text:
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
24 Related JoVE Articles!
A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures
Institutions: Cedars-Sinai Medical Cente, Southern California Permanente Medical Group, Detroit Medical Center, AdvanDx.
Enterococci are a common cause of bacteremia with E. faecalis
being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis
is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis
OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ
hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis
/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.
Immunology, Issue 48, PNA FISH, Enterococcus, Blood Culture, Sepsis, Staining
A Novel Ex vivo Culture Method for the Embryonic Mouse Heart
Institutions: University of North Carolina at Chapel Hill .
Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.
Developmental Biology, Issue 75, Cellular Biology, Molecular Biology, Biomedical Engineering, Bioengineering, Medicine, Anatomy, Physiology, Cardiology, Embryology, Embryonic Structures, Cardiovascular System, Cardiovascular Diseases, Surgical Procedures, Operative, heart, mouse, embryonic, organ culture, coronary plexus, ex vivo, cell culture, transgenic mice, animal model
A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors
Institutions: Texas A&M University (TAMU), Texas A&M University (TAMU).
Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus
(Canestrini), and the mosquito Aedes aegypti
(Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al
., 2002 and Stables et al
., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.
Immunology, Issue 50, Aequorin calcium reporter, coelenterazine, G protein-coupled receptor (GPCR), CHO-K1 cells, mammalian cell culture, neuropeptide SAR studies (SAR= structure-activity relationships), receptor-neuropeptide interaction, bioluminescence, drug discovery, semi-throughput screening in plates
Isolation and Culture of Human Fungiform Taste Papillae Cells
Institutions: Monell Chemical Senses Center, New York University College of Dentistry, AFB International.
Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4
. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8
. We recently reported on a successful method for the extended culture of rodent taste cells9
. We here report for the first time the establishment of an in vitro
culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C β2, (PLC-β2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-β2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al
Neuroscience, Issue 63, Fungiform, taste cells, cell culture, gustducin, calcium imaging, molecular biology, human fungiform papillae
Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression
Institutions: University of Southern California, University College London.
Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2
. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5
. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g.
age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT
) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC
oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.
Cancer Biology, Issue 66, Medicine, Tissue Engineering, three-dimensional cultures, stromal-epithelial interactions, epithelial ovarian cancer, ovarian surface epithelium, ovarian fibroblasts, tumor initiation
The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Institutions: Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e.
endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
Basic Protocol, Issue 82, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
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
V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting
Institutions: Bio-Rad Laboratories.
The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc.
The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.
Basic Protocol, Issue 82, Biotechnology, Pharmaceutical, Protein electrophoresis, Western blot, Stain-Free, loading control, total protein normalization, stain-free technology
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Protein Isolation from the Developing Embryonic Mouse Heart Valve Region
Institutions: University of North Carolina at Chapel Hill, New York-Presbyterian Hospital/Weill-Cornell Medical Center.
Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 μg of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.
Developmental Biology, Issue 91, heart, valve, embryonic, mouse, development, protein, western blot
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
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Institutions: University of Exeter.
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
Biophysics, Issue 91, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ
and in vivo
express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+
indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+
events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
Viability Assays for Cells in Culture
Institutions: Duquesne University.
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.
Cellular Biology, Issue 83, In-cell Western, DRAQ5, Sapphire, Cell Titer Glo, ATP, primary cortical neurons, toxicity, protection, N-acetyl cysteine, hormesis
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages
Institutions: Kingsborough Community College, University of Texas at Austin, Kean University.
The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae
. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages.
We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.
Infection, Issue 75, Medicine, Immunology, Infectious Diseases, Microbiology, Molecular Biology, Cellular Biology, Biochemistry, Bioengineering, Bacterial Infections and Mycoses, Mucosal immunity, oleic acid, linoleic acid, linolenic acid, cholera toxin, cholera, fatty acids, tissue culture, MTT assay, mouse, animal model
Mouse Adrenal Chromaffin Cell Isolation
Institutions: University of California, Irvine (UCI), University of Southern California, Keck School of Medicine, University of Southern California, Keck School of Medicine, University of California, Irvine (UCI).
Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.
Developmental Biology, Issue 2, Neuroscience, mouse, adrenal
PuraMatrix Encapsulation of Cancer Cells
Institutions: Harvard Medical School, Dartmouth College, Harvard Medical School.
Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these models utilize reconstituted basement membrane derived from animals which contain a variable amount of growth factors and cytokines that can influence the growth of these cell culture models. Here, we describe in detail the preparation and use of PuraMatrix, a commercially available self assembling peptide gel that is devoid of animal-derived material and pathogens to encapsulate and propagate the ovarian cancer cell line, OVCAR-5. We begin by describing how to prepare the PuraMatrix prior to use. Next, we demonstrate how to properly mix the PuraMatrix and cell suspension to encapsulate the cells in the hydrogel. Upon the addition of cell culture media or injection into a physiological environment, the peptide component of PuraMatrix rapidly self assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 5-200 nm1
. In addition, we demonstrate how to propagate cultures grown in encapsulated PuraMatrix. When encapsulated in PuraMatrix, OVCAR-5 cells assemble into three dimensional acinar structures that more closely resemble the morphology of micrometastatic nodules observed in the clinic than monolayer in vitro models. Using confocal microscopy we illustrate the appearance of representative OVCAR-5 cells encapsulated in PuraMatrix on day 1, 3, 5, and 7 post plating. The use of PuraMatrix to culture cancer cells should improve our understanding of the disease and allow us to assess treatment response in more clinically predictive model systems.
Cellular Biology, Issue 34, PuraMatrix, OVCAR-5 cells, Cancer, in vitro models, 3D, Encapsulation, PuraMatrix,
Preparation of 2-dGuo-Treated Thymus Organ Cultures
Institutions: University of Birmingham .
In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo
can limit analysis of individual cellular components and particular stages of development. In vitro
culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro
. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro
with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
Immunology, Issue 18, Springer Protocols, Thymus, 2-dGuo, Thymus Organ Cultures, Immune Tolerance, Positive and Negative Selection, Lymphoid Development


Sacramento River Blues – Piano Del Cuore
This arrangement consists of 2 pages. Chords have been added to this sheet.
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ROBERT MAPPLETHORPE at Olga Korper Gallery (17 Morrow), today (Thursday, September 8) to October 1, reception 6-9 pm today. 416-538-8220. Rating: NNN Rating: NNN
Sixteen years after his death, art history is still sorting out the place of American photographer Robert Mapplethorpe . His beautiful, sexually charged black-and-white photography recalls the 70s and 80s yet seems neither dated nor retro.
The Olga Korper show has 38 large, well-chosen limited-edition prints produced during his lifetime, all in splendid off-white frames. Extraordinary compositions and contrasts bring the nudes, still lifes and portraits of the famous to life. The magic gaze of Mapplethorpe's lens can render virtually anything erotic.
The photo Livingston zones in on a man's upper chest and throat. A master at depicting masculinity, Mapplethorpe captures the shadows rippling in minimal light across his subject's muscular form and textured skin.
In Legs/Melody, a shapely pair of crossed legs in fishnet stockings stands out from a black background. Playful use of depth of field leaves some upper thigh out of focus, suggesting the viewer's close proximity.
Anyone can make a flower look sexy, but Mapplethorpe didn't restrict himself to floral salaciousness. Extending the same gaze to a pair of hairy coconuts draped in fabric, he coyly turns them into a magnificently lit scrotum.
His portraits celebrate the male or female form while also intimately depicting their subjects' character. Among others, there's a fabulous shot of sculptor Louise Nevelson drawn up to her full, scary height, and a few engaging looks at Mapplethorpe's close friend Patti Smith.
Unfortunately, this selection does not convey a sense of Mapplethorpe's complete oeuvre. Missing are the notorious sado-masochistic works that incurred the wrath of conservative groups in the U.S. The decision to exclude the hardcore to avoid misrepresenting the artist as shock-value-dependent seems misguided.
Rather, an overview of Mapplethorpe should include all his themes without prejudice, or fear of it.

domain: artistic


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At Live Gourmet we have a saying when it comes to adding our fresh lettuce to your meals, “It’s better with butter – butter lettuce that is.” Salad Bar favorites such as Iceberg Lettuce or Romaine Lettuce contain less vitamins and nutrients in comparison to these leafy greens. Did you know that Butter Lettuce is rich in calcium and a good source of iron, vitamins A and C?
|Some bunny loves lettuce!|
When you buy our Living Butter Lettuce you’ll notice that the roots are still attached – this is because it’s a living plant. We use hydroponic growing methods which help reduce contamination and dirt, bringing you cleaner and fresher produce. In addition to Butter Lettuce we grow Upland Cress, Red Butter Lettuce and Arugula.
This week we’ll examine each of these types of greens to get the nutrition facts straight. Read these tips before you reach for the Iceberg!
Nutrients: Arugula has many vital phyto-nutrients, and antioxidants that can immensely benefit health. It contains the minerals copper and iron. It’s also a great source for folic acid.
Vitamins: Contains vitamin K – great for bones, vitamin A – great for skin and vitamin C – fights infection.
Calories: A serving of fresh leaves totals around 10 calories.
Fresh Tip: Add smaller leaves to your burger or sandwich. Cook leaves in a soup, sauce or stew.
The next time you are tossing together a lunchtime salad or wrapping up a dinner you’ll be able to answer why your leafy greens are better!

domain: agronomy


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Projekt wnętrz mieszkania - Małe wąskie schody jednobiegowe drewniane, styl nowoczesny - zdjęcie od ARCHISSIMA
Aranżacje wnętrz - Schody: Projekt wnętrz mieszkania - Małe wąskie schody jednobiegowe drewniane, styl nowoczesny - ARCHISSIMA. Przeglądaj, dodawaj i zapisuj najlepsze zdjęcia, pomysły i inspiracje designerskie. W bazie mamy już prawie milion fotografii!
Step inside a canalside London home | Ideal Home
With a waterside location, this refurbished 1970s London home is a tranquil spot in the centre of town. Homes & Gardens takes you around a contemporary canal-side home. For more house tours, room ideas and expert decorating advice, visit housetohome.co.uk
Hallway ideas, designs and inspiration | Ideal Home
Hallway design ideas for whatever space you have to play with. Find out how to make the most of your home's entrance hall with these looks and styles
Classic Glass Staircases | Traditional Glass Staircases | Jarrods
Modern mix of glass & natural oak with carpeted treads that sweep around the first-floor landing. Allowing for a contemporary style staircase with half landing. To add to the modern finish, glass has been inset into the wooden staircase frame (alternative to traditional spindles). JARRODS STAIRCASES We pride ourselves on creating beautiful handmade staircases for your home. Covering England, Wales & the Channel Islands. For all enquiries, please visit our website or call 02920 529797.
HOUSE FIVE — THREE BIRDS RENOVATIONS
Most developers would have chosen to detonate rather than renovate this Fibro shack, but we wanted to prove you don't need to knock these homes down, you can renovate them into your dream home and fast! Moreover, this reno holds a very special place in our hearts, especially Bonnie's - watch Episode 2 to learn of the heartbreak behind this house.
fun with “Accent” lighting, you can use to emphasize certain aspects of your room and add drama. Even tho yr table lampsdo this, is great to bring in more. Custom shelving with integrated lighting is striking. Niche lighting is fantastic at highlighting displays adding depth to a room with the play between light and shadow on the objects. A great and affordable way to bring focus to a corner is a display of the light up twigs, another fun thing 2 add is fairy lights n glass bowl
23+ Most Popular Small Basement Ideas, Decor and Remodel
23+ Most Popular Small Basement Ideas, Decor and Remodel | Tags: small basement ideas low ceilings,small basement ideas layout,small basement ideas on a budget
Evening Standard Page 1 15022017
Creative Direction and Interior Design British Designer Tara Bernerd is founder of the established interior architectural practice, Tara Bernerd & Partners. Working with an executive team of highly experienced architects and designers based out of the London Headquarters in Belgravia the focus for Bernerd is on the relevance of creative direction and interior design. Bernerd's business interests continue to grow, working increasingly on a global platform with projects in New York, Chicago…


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I gotta say, I don’t even have a cat…but these are KOOL!
Last November, i-Tunes made a LOT of people happy with their long-awaited release of the [lastfm link_type=”artist_info”]Beatles[/lastfm] catalog for download. But, there were some missing pieces. One of which was the Anthology series from the […]
If you love music and want to help with the Japan relief efforts, I’ve got great news for you. “Songs For Japan” is available today from Universal on i-Tunes. No matter what you’re style is, […]

domain: hobby


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LONDON, Nov 25 (Reuters) - Uber was stripped of its London operating licence on Monday for the second time in just over two years as the city’s regulator said the taxi app was not “fit and proper”, having put passenger safety at risk.
A change to Uber’s systems allowed unauthorised drivers to upload their photos to other drivers’ accounts, meaning they could pick up passengers as if they were the booked driver, which happened in at least 14,000 trips, Transport for London (TfL) said.
“It is unacceptable that Uber has allowed passengers to get into minicabs with drivers who are potentially unlicensed and uninsured,” Director of Licensing, Regulation and Charging at TfL, Helen Chapman said on Monday, the day the firm’s licence expires.
The Silicon Valley-based company has 21 days to appeal the decision and can continue to operate throughout the process, which is likely to include court action. (Reporting by Costas Pitas; editing by James Davey)
Our Standards: The Thomson Reuters Trust Principles.

domain: law


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Payday loan offer alternative economic options for buyers requiring fast money.
Whether you are facing a financial challenge or wanting to stay away from one, rapid pay day loan debts may possibly provide the financial reaction you’ve been looking. The choice web financial products enhance other sorts of money, furnishing quickly funds for employees having difficulties to make stops meet between paydays.
When you need assist encompassing monthly outgoings or an unanticipated expense affects finances, responding to a couple of pre determined questions aids you to definitely determine whether or else perhaps not an instantaneous pay day loans may be the foremost way to investments requirement.
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Whenever is it possible you payback the mortgage rapid wage advance financial loans are not continuous people this funds have to be repaid within weeks or several months. If you require many payback, different financial products are available, which may much better serve your requirements 12 months. However, if you’re ready for rapid repayment, through a payday I am pending, a instant money debt will be the versatile cash solution needed to help remain latest.
Can you pay for cost payday advances give rapid revenue needing appropriate repayment. The loans which versatile helps workers with steady money and area in their paying plans for fees. You have to do their maths prior to making obligations, ensuring your wages stream is sufficient to produce repayments which are appropriate.
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As soon as you see a normal debt, standard creditors might require directly check outs, decreasing the to funds. And you should plan to supply considerable paperwork, helping the financial financing system. Smooth financial obligation companies have a approach which different.
Starting a loan application that is online the 1st step toward money acceptance are a really finance dealer, so an individual application when it comes down to financing opens doorways to a few leading lenders. The easy on-line program asks for details about your earnings and operate, as well as guides such as for example their appropriate label including to take into account for any internet based that’s immediate you
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The busy funding markets contains lots of strategies from credit card bills to https://signaturetitleloans.com/payday-loans-hi/ house financing you should coordinate the sort of financial support you opt to their task close by. Do you actually require a several 12 months present to get a property or car purchase as well as an easy retail credit score rating option, for instance a revolving store card the sort of financing you select not simply impacts your personal expenses of accounts, but it also has an effect on exactly how quick you might finances.
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Simple on the web Application quick financing supply payday reduction, embellishing funds to hold one to your personal future fee. You do not have time to hesitate with instant purchasing the product range timely solution is a straightforward software which online the ball moving, utilizing simply minutes to try permitting that select the finances from the financing quicker.
Quickly suggestions and quick Loan Turnaround Times When time period is just solid, material and mortar creditors may slow down we lower. Banking institutions, constructing forums, and financing unions feed diverse funding possibility, though the funding acceptance may necessitate in individual program along with other time mandates being taking in. Quick pay day loan specialists have the ability to provide quick notice and affirmation. The work obtains interest which fast once you be prepared for a favored financial institution, money is moved on to your present levels, right away.

domain: economics


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A collection of short stories from the USA Today bestselling Shifter Academy world!
by Angel Leya, Tricia Barr, Joanna Reeder, and Jesse Booth
Genre: Short stories, young adult, paranormal romance, urban fantasy, fairy tale, fairytale retelling
Delve deeper into the USA Today bestselling world of Shifter Academy with four incredible short stories.
Making Waves (by Tricia Barr)
Mermaid seer Delphine has foreseen the destruction of the Mer if they choose to stay in the ocean. But she’s also seen another path, one that can save them: migrating to land.
She’ll need a plan, and a ton of money to facilitate their move to the human world. But a rival Mer who wants to claim her as his bride, a significant lack of seed money, and a few pesky human rules are standing in her way. Can Delphine push herself and her visions further than they’ve ever gone before to save her people, or will her great mer kingdom meet its end?
Blood Heritage (by Jesse Booth)
Join Draven on his twentieth birthday as he finally becomes a vampire and takes the mantle of the Denholm Heir.
His first task? Prove his abilities and worthiness.
But Draven’s ambitions are lofty, even for the power-hungry vampire race. Will his ambitions jettison him into the ranks of the revered, or will they be his undoing?
Dante & Calliope (by Joanna Reeder)
This shifter fairy tale, told in the style of The Princess Bride, follows a young couple who find each other irresistible, yet they are at impossible odds.
What is keeping them apart, and what lesson does Lady Victoria Dracul wish her children, Kol and Tatiana to learn through such a story?
In Cinders (by Angel Leya)
Ella is a phoenix shifter who has yet to shift. When her widower father finds a new bride, she’s reluctant to accept the change. But change proves to be too much when the night of his wedding, her father has a heart attack and dies. Ella is reeling from the loss, but her step-mother and step-sisters are determined to carry on–with or without her.
In Cinders is a fairy tale retelling of Cinderella, set in the victorian South of the Shifter Academy world.

domain: library


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By Albert Makendenge
In most African countries, agriculture accounts for 70 percent of the labor force, over 25 percent of GDP and 20 percent of agribusiness. The industry remains largely traditional and concentrated in the hands of small holder famers and pastoralists. There are a number of characteristics common to all smallholder farmers, whether or not they control the land they farm or the commodities they produce.
- They produce relatively small volumes on relatively small plots of land.
- They may produce an export commodity as a main livelihood activity or as one of many activities.
- They are generally less well-resourced than large scale commercial farmers.
- They may depend on family labor and/or may hire workers.
- They are usually considered part of the informal economy (because they may not be registered, tend to be excluded from aspects of labor legislation, lack social protection and have limited records.
Smallholders face a mix of risks and challenges which threaten their livelihoods, food security and nutrition – usually not getting the same kind of support and not getting the same level of access to finance and technologies as the bigger commercial farms. They form a vulnerable group that is often neglected by development policy and they account for most of the world’s poor and hungry. But however small they may be, smallholders in their numbers remain the key to ending hunger and malnutrition worldwide.
If the situation is to change and the plight of the small holder farmers is to end, world agriculture and every sector connected to it will need to undergo some major changes to meet the food demands of an increasingly growing population. And while these changes occur, most importantly policy changes, smallholder agriculture which can contribute even more than it currently is even in the face of an unfair playing field, cannot afford to be left out of the equation. Their inclusion is clearly key on the path to achieving the world’s sustainable development goals. At vaMudhumeni our goal is to help all farmers by sharing farming advice on several multi-media platforms like Facebook, WhatsApp, Instagram, YouTube etc .We also have a wide range of agricultural experts and consultants on our team as well who are willing and able to assist all farmers. Visit our blog at www.vaMudhumeni.org to learn more.


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Bronchoscopy is useful in both the diagnostic work-up as well as a therapeutic modality.
The timing of performing bronchoscopy is controversial. One suggestion is to perform urgent bronchoscopy when there is rapid deterioration and elective bronchoscopy within 24–48 h in stable patients.
In patients with massive hemoptysis, rigid bronchoscopy is the method of choice due to its better suction ability. The major limitation of rigid bronchoscopy is that it is difficult or even impossible to visualize the upper lobes or peripheral lesions
Selective intubation of one lung can be performed by a rotational technique. After intubating the trachea, the tube is rotated through 90 in the direction of the desired placement until resistance is felt. The tube placement should be confirmed both clinically and radiologically.
Alternatively, a double-lumen endotracheal tube can be passed to protect the unaffected lung.
Currently, surgery represents one of a few treatment options, but still represents the only definitive one.
Surgical mortality ranges 1–50%.
Surgery remains the procedure of choice in patients with localised bronchiectasis, trauma, hydatid cyst, arteriovenous malformations, thoracic aneurysm and aspergilloma, because it is curative for these underlying diseases.
A patient with an aspergilloma should undergo surgical resection. Unfortunately, such patients often have significant concomitant bronchiectasis that may preclude them from surgery due to insufficient pulmonary reserves. In these patients intracavitary Na of K iodide is curative. In some series external beam irradiation was used .
Some of the immunological diseases, such as Goodpasture’s
disease, can present with massive haemoptysis. These diseases do not need invasive procedures and are usually treated with high-dose corticosteroids, cytotoxic agents or plasmapheresis.
In practice tailored management for hemoptysis is needed

domain: literature


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Human Nature: Fate vs. Free Will
For centuries, it has been human nature to strive for freedom. We struggle to create our own futures, regardless of our presumed fate. Songwriters, artists and even play writes constantly include stories of free will and destiny. Sophocles, a famous Greek playwright, beautifully includes fate and free will as themes in his plays Oedipus the King and Oedipus at Colonus. This motif is so abundant in Greek literature because oftentimes characters try to avoid an oracle that they don’t want to accept to be true; this opens up an opportunity for human nature to fight fate, which is what Sophocles sets the basis for his plays. Although each character’s fate is chosen for them, they try to fight their destiny because they want a brighter future. In Oedipus the King, there are two major oracles that cause Oedipus to fight for a different fate. The oracle declared, ”…doom would strike him down at the hands of a son, our son, to be born of our own flesh and blood” (Sophocles, Oed 786-788) this is in reference to his father, and also that Oedipus was, “ fated to couple with your mother, you will bring a breed of children into the light no man can bear to see- you will kill your father, the one who gave you life” (Sophocles, Oed 873-875). These two events happen very quickly in the play and from then on he tries to fight his fate. As he struggles, his destiny is becoming more of a reality without him knowing. This makes Oedipus a tragic character, because he purposefully tries to make only good, but ends up killing his father, and marrying his own mother. One could argue that if Oedipus never heard the fate from the oracle, he wouldn’t have tried to avoid it the way he did, and could have possibly changed his own destiny. This makes the oracle that Oedipus received a self fulfilling prophesy, meaning that the existence of the fate created such a distaste in the mind of Oedipus that it consumed him and made it a reality. Oedipus is a man...
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Nowadays, even Polish people are travelling whole around the world. It is possible thanks to the cheap airline companies, which arrived in here dozen years ago. Year after year, there are more and more different travel destinations available, even to the very distance continent. You like to visit your uncle in New York? No problem, you can get there from Warsaw. You wish to have a vacations of your live in China? A lot of different flights are available to this country from Poland. There is a lot of options, you just need to look for it.
Going to the United States is simpler then ever. But even if we are a member of European Union, we still need to have a visa to go there. You can get it in Krakow or in capital, but make sure to arrange your appointment first. After you get this paper, you could book your flight. You don’t have to go to another country just to get to the Sates, there are plenty flights from Warsaw available to the new York. Even if you like to go to another city in there, this option is the cheapest. After you arrive in NYC, you can catch another, domestic flight to different city, it is very cheap in there. But before you get to the plane, make sure you don’t have any kind of food in your baggage, also in this checked one. Because it is forbidden to bring any type of meal to this country.
Another interesting destination is China. If you like to go there from Poland no problem, because there are a lot of flights from Warsaw to Beijing. Most of it are in very reasonable price, if you book it couple months before your journey. Beijing is very interesting place to visit, so even if you are planning to explore another part of China after you arrive in there, you should have a tour around this town. To any other part of a country you can grab a domestic flight or a train. This second option is longer, but you will have an opportunity to see a lot of a country. But before you go to the airport in Warsaw, prepare to your flight properly. This is long trip, so make sure you pack a lot of things for entertainment. Some interesting book or nice magazine should be fine.
There are a lot of various flights from Warsaw available to another continents. If you like to go to the State, you don’t have to travel to Berlin first. The same is with China. To get to the Beijing, you just need to book a flight from Polish carrier. This is nice option, because you don’t need to change planes during your trip, which sometimes could be very frustrating.

domain: geography


text:
Discover more from Mama Dentata
Women Talking About Women Talking
A field report from the Education For Women's Liberation conference in London
Last weekend, I was one of many women who travelled to London to attend the Education for Women’s Liberation conference. Cohosted by Woman’s Place UK and University College London’s Women’s Liberation group, the event page set out the theme for the day as such:
Focusing on education in feminism and women’s lives, the conference will address interconnected themes including: women’s voices in education; sexual harassment in schools and universities; the history of women’s access to education in local and global contexts; the ways in which women’s entrance into education and research has changed workplaces and academic disciplines; the impact of gendered stereotypes in educational spaces, and sex and relationship education in schools.
To be honest, nothing about this description sounded exceptionally exciting at first blush. Bog-standard feminist fare, this would have seemed to me, when I finished my Women & Gender Studies degree nearly sixteen years ago. Back when you could use the word ‘women’ and no one batted an eye, because everyone knew exactly what that word meant, and had always meant (whereas almost no one I spoke to outside of my major seemed to have even heard of ‘gender’). But somehow, speaking about the female experience has been shunted out of the centre of feminism since then. It now tends to get labeled as ‘radical’. And therefore, something to get excited about after all.
Which meant two things. First, that the conference was sold out; all 900 tickets had been snapped up. And second, that when I rocked up at 9am with my own ticket in hand, I had to get past protesters. Of both sexes - and of various genders, I’m sure. There weren’t many of them, and they weren’t up to very much when I walked past (though apparently things did kick off more later in the day), so I didn’t pay them too much heed. But the overall impression I got at that early hour was of young-ish women. Women protesting against the right of women to meet and speak to each other about women’s issues and rights. Who could apparently only conceive of an organisation that would put together all-female panels on women and education as a ‘hate group’, and therefore stood outside chanting “Shame!” at me as I walked past, and at the university for allowing such an event to take place on their campus.
On that very same day, and every other day I traveled around that massive metropolis, I saw posters all over the underground for the film Women Talking. It’s based on a novel that was based on actual events. Where a group of men in a Mennonite community decided it would be a swell idea to source animal anaesthetic from a vet so they could knock out and rape over 100 women in their colony. I mean, it says ‘women’, but amongst those brutalised were girls as young as three. Three.
And indeed, the trailer for the film is narrated by a little girl’s voice. As it opens, she says, “Where I come from, where your mother comes from, we didn’t talk about our bodies.” As she says this, we see Rooney Mara’s character waking up to discover bruises across her thighs that look far, far more like the markings of a car crash than anything I’ve ever woken up to in the twenty years since losing my virginity. The rest of the trailer shows the women of the colony discussing whether they should leave their home, or stay and fight the men.
So. On the one hand, a film about women talking. That is explicitly called Women Talking. Featuring almost nothing but women talking. About the fears and responsibilities and vulnerabilities and choices that are distinct to their fate of being female. Exploring their options together, with minimal input from men. In the context of a Hollywood production, such activities are deemed worthy of a premium marketing push, and widespread acclaim.
And on the other hand, a real life gathering of women talking. Women who work as writers, lawyers, teachers, health workers, researchers, politicians, organisers, artists, and in all manner of other occupations. Talking. About our fears and responsibilities and vulnerabilities and choices as females. Exploring our options together, with minimal input from men. In the context of a university campus, such activities are deemed ‘hate speech’ by some locals, and require a small security detail to make sure women aren’t accosted as we come through the doors.
It seems a strange juxtaposition to me.
Women. Using words. Clearly, as a woman writing, this is something I care about very much. But it’s much more than vocational. I’ve recently come to realise it has probably been the preeminent preoccupation of my life. I tend to think of words as what I am best at, but, in truth, I struggle with them terribly. When there’s something I really care about expressing, my jaw clenches, my stomach turns into a ball of acid, and I shake so much I can’t sit still. My whole body conspires to steal my fingers and mouth away from my mind, and leave me mute. Classic signs of a fight-or-flight system hijacking.
I used to assume this was pretty normal. And I think, unfortunately, some version of it is. But lately I’ve been wondering whether I’m actually a particularly reactive case. I grew up in a household where having a ‘smart mouth’ was punished, sometimes quite physically, and sometimes with words hurled so violently, their impact felt physical. Both hurt, and both were terrifying. Again, I don’t think there’s anything that unusual about such an upbringing, and know many homes are much more oppressive. But I find it unlikely that it wouldn’t be at the root of whatever has trained my body to be so very fearful of upsetting anyone by speaking. And wonder how much other people - particularly women - struggle against this same conditioning.
When I first escaped to university, and discovered Women’s Studies, and feminism, I was captivated, and elated. I thought I had found the tools that would save me, and other young women, and possibly humanity altogether: words. Like patriarchy, and misogyny, and coercion. Like consumerism, and objectification. Like structural, and complicit. Like vindication, and liberation. And praxis. Words that could spell out why the world was so scary and sordid and astonishingly unfair, and words that could sketch out plans for us to make it better.
However, I did not foresee that these very words would become feminism’s great quagmire. I mean, I knew women could be silenced by preventing us from using our words - by barring us from places of learning and discourse, or by physically strangling our words as we tried to push them out of our throats. What I didn’t realise was how silencing could be achieved just by stealing our words away from us; by gutting them of meaning. That we would get to a place where a preeminent medical journal would think it was better to refer to my sex as “bodies with vaginas” rather than ‘women’. That just as I became brave enough to take on the ultimate transformation and responsibility of becoming a mother, certain activists and politicians would try to steal that brutally hard-won and most meaningful title from myself and mothers like me, and try replacing it with the much diminished term ‘birthing parent’ instead. That the unrelenting marketing of cosmetic products and procedures that my professors had taught me to describe as ‘exploitation’ would be rebranded and celebrated as ‘empowerment’. That the term ‘adult human female’ would, for many, become synonymous with ‘terf’, which would in turn become synonymous with ‘bigot’. And that ‘feminist’ and ‘patriarchy’ would each get bandied about so wantonly as to almost become gibberish. And so on, and so on. So that the so-called feminism of the so-called mainstream would become the stuff of Orwellian Newspeak, stopping women’s gains of the last century in their tracks, and sometimes shoving them backwards. Because how can you advocate for a thing that you can’t properly speak about?
And it was this perversion of the language, this stealing of words, this silencing, that lay at the heart of the conference. Though formally about education, the thing that the speakers and panels and attendees all kept returning to was words. They spoke of many other important things, too, like civic education, public policy, incarceration, assault, safeguarding, and class. But over and over again, what all these disparate issues kept coming back to was the struggle around language. That was the real matter at hand; a reclaiming of what women are allowed to think and research and say. The necessity of having words to accurately describe our lives and bodies, and thus value and understand and protect them. Including, crucially, the word ‘No.’ From the opening remarks of barrister Akua Reindorf, stating that debate is how bad ideas are “exposed to the disinfectant of sunlight,” and that, “There is no inclusion where people are silenced.” To the closing remarks of organiser Ali Ceesay, calling out the “demonisation of girls who say ‘no’ to misogyny,” and insisting that, “talking about our lives will never be an act of hate.”
And in between the opening and closing it was the same; words, and what they mean, and why it matters how we use them. The morning offered an option of five panels. It was a hard choice, but I ended up going to the panel on women’s voices in education - in part because it was going to be chaired by Kathleen Stock. I only found out after I’d taken my seat that she was not able to be there that day after all, and that was a disappointment, to be sure. But the panel ended up being great all the same. The highlight being, in my eyes, Professor Judith Suissa, who spoke with a combination of intelligence, clarity, and vehemence that I can only marvel at and aspire to. “Telling us not to speak of and name our bodies,” she concluded, “is of a piece with trying to control our bodies.” Which should hardly need saying. And yet, it does. In real life just as much as in movie trailers.
In the afternoon, we could choose just one of the twenty-one workshops. That took some fierce agonising, but in the end I decided I really ought to go to the one run by a group called With Woman, about language and maternity. Because that is obviously very much in the realm of what I’m trying to work on with this Substack. The workshop was interesting, but a bit wide-ranging for such a short time slot. However, it did lead me to a couple of exciting resources. The first is a group called All Mothers Work, which no longer seems to be active, but for a while produced glorious statements such as this:
The other was something the presenters kept calling ‘The Paper'. ‘THE Paper’. Which I have since downloaded, and read, and highlighted. And will be referring to a lot going forward, I am sure, and I may even do a whole post on it. Because it deals very thoroughly with the recent push to de-sex the language used in reproductive, maternity, and newborn care. And how, in an effort to make their language more ‘inclusive’, providers are actually making it less clear, less accurate, and less helpful - and indeed, making it dehumanising, and sometimes dangerous to the point of life-threatening. For the children involved, as well as the mothers.
The women who organised the conference, and many in attendance, tended towards the purple, white, and green colour scheme of the Suffragettes. The programmes and lanyards we received were in these colours, and many women were wearing scarves in this combination. I have no such scarf, but I do own a small book called Suffragette Manifestos. Its cover proclaims:
Which sounds nice and punchy, but is actually a chopping up of something Emmeline Pankhurst said in a speech she gave at the Royal Albert Hall in 1908, the transcript of which is contained in said small book:
“They said ‘You will never rouse women’. Well, we have done what they thought, and what they hoped impossible - we women are roused. Perhaps it is difficult to rouse women, and they are longsuffering and patient, now that we are roused, we will never be quiet again.”
I wonder if Ms. Pankhurst really believed that patience was what made women hard to rouse. I, for one, have never been known for my patience. Nor have I ever looked into the swirling mass of feelings inside me and thought, Ah, that’s what’s bothering me. I’m too patient today. Nope. I reckon that if women then were hard to rouse, it would have been for the same reasons as today. Most of which boil down to one thing: fear. Fear of being seen as selfish, or unkind, or on the wrong side. Fear of being kicked out of social groups. Of losing our jobs, and being unable to support ourselves and our loved ones. Of threats to our children. Of being attacked. Arrested. Ostracised. Silenced.
Most of the above did happen, in varying combinations, to the women speaking at the conference. Except the silencing part. Silencing was the intended effect of all the other crap thrown at them. But they were not silenced. And instead, they received thunderous applause from the better part of a thousand women last Saturday. Over and over and over again.
Truly, the real benefit of going to the conference was just to hear these fantastically unsilencable women speak about how bizarre (or as Helen Joyce perfectly put it, “incredibly stupid”) this situation is that we find ourselves in. And for those of us who are less assuredly vocal to have the chance to talk amongst ourselves, too. Judging by my own experience, the handful of women I had good conversations with, the many other women who asked questions in the panels and workshops, and the wave after wave of applause across the main hall, there was a huge amount of catharsis taking place, along with a replenishing of courage. Which I needed as much as anyone. And I felt that the lockdown years, and the disconnection that came with them, really are behind us now, at last. And that a new phase, inviting new words and interactions and choices, lies ahead.
If you’re interested in hearing more about the conference, you can find quite a lot of other women’s coverage of the day by using #Ed4WomensLib. Or if you were there yourself, and want to say hi, by all means - let’s talk.

domain: sociology


text:
Kienbock's disease is the death and deterioration of the lunate, one of the small bones in the wrist. It usually occurs in young adults and causes wrist pain, weakness, and loss of motion.
The exact cause of Kienbock's disease is unknown. It may result from a compromise in the blood flow that supplies the lunate. Theories suggest that an increase of pressure within the lunate leads to congestion of the blood vessels that supply the bone, causing death of the bone cells. Some believe that it may be caused by trauma, although in many cases the disease cannot be tied to a specific traumatic event. A shortened ulna may also contribute to development of the disease, because a shortened ulna can cause the lunate to receive higher loads of pressure as the wrist is used.
Wrist pain, limited motion, and a weak grip are very common. Swelling and pain over the lunate on the top of the hand may also be present. The intensity of these symptoms may range from mild to severe.
Kienbock's disease is diagnosed with x-rays of the wrist, which may show increased density of the lunate, cystic changes, fragmentation, bone collapse, or arthritis, depending on the stage of the disease. A bone scan or MRI may also be needed to confirm the diagnosis.
The ideal treatment for Kienbock's disease is still debatable. Prolonged casting or bracing to rest the wrist and anti-inflammatory medications are usually the first recommendation for pain relief, but they do not necessarily halt disease progression. Surgery is usually recommended if conservative treatment does not resolve the pain. Surgical options may include shortening of the radius, vascularized bone grafts, or fusion of the carpal bones in the wrist.

domain: health


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