, love, and will make you feel like you knew them for a long time. Scarlett and Halley's friendship will reach out, wrap you around, and make you feel so much better than when you started this novel. Sarah Dessen is one of the most well-respected and beloved YA authors for a reason and if you haven't read one of her novels yet, well, what are you waiting for?


text:
Judge Richard Posner, who recently dismissed the Apple-Motorola patent case, is again speaking out on the issue of software patents.
In a recent interview with Reuters, Judge Posner questions the utility of applying patent law to computer software. Citing the relatively lower cost of R&D for software (vs. other industries, like pharmaceuticals) and the growing complexity of electronic devices, such as smart phones, which may incorporate thousands of components, each of which may be the subject of multiple patents, raises the specter of courts clogged with endless patent litigation .
While there are strong arguments available both for and agasint applying patents to software, the increasing discourse, even among jurists, of the implications of the current course of patent law and patent enforcement makes it clear that there is clear need for reform in the system. Rationalizing the competing interests of incentivizing innovation by protecting intellectual property and preventing the creation of technological roadblocks to further innovation is an acute need. Nor can we move towards a balancing of these interests too quickly, as the other thing that rational markets need are predictable rules by which to play. Currently, the rules are anything but predictable, and this retards investment in technology.
In a still-sputting economy, disincentives for new investment and new innovation are the last things that we need.

domain: news


text:
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Freeze Dried Food Storage
Dehydrated Potato Dices in a 1.75 pound can.
Add to Wishlist
Dehydrated Potato Dices are full of flavor and can be used in any recipe calling for potatoes, including soups, casseroles, side dishes, and pot roasts.

domain: food


text:
I put myself into a lot of swaps. Swapping is a lot of fun and it gives me a reason to start crafting again and again. Today I made four cards, yesterday I made three. Fun, fun, fun and such a suprising outcome. They are all beautiful. I have to admit that the cards I made today are (for me) simple, but I am still stunned. At night, yesterday, I start coloring with my copics. Nearly all images I once printed or stamped are colored.
There are four six left to color, I think ... I have a bad habit, yes I have to admit it ...
When I have colored an image and I like it very much, I tend not to use it. Why ... overwhelmed I think ... I'll get over it. Have to, because I can not keep all those images, just because they are so beautifully colored. But we'll see!
I have to make four cards for four partners from ourscrappingclub.ning.com. It is sender's choice, so I just went for it.
Card # 1
I love this card. Do I have to sent it? I can't keep all the nice cards I make?!? Okay, this one (all those four cards, by the way) will be sent to the USA. The scalloped square and the red square are cut with the help of the Make the Cut and my Cricut expression. The circle on which the girls is, is cut with a nestability on the big shot. And, how silly, I just noticed that I forgot to put a sentiment on the card. Will be done in a minute:) The little girl is an image from a coloring page. With a Viva decor pearl pen I attached three pearls; the color is white.
Card # 2
Why not make a masculin card? Most of the time it is hard to come up with inspiration for a masculin card. But today (I don't know where it came from??) I just went on, as I said earlier. I love this KennyK image. He is so handsome and I love American football. Lively and full of energy ... Also colored with copic markers, the helmet and other green parts are colored with prisma color markers. With my Sizzix big shot and a cuttle bug embossing folder I embossed the blue background. A red ribbon it attached under his feet. With the program Make The Cut and my Cricut expression I cut the tag and stamped a sentiment on it. The sentiment is from the set "Creepin Kit" from The Greeting Farm. Hehehehe, his pants are so dirty. Doesn't he rock?
Card # 3
Did I had a KennyK moment? I think so! Again a KennyK digital image. She is colored with prisma color markers. The tag is cut with my Sizzix big shot and a Spellbinder's nestability.
Card # 4
Months ago I downloaded a few images from coloring pages. They were meant to experience digital images. Notice that the images on coloring pages have thicker drawing lines than the digital images you can buy.
Well, I do prefer rubber stamps over acrylic stamps and digital stamps. It gives me a certain crafty feeling, I can see and touch my rubber stamps, for sure when I am searching for creativity.
Still I have to admit that digital stamps have some advantages such as enlarging the image, the fine lines on the image and it's also possible to print the image on an exact spot.
So this image is colored with copics on marker paper. The patterned paper is from a patterned paper block, but it has no brand name on it. I used a nestability die for the sentiment. The stamp used is from a set and this one has no brand name either.
I would love to know what you think. And if you have any questions, please leave a comment. Thank you for stopping by.

domain: design


text:
A movie Night Flyer Template with Popcorn Illustrations
This Movie Night Flyer Template is perfect for promoting movie, TV, cinema and film nights. The flexible design is suitable for nightlife venues, schools and community centres.
- The templates come in Photoshop PSD & Illustrator Ai Vector format.
- To edit this template, you will need Adobe Photoshop or Adobe Illustrator
- The design is print-ready in 300ppi CMYK colour format.
- All layers are organized & clearly labelled for fast and efficient editing.
- Stock photos are included, though using your own is also easy.
- Final printed size: 5×7″


text:
Spain joins NATO missile shield. Another Bailout precursor ?
The Spanish government made the announcement on Friday after sealing a deal with the US to participate in NATO’s anti-missile shield.
According to a statement by the government, Spain authorized the signing of the deal at a cabinet meeting on Friday.
“Its principal activity will be to contribute to defending against ballistic missiles,” the statement said.
Following the decision, Spain’s Deputy Prime Minister Soraya Saenz de Santamaria told a news conference that the deal would help boost the economy in the region.
This comes as the left-wing political coalition in Spain has leveled criticism at the move, saying it makes the country “a military target.”
Leaders of the 28-member NATO alliance expressed their support in 2010 for the Europe-wide ballistic missile shield.
The system will later expand to include land-based interceptors in Romania, Poland, Turkey and The Netherlands.
- U.S., Spain to Enter into Missile-Defense Pact (hispanicallyspeakingnews.com)
- Russia Warns U.S. Against Deploying Final Phases of Missile Shield (nti.org)
- PressTV: 4 US warships to head to Spain as Madrid joins NATO missile shield (jhaines6.wordpress.com)

domain: politics


text: BAZF, a member of the Bcl6 gene family, acts as a sequence-specific transcriptional repressor in association with Bcl6. However, the tissue expression pattern of BAZF differs from that of Bcl6, suggesting a Bcl6-independent function of BAZF. In order to examine the physiological function of BAZF, we generated BAZF-deficient mice and transgenic mice with BAZF-cDNA under the control of the lck proximal promoter (lck-BAZF). These mice were viable and no gross anatomical abnormalities were observed after birth. Since Bcl6 is a key molecule for the generation of memory T cells, we examined the function of T cells of these mice. We show here that cell proliferation of naive CD4 T cells, but not memory ones, of BAZF-deficient mice to anti-CD3 antibody stimulation was impaired. Conversely, cell proliferation of naive CD4 T cells, but not memory ones, of lck-BAZF mice was augmented. Since cell proliferation of naive CD4 T cells of lck-Bcl6 mice to anti-CD3 antibody stimulation was severely impaired, BAZF may attenuate the regulatory effect of Bcl6 on antigenic activation of naive CD4 T cells by Bcl6/BAZF heterodimer formation. These results suggest that BAZF is necessary for activation of naive T cells to antigenic stimulation.

domain: medical


text:
It’s been a year since my first post and what a year it has been. I have had the pleasure of reading many great stories and have vastly expanded my taste in books. My voracious appetite for reading has expanded beyond measure but has been equally satiated by the experiences I’ve had. I am so thankful for the opportunities this blog has given me, most specifically to connect with other readers on this platform, to experience new books via Netgalley and to humbly interact with authors via Twitter (little compares to the thrill of seeing an author has liked and/or retweeted my review!). I wanted to put together a short list of my most favorite books that I reviewed this past year to highlight their merits and to help me to reflect on what I accomplished. These are in order of date reviewed and all rank pretty equally at the top of the list of my favorites on Read Voraciously. Take some time to check them out and I would love to hear your opinions! Here’s to year one and many more to comes!
A unique perspective on the complexity of life that highlights how small experiences and choices can have a large impact. I loved the way this was written, where each version was explored deeply with the narratives alternating as time continues to pass.
I didn’t realize how much I liked this novel until well after I finished it. Only now, after experiencing a maturing in my appreciation of well writtin novels, can I fully understand just how well Haslett handles mental illness and the effect it has on families and the afflicted.
If you haven’t read this yet you need to, as simple as that.
Books are meant to allow you entrance to otherwise unexplored avenues. As a wife and mother this story did that for me. While the subject is highly taboo in the family circle, it is something many wives and mothers fantasize (and often only that) about so it was enjoyable to watch that play out in the story.
I am and have been a long time Picoult fan so I will forever include her novels on my top lists. This one was particularly relevant, especially given the fact that her audience is made up largely of white, privileged women though of course not exclusively. Her authors note at the end was the most powerful piece of this story, in my opinion and I covered it in my review for that reason. Race needs to be talked about and our privileges, and at what or whose expense those exist, need to be acknowledged and understood.
This little known story of Albert Einstein’s first wife, Mileva Maric, is about the struggle of women to over come the constraints of society. This topic is still relevant today and one that resonated with me as a women who works in the sciences.
In the same vein as The Other Einstein this story examines what happens to a woman’s dreams when the expectations of society come to call. Set in a more recent time, during the creation of the A-bomb, we see how the male agenda dominates and what that does to a marriage.
A well-written tale of the struggles of adolescence. This resonated deeply and for anyone who has been bullied or been an accessory to that cruelty, or, really, went through high school, it will too.
A lifetime friendship between two weirdos trying to sift through their experiences. Dark and humorous in equal measure.
A uniquely surreal examination of the refugee crisis. The writing is so beautiful, for a lack of a better word. His lengthy sentences infuse a fluidity to the text which stands in deep contrast to the difficulty with which certain people move through this world these days. A timely read that is absolutely worth your time.


Welcome to the Hooniverse News! As always, this is a weekly recap of the biggest stories in the automotive industry without the fluff or bull. This week, Acura stuns with the Type S Concept, Shelby GT500 tech starts sharing hardware with the GT350R, Dodge celebrates 50 years of the Charger Daytona, Ford adds FX2 off-road package for the Ranger, and Chevrolet reveals official pricing for the C8 Corvette.
Acura Type S Concept
As promised, Acura took the covers off a concept that will help usher in a new era of Acura performance. And it’s stunning. Making its public debut at The Quail, a Motorsports Gathering (which I have to say is still one of the dumbest names for a car show I’ve ever heard) today is the Acura Type S Concept, a design study that will set the stage for the return of the Type S and heavily influence the character of future production models bearing the name.
The Acura design team set out to express the essence of “Precision Crafted Performance“, which has been the brand’s tagline since the 2nd-generation NSX was introduced. They did that through the car’s stance, proportions, and a silhouette that is “powerful and expressive, yet clean and cohesive”. It has the sports car essentials like a wide track, low stance, a long hood, and short overhangs. The concept introduces two reworked design traits from Acura’s current lineup, those being the Diamond Pentagon grille with a new intricate pattern and next-gen four-lamp “Jewel Eye” LED headlights. Some of the details go beyond sports cars though, like the “chicane” daytime running lights and taillights which were influenced by the ARX-05 Daytona Prototype car that’s currently one of the top players in IMSA.
The styling of the front fascia is aggressive and outrageously beautiful but also functional. Large air intakes would be sized appropriately for routing air to “a high-performance engine” (no details were given) and Brembo brake package. At the business end is an upswept decklid with a sharply creased centerline drawn from the hood, an integrated forged carbon spoiler, rear diffuser, and chrome quad-tip exhaust. The 21″ multi-spoke wheels wear high-performance summer tires in a square 285 setup which would be ideal for handling on and off the track.
There’s really no sure indication as to whether we’ll see this car in production, but they have promised that two Acuras will debut with the Type S badge in the next two years with heavy influences from this thing. Those cars should be a TLX and NSX (if they don’t want to call it a Type R instead). They will most likely just be the RDX and MDX because the wrong suits will get involved.
2020 Mustang Shelby GT350R
The GT500 isn’t the only Shelby getting love as we roll into the 2020 model year. The wonderful Shelby GT350R is getting a few tweaks to enhance its driving characteristics even more, and it’s done so using a few things from the GT500 itself. Ford says this won’t be the last time that lessons learned from the GT500 trickle down into other parts of the Mustang lineup.
The GT350R’s 2020 refinements start with new front suspension geometry with a redesigned high-trail steering knuckle taken from the GT500. Expect better front end bite and responsiveness out of that. Ford also swapped in a new steering rack and recalibrated electric power steering for greater steering precision. That’s it for the new hardware – a short list but with big improvements.
Meanwhile, some new paint colors from the GT500 also get added to both GT350s – Grabber Lime, Twister Orange, Iconic Silver, and Red Hot Metallic. One thing that hasn’t changed is how awesome the GT350 lineup is.
Charger SRT Hellcat Widebody Daytona 50th Anniversary Edition
Meanwhile, at the Woodward Dream Cruise, Dodge revealed another price gouging opportunity for dealerships. It’s the Charger SRT Hellcat Widebody Daytona 50th Anniversary Edition, which is one of those names that tells you everything there is to know about it right up front. It’s a Charger SRT Hellcat Widebody. It celebrates 50 years of the Charger Daytona which was a groundbreaking moment for Dodge, being the first car to break 200 mph average lap speed at Daytona. And it has the words Anniversary Edition at the end, so the dealer markups just shot up about 15%. On top of that only 501 examples are being built (just like the original Charger Daytona) so that means dealer markups are now approximately 50%. It goes on sale in the fall.
Ford Ranger FX2
Ford is adding another off road trim level to the revived Ranger later this year. Based on the popular FX4 package, it gets an FX2 package for those who want some extra off road capability in a rear-wheel-drive Ranger for those who don’t need 4WD to have fun off the pavement.
The FX2 package rides on 17″ or 18″ wheels with off road tires and off-road tuned suspension which helps it adapt to whatever terrain you have the guts to throw at it. It also rocks a front underbody guard and air dam-delete for extra protection and approach angle clearance. Crucially, there’s also an electronic-locking rear differential and an off-road gauge cluster screen with pitch, roll, and yaw data shown in real time.
As if Ford needed to justify adding another off road package for truck buyers to throw money at, they claim one in every three Rangers is equipped with the FX4 package. By adding an off-road-ready package with a more common drivetrain and a bit of a price difference ($595 as opposed to $1,295 for the FX4), Ford is expecting to tap into even more buyers.
Meanwhile, Hooniverse anticipates that nine out of ten Ranger FX2 buyers will be looking for aftermarket FX4 stickers.
2020 Corvette Official Pricing
When Chevrolet was wrapping up their live debut of the all-new C8 Corvette a few weeks ago, one of their suits stood up on stage and claimed that the new mid-engine sports car would start under $60,000. Assumptions were made almost immediately that it would start at $59,999. “Don’t be silly”, Chevrolet execs must have said. “We’re not gonna sell it for $59,999….”
“We’re gonna sell it for $59,995“.
That is the official starting price of the base C8 Corvette and that includes destination charges. The Corvette at that price range is the 1LT trim level which includes the basics – 490-horsepower V8, 8-speed dual clutch, and other tech features. It’ll fit the bill for most casual buyers, as demonstrated by their choice of tires – all season run flats. The next trim levels add more luxury and tech, like the nose lift system which stores locations for automatic activation which can only be had on the 2LT and 3LT trims. Those start at $67,295 and $71,945 respectively.
Then there’s the package we would all want. The Z51 performance package (pictured). That adds 5 horsepower, performance exhaust, performance suspension, electronic performance limited slip differential, performance front and rear spoilers, boosted performance brakes, Michelin Pilot Sport 4S performance summer tires, and extra performance cooling for all that performance is a $5,000 option and can be had on any trim level. So at a minimum, buyers looking for more performance out of their Corvette will be spending at least $64,995 for a 1LT Z51. Any more money would just make you more comfortable… until the Z06 arrives.
I know a lot of people are excited about this car and its price. But if you want some advice, just wait a little bit. Once the first production C8 sells at Barrett-Jackson to a Hawaiian shirt enthusiast and all the dealerships have sold their first allotments for $200,000 because they’re assholes who want to ruin things for everyone so their manager can get a bonus, maybe then you’ll have a chance to buy a C8 for its true MSRP. And if you still can’t, we start an uprising.
What’s Your Automotive News?
That’s all I’ve got for you this week, so now it’s your turn. If you saw anything, fixed something, broke everything, or otherwise did anything even remotely car related that you want to share with your fellow hoon, sound off in the comments.
Have a good weekend.
Leave a Reply


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This page shows the publications co-authored by Ebrahim Barkoudah and Naomi Fisher.
Maximum renal responses to renin inhibition in healthy study participants: VTP-27999 versus aliskiren. J Hypertens. 2016 May; 34(5):935-41.
Renal responses to three types of renin-angiotensin system blockers in patients with diabetes mellitus on a high-salt diet: a need for higher doses in diabetic patients? J Hypertens. 2011 Dec; 29(12):2454-61.
The connection strength for co-authors is the sum of the scores for each of their shared publications.
Publication scores are based on many factors, including how long ago they were written and whether the person is a first or senior author.


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156 search results
Investigating and Comparing Life Cycles
A Tier I life science instructional resource for grade 3
Weathering and Decomposition
A Tier 1 earth science instructional resource for grade 3
Interactive Math Glossary
A Tier 1 energy instructional resource for grade 3.
Getting Started Guide
New to the Texas Gateway? This interactive guide will help you get started today!
Target 2% Lessons to Support Comprehension for Grades K through 5
These comprehension activities are designed to enhance the instruction of all teachers of reading and to meet the learning needs of all students, as indicated by data.
Target 2% Lessons to Support Vocabulary for Grades K through 5
Vocabulary knowledge is an essential component of comprehension, language proficiency, and reading growth for all students.These vocabulary activities are designed to enhance the instruction of all teachers of reading and to meet the learning needs of all students, as indicated by data.
Target 2% Lessons to Support Phonics for Grades K through 5
Phonics is the relationship between the letters (graphemes) of written language and the sounds (phonemes) of spoken language. These phonics activities are designed to enhance the instruction of all teachers of reading and to meet the learning needs of all students, as indicated by data.
Target 2% Lessons to Support Fluency for Grades K through 5
Fluency is the ability to read text effortlessly, allowing the reader to concentrate on meaning. Fluency includes reading accurately, quickly, and with prosody (appropriate phrasing, expression, and attention to punctuation).
Unit 2. Why Use the TPRI?
TPRI Module 1: Unit 2 Used in Grades K-3
Interactive Science Glossary
The Interactive Science Glossary is a resource that supports students who are learning the language of science. This resource includes written and spoken definitions and sentences in both Spanish and English. Students can even create, download, and print their own vocabulary cards.
Colombian Folktale: Pastorcita
This video features the Colombian story Pastorcita in both English and Spanish. Pastorcita has many elements that are similar to “Little Bo Peep.
Familiarize your children with the alphabet with this fun chalk activity from Arthur.
This resource group teaches students a wide variety of reading strategies. It touches on such issues as careful reading and follow-through, context clues, capitalization, transition words, features of printed text, and the relationship between text and illustration.
Words from the Wild: R
Jahreese identifies words containing the letter "r" around his neighborhood in Harlem. This resource teaches reading, decoding, spelling, and vocabulary.
Road Trip—Long U
Knowing that "ew" and "u" produce the same sound, Henry and Charlotte play a game collecting words using those letters/letter combinations from signs and billboards along the road.
Martha's Memory—Martha Speaks | PBS KIDS Lab
Help children understand point of view in storytelling and build vocabulary using this Martha Speaks video! Martha and friends discuss what the words "certain" and "sure" mean, and Martha shares her version of how a game they played ended.
Martha's Perspective—Martha Speaks | PBS KIDS Lab
Help children build vocabulary using this Martha Speaks video! Martha defines the word perspective.
Screen reader support enabled.
Forming and Using Possessive Nouns | No Nonsense Grammar
Possessives show when a noun belongs to someone. It is often indicated with an apostrophe "s," but when words end in "s" only an apostrophe is added.
Screen reader support enabled.
Music Video: Capitalize
Lisa sings "Time to Capitalize" to teach children the rules of capitalization. This resource teaches capitalization, punctuation, spelling, and the concept behind proper nouns.

domain: library


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USA -(Ammoland.com)- Do you have children or students between the ages of 12 and 16? We are pleased to announce an exciting opportunity for Arizona youth to participate in a free outdoor adventure camp this summer!
The Arizona Desert Bighorn Sheep Society and the Arizona Deer Association, with support from multiple partners, are hosting their first annual “Youth Outdoor Adventure Camp” this July.
The camp is intended to provide a fun, outdoor experience for youths ages 12-16, with an emphasis on those who have limited experience with outdoor activities.
And it is FREE to participants!
For more information, including registration and contact information, please check out the camp application (two versions provided):
Feel free to distribute this information to any potential students. If you are interested in assisting, please reach out to the contact information included in the application materials.

domain: hobby


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Whether you’re into the classic games or the latest video slots, Pragmatic Play has a variety of options to keep you entertained. With more than 150 video slots, the studio offers a broad variety of titles to suit a variety of players. The company also offers an unlimited free trial and a demo mode, which allows you to play games without signing up.
Pragmatic Play offers an extensive selection of slots with a traditional theme. The company’s game portfolio features three-reel games, fruit symbols and a three-dimensional feel. The company also promotes their slots through a variety of channels, such as promo campaigns, streamers and reviews. They have also recently acquired the license for the Megaways platform, which adds value to their portfolio. The Megaways platform is used to create adaptations of older hits.
Pragmatic’s portfolio features some hits that players love, such as the slot 888 dragons. The game has a classic design and is easy to play. Its high RTP offers players a chance to win big. It also features a hold&spin feature, which allows players to spin the reels repeatedly for bigger wins. During the hold&spin feature, players receive credits for special symbols that land during the feature.
Pragmatic’s slots feature striking graphics and sound effects. Their game portfolio features over 100 titles, and the studio offers an extensive demo mode for players to try out the games without risking any money. They have also partnered with several gaming sites. Pragmatic has also acquired the license for the Megaways platform, so you can play new Megaways titles.
Pragmatic’s portfolio also features titles with Hold&Spin, a feature that allows players to spin the reels repeatedly for big wins. During the feature, symbols stay on the screen until a new symbol lands, giving players the chance to make more winning combinations. The company also offers a battery-saving mode, which allows players to play while they’re on the go.
Pragmatic’s slot games are easy to play, and they offer players a lot of features. The studio has a wide range of slots, including games with a hold&spin feature, which lets you create extra winning combinations during the feature. Some games have two or three reels, while others have just a single reel. Players can also choose to customize the settings to suit their preferences.
Pragmatic Play is a relatively new company that owes much of its success to maximum exposure and affiliation with traditional affiliates. Its slots are reminiscent of the classic slot games of old, but it does not care too much about the individuality of each game. Instead, the company relies on promotions, promo campaigns, and streamers to get their name out there. Its slots have striking graphics and a three-dimensional feel, and the studio offers an extensive demo mode to test out the games.
Pragmatic’s slots are available on many different platforms, including mobile. Its slots feature a number of different symbols, which vary according to the theme. Some symbols are stylized lucky sevens, while others are fruits and bells.


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Cardinology: Ryan Adams and the Cardinals
I’m a pretty big fan of this man and his band, the Cardinals.
For a long time, Ryan Adams (not BRYAN Adams) developed quite a reputation not only for his music, which has always been well received critically, but also for his behavior on stage during shows. Many of those problems have been attributed to drinking and whatever, but those days seem to be gone now.
I was able to catch Ryan Adams and the Cardinals in Louisville in 2007, and then later that year in Columbus, and both shows, while extremely different in style, were incredible. After seeing them in Cincinnati this past year, I’m excited to see them yet AGAIN in Louisville in March.
Their shows usually feature a mixture of material both from the band and from Ryan Adams’ solo time, and it is likely that the upcoming tour will showcase the new album, Cardinology. I was lucky enough to receive this album (yes, vinyl) from my wife for Christmas. It included a code for a digital download (something several artists are doing these days), and I have it loaded up in the iPod where it has been in pretty much constant rotation.
From the opening “Born Into a Light” to the closing “Stop”, this record is solid throughout. Ryan Adams seems to be trying out a little different vocal style on this one, and I really do like it. I hear shades of Warren Zevon…a touch of Neil Young…but it is all just perfect. Add to that the beautiful backing vocals by guitarist Neal Casal, and it makes for an awfully nice sounding record.
So give it a shot, and if you like what you hear, head on up to Louisville on March 16th at the Louisville Palace…it should be a great show!

domain: entertainment


text: Barbie loves being fashionable, and there's no way she could have missed a brand new trend that's filling the fashion scene. That's right: we're talking about a new trend alert: Serenity and Rose colors. In Barbie Trend Alert: Serenity Vs Rose, Rose Quartz and Serenity join together to create a unique balance between the warm tones and the cooler ones. Barbie absolutely adores this new combination, and she is planning on using it throughout her wardrobe. Your important task in this exciting new game will be to make sure Barbie's new closet looks really amazing by helping her mix and match the cutest outfits, all in these gorgeous colors. All you have to do is mix and match the clothes and then decide upon the winning combination. Once that is done, in Barbie Trend Alert: Serenity Vs Rose you will also have to choose a new hairstyle for Barbie, and then do her make-up so that it emphasises her natural beauty. Have a really amazing time playing our brand new dress up game called Barbie Trend Alert: Serenity Vs Rose, on Capy.com!


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You love your cat and your cat loves you. That’s why separation can be quite hard on our kitties (and us!). So if you come back to collect your pet and it is acting a little differently, it’s probably down to a few simple things. Cats don’t like change, they are creatures of habit and are very comfortable in their routine. Your separation and having been in a new location may have had an effect on your pet. If the abnormal behaviour persists the reason may be medical and it would be best to get your pet checked out by a vet, however, before you panic, give your cat time to settle back down at home and back into a routine. Cats need time to adjust to change, just like us!
Before you go away
If you are planning to go away on holiday, leaving your feline friend in a cat hotel, it’s important to keep your schedule as normal as possible beforehand so you don’t alarm your kitty! Remember to feed your cat at the usual times and make sure to keep your own stress at a minimum. Cats are very clever and they can sense if you are anxious or upset. If they sense something is wrong they will pick up on your stress, and it will stress your cat out too, and that wouldn’t be good terms to part on. Do your best to keep your own schedule as normal as possible Kitty will notice all the extra shopping trips and the unavoidable chaos that accompanies the preparation to go away. Another tip to use in order to keep your cat as calm as possible before you go on holiday is to pack slowly. Up to 2 weeks before you go away, set out your suitcases, then slowly over the two weeks fill them with everything you will need. This gives your pet plenty of time to investigate and accept the change in their own time. It also means you will avoid frantic packing as your vacation date approaches, minimising the stress on both you and your cat.
Returning your cat to normal after being on holiday
When you return from your (hopefully wonderful) holiday, you will be ecstatic to see your best feline friend for a reunion. However, do not be surprised if your cat doesn’t reciprocate your feelings. Cats take a while to adjust to new situations Whilst you may be ready to go back home, your cat has just become used to being at the cattery, and it will take them a few days to settle back down into a routine.
Speaking of routine, it’s important that you don’t overfeed your cat or give him/her excessive amounts of treats. While their unusual reaction to you may have made you feel bad, don’t overcompensate by overfeeding, although a few extra treats to make kitty feel appreciated is fine. If you feel your cat has lost weight due to stress, (this is hard to tell without going to the vet), you needn’t worry. Feed your cat at normal times with their usual amount of food and your cat should bounce back within a few weeks. If the weight issue continues to concern you after this time, please seek advice from your vet.
Give your cat undivided attention for a few minutes every now and then during the days when you return from holiday. While you are busy unpacking and getting everything straight, your cat will also be busy exploring and reacquainting his/herself with the house again. It’s important to help build your cat’s confidence with affection and play, and to let them know that things will be returning to normal now that you are both back at home. Playtime will be essential in distracting your kitty from the fact you were separated, helping them to more quickly re-adjust to being back with you.
If you feel your cat is still stressed after a few days, don’t worry, they probably just need a little extra support in knowing that your bond is still strong and that they are safe. For extra help in calming kitty, you can purchase calming drops or plug-in scents at your local pet store to help relax your feline friend. Additionally, you could put on some calming or natural music. There is scientific evidence behind a cat’s mood being influenced by music. Soothing music will also help you to relax too, which kitty will sense and take on board, helping reduce stress for both of you.
Take a look at how you’re feeling after your return from holiday, as you may have noticed by now, our cats are super sensitive to their owner emotions and mood changes. Perhaps find time during the day for some self-care, whether it’s meditation, reading a book or even partaking in your favourite indoor hobby. You may find that if you are relaxed and calm your cat will soon follow your mood. Keep these things up alongside your normal routine and soon enough your cat will readily reacquaint itself with both you and his/her surroundings.
All in all, remember to prepare for the worst and hope for the best. Everyone’s cat reacts differently to their time in a cattery, and the experience can differ each time. Be patient, prepare to give your cat time to regain confidence. Get back into a normal routine, and remember, the more stressed you are, the more stressed your cat is going to be. You know your cat best at the end of the day, so if you feel something is further amiss with your cat, please take them to a vet as soon a possible.

domain: pet


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College's student computer programming team The Transformers
won first place in Division II of the Association for Computer Machinery
Southeastern Regional Programming Team competition.
The annual competition was held Oct. 12 at Embry-Riddle
University in Daytona Beach, Fla. Eighty-four teams from 40 Southeasterncolleges
and universities participated. Division II included 31 teams from
institutions that do not have graduate programs in computer science.
The BSC Transformers team placed 15th out of 84 teams
overall.Another BSC team The Horde placed 12th in Division
II and 45th overall.
The BSC student teams are advised by BSC Assistant
Professor of Computer Science Dr. Marietta Cameron and BSC Associate
Professor of Computer Science Dr. Lewis Patterson.


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"Little Miss Sunshine." "500 Days of Summer." "Midnight in Paris." Titles like those are surely proof that summer audiences have appetites for something beyond superheroes. Add up the right…
Breakup at a Wedding
Periods.Films creators Victor Quinaz and Anna Martemucci are partnering with the Nerdist Alliance, Nerdist Industries' YouTube network.
Oscilloscope Laboratories is getting married to "Breakup at a Wedding," acquiring North American rights to Victor Quinaz's debut feature and setting a June 18 release date for VOD and digital…
A pregnant Dutchwoman is caught between two very different brothers in "The Family Way," another well-acted, glossy mainstream romantic comedy from helmer Joram Lursen, thesp-scribe Kim van Kooten…
A film so full of explanatory flashbacks and animated sequences visualizing its characters' invented yarns that their real dramas are almost obscured.
Diversity is the star of Ernesto M. Foronda and Silas Howard's "Sunset Stories," a deliberately nontraditional romantic comedy that embraces the sort of actors -- and characters -- typically…
There's no denying that writer-director Jordan Roberts earns a good number of laughs, and even some extended guffaws, with his crass romance about sex, lies and digital video.
World preem features -- including the narrative pic written by and starring Chris Colfer of "Glee" and a new doc from Morgan Spurlock -- are among the more than 40 movies joining the Tribeca Film…
The old fortune-cookie wisdom about the journey being more important than the destination is emphatically underscored by Brazilian Sundance entry "Father's Chair."
"I'm almost starting to think a bad economy makes for better artists," John Cooper noted as he unveiled the lineup of the 28th annual Sundance Film Festival, marking his third year as director of…
Getting thrown out of a car by Scott Rudin on a London highway has its benefits.


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Let's Play Music : Playlists - 14 Best Classical Music Tracks to introduce Kids…
Best Classical Music Tracks for Kids - perfect for encouraging creativity
Classical music is a great way to encourage imagination, movement and concentration. Here are our best classical music tracks for kids.
Marching is a fantastic musical activity for kids. It combines music, movement, listening. preschool music, music and movement, classical music for kids.
Music Notes in White -- Manufacturer: Windham -- Designer: Whistler Studios -- Collection: Good Vibrations -- Print Name: Music Notes in White Also music staff design
Let's Play Music: 10 Best Classical Tracks for promoting calm & tranquility
Collection of Free Pirate Music - 5 Classical Music Tracks Perfect For Pirates & Adventures PIRATES
Classical music for toddlers at home - 40 Best classical tracks …
5 Best Classical Music Tracks for Kids : Fairy Dancing - Let's Play Music. I'm not sure what to classify "Fairy Dancing" as. but I do know that these are great songs for creative movement