text:
Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes’ association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.
21 Related JoVE Articles!
Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
Institutions: University of Missouri, University of Missouri, University of Missouri.
As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.
Genetics, Issue 67, Molecular Biology, Cellular Biology, RNA, mRNA, Ribonucleoprotein, immunoprecipitation, microarray, PCR, RIP-Chip
Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
Institutions: University of Ottawa, Montreal Neurological Institute, University of Ottawa.
Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.
Cellular Biology, Issue 92, cellular stress, translation initiation, internal ribosome entry site, polysome, RT-qPCR, gradient
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
Institutions: The Scripps Research Institute, City College of New York.
The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32
P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro
, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs.
Infectious Diseases, Issue 87, Cleavage, Polyadenylation, mRNA processing, Nuclear extracts, 3' Processing Complex
Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Institutions: McGill University, Karolinska Institutet, McGill University.
mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed “anota” algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.
Biochemistry, Issue 87, Cells, Eukaryota, Nutritional and Metabolic Diseases, Neoplasms, Metabolic Phenomena, Cell Physiological Phenomena, mRNA translation, ribosomes,
protein synthesis, genome-wide analysis, translatome, mTOR, eIF4E, 4E-BP1
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
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
Institutions: University of Pennsylvania, Integrated DNA Technologies, Inc..
The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3’-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.
Genetics, Issue 90, RNA, imaging, single molecule, fluorescence, living cell
In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells
Institutions: University Hospital Tuebingen.
The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.
Genetics, Issue 93, mRNA synthesis, in vitro transcription, modification, transfection, protein synthesis, eGFP, flow cytometry
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation
Institutions: German Cancer Research Center (DKFZ).
Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting 'translatomics' to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo
samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.
Neuroscience, Issue 86, central nervous system, CNS, translation, polyribosome fractionation, RNA, Brain, spinal cord, microarray, next-generation sequencing, gradient, translatome
Analysis of Translation Initiation During Stress Conditions by Polysome Profiling
Institutions: Laval University, CHU de Quebec Research Center.
Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.
Cellular Biology, Issue 87, Translation initiation, polysome profile, sucrose gradient, protein and RNA isolation, stress conditions
The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation
Institutions: University of Iowa Carver College of Medicine.
oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro
development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro
development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila
follicles provides a rich source of information into widely conserved processes of development and morphogenesis.
Developmental Biology, Issue 82, Drosophila melanogaster, Organ Culture Techniques, Gene Expression Profiling, Microscopy, Confocal, Cell Biology, Genetic Research, Molecular Biology, Pharmacology, Drosophila, oogenesis, follicle, live-imaging, gene expression, development
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Institutions: University of Alabama Huntsville, Stanford University .
Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7
. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13
. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14
. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro
translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli
strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16
. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1
. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14
and/or ii) alkylation protection assays1,14,17
Molecular Biology, Issue 48, Ribosome stalling, ribosome isolation, peptidyl-tRNA, in vitro translation, RNA chemical modification, puromycin, antibiotics.
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
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
Institutions: SwitchGear Genomics.
MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.
Genetics, Issue 55, MicroRNA, miRNA, mimic, Clone, 3' UTR, Assay, vector, LightSwitch, luciferase, co-transfection, 3'UTR REPORTER, mirna target, microrna target, reporter, GoClone, Reporter construct
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
Institutions: Cleveland State University.
The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1
. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1
. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2
. Codon bias is organism as well as tissue specific2,3
. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4
. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8
. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9
. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo
) protein folding1,7,10,11
. To understand the process of protein folding in vivo
, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8
. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro
translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro
transcribed mRNA is used for in vitro
translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.
Genetics, Issue 65, Molecular Biology, Ribosome, Nascent polypeptide, Co-translational protein folding, Synonymous codon usage, gene regulation
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
Institutions: Cleveland State University.
Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5
. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli
protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA
. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAG
P-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9
. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7
. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo
in E. coli
cells and in vitro
in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11
. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro
Molecular Biology, Issue 64, Ribosome, nascent polypeptides, co-translational protein folding, translational arrest, in vitro translation
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
Institutions: Max von Pettenkofer Institute, University of Cambridge, Ludwig-Maximilians-University Munich.
The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e.
total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila
, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.
Genetics, Issue 78, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
In Vivo Modeling of the Morbid Human Genome using Danio rerio
Institutions: Duke University Medical Center, Duke University, Duke University Medical Center.
Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo
complementation in zebrafish. Zebrafish (Danio rerio
) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo,
and can be genetically manipulated.1
These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.
Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model
Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Institutions: University of Toronto.
In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e.
"free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ
hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.
Cellular Biology, Issue 70, Biochemistry, Genetics, Molecular Biology, Genomics, mRNA localization, RNA, digitonin extraction, cell fractionation, endoplasmic reticulum, secretion, microscopy, imaging, fluorescent in situ hybridization, FISH, cell biology
Microinjection of Zebrafish Embryos to Analyze Gene Function
Institutions: Harvard Medical School, Children’s Hospital Boston.
One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo. Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product. Conversely, processed mRNA can be added to the embryo to increase levels of a gene product. The vehicle for adding either mRNA or morpholino to an embryo is microinjection. Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour. This video shows all the steps involved in microinjection. Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish. Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume. Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.
Developmental Biology, Issue 25, zebrafish, morpholino, development, microinjection, heart of glass, heg

domain: medical


Geothermal Electricity: The Earth's warmth-named geothermal Vitality-escapes as steam at a warm springs in Nevada.
The Earth's warmth-referred to as geothermal Power-escapes as steam in a incredibly hot springs in Nevada. Credit score: Sierra Pacific
Geothermal Power is the warmth with the Earth. It really is thoroughly clean and sustainable. Means of geothermal Strength range from the shallow floor to sizzling water and incredibly hot rock identified a few miles beneath the Earth's area, and down even deeper into the really higher temperatures of molten rock termed magma.
Practically almost everywhere, the shallow ground or higher 10 ft from the Earth's floor maintains a nearly consistent temperature in between fifty° and 60°File (10° and sixteen°C). Geothermal heat pumps can faucet into this resource to heat and funky structures. A geothermal warmth pump program consists of a heat pump, an air delivery program (ductwork), and a heat exchanger-a system of pipes buried while in the shallow ground near the setting up. While in the Wintertime, the heat pump gets rid of heat through the warmth exchanger and pumps it to the indoor air supply program. In the summertime, the process is reversed, and the warmth pump moves warmth from your indoor air into the warmth exchanger. The heat removed from the indoor air in the summer time can even be utilized to offer a no cost source of very hot h2o.
In the United States, most geothermal reservoirs of very hot h2o are located while in the western states, Alaska, and Hawaii. Wells is usually drilled into underground reservoirs for your era of energy. Some geothermal electrical power vegetation utilize the steam from a reservoir to electricity a turbine/generator, while some use the very hot h2o to boil a working fluid that vaporizes then turns a turbine. Incredibly hot water near the surface area of Earth may be used directly for heat. Direct-use programs include things like heating properties, expanding vegetation in greenhouses, drying crops, heating drinking water at fish farms, and several other industrial procedures such as pasteurizing milk.
Sizzling dry rock assets come about at depths of three to five miles everywhere beneath the Earth's surface area and at lesser depths in specified parts. Usage of these means involves injecting cold water down a single properly, circulating it by way of incredibly hot fractured rock, and drawing from the heated h2o from More information A further well. Presently, there aren't any industrial programs of this engineering. Current technological innovation also won't still make it possible for Restoration of warmth straight from magma, the quite deep and strongest source of geothermal Strength.
A lot of technologies have already been produced to reap the benefits of geothermal Strength - the heat from your earth. NREL performs analysis to build and advance technologies for the subsequent geothermal purposes:
Geothermal Electricity Systems:
Geothermal Electrical power Creation
Creating electrical energy in the earth's heat.
Geothermal Immediate Use
Manufacturing warmth directly from scorching water throughout the earth.
Geothermal Warmth Pumps
Using the shallow ground to warmth and funky structures.


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James Pulham and Son was a firm of Victorian landscape gardeners and terracotta manufacturers which exhibited and won medals at London's Great Exhibition of 1851 and 1862 International Exhibition.
James Pulham and Son Wikipedia
James Pulham and Son was founded by James Pulham (1793–1838) of Woodbridge in Suffolk, who was succeeded by his eldest son James (1820–1898) and then by two more generations of eldest sons, both also named James. The firm went out of business in 1939.
The firm was best known for the construction of rock gardens, follies and grottoes in which the firm used both natural stone and their own invention, Pulhamite artificial rock. Pulham and Son also manufactured a wide range of terracotta and Pulhamite garden ornaments, originally at their works in Tottenham, but after 1840 at Broxbourne in Hertfordshire. In 1895 the firm was granted a Royal Warrant by the Prince of Wales, later King Edward VII and some of their work survives at Sandringham House and Buckingham Palace.
After exhibiting at the Great Exhibition of 1862. One of the two works exhibited there was the upper section of what is now Dunorlan Fountain (from Dunorlan Park, Tunbridge Wells). Originally called the 'Hebe Fountain' (due to the image of Hebe - daughter of Zeus, at the top). James Pullham II (the son) received a recommendation for the architectural decoration in terracotta.
At the 1867 Paris Exhibition the company showed the "Preston vase" (several of which were made for the People's Park, Preston), and the Mulready monument. The latter, modelled by Godfrey Sykes consisted of a pedestal 15 feet long and 10 feet wide, supporting an effigy, seven feet long, on a raised bier. It won a silver medal at the exhibition, and was installed at Kensal Green cemetery on its return from Paris.

domain: design


First Look: The Flashy, Fabulous Ladies of the
Young Dolly Parton sitting with a guitar writing a song. Parton wrote every day, and had more than a 100 songs at her disposal. A multi-instrumentalist, Parton was more than equipped to.
based on the song jolene by dolly parton artist info unknown. daily needs of an art bitch


It’s interesting to chart changes in expectations around the average working week. In a recent article in the New Yorker magazine, Tim Wu (Professor at Columbia Law School) was reflecting on what became of the ambition for a 30 hour working week. In the 1960’s, the prevailing wisdom was that with advances in science making routine tasks easier, we should all have been able to look forward to less work and more leisure. Now according to those fine people at the Office of National Statistics, the average working week in the UK is around 39 hours – it’s higher elsewhere in Europe, and considerably higher in the States. What’s happening? Well as longer tasks got easier to complete we have, says, Professor Wu, been overrun by a “plague” or “tyranny” of what he calls “tiny tasks, individually simple but collectively oppressive.” He goes on “And when every task in life is easy, there remains just one profession left: multitasking.”
So the utopia of a short working week is in danger of being replaced with the dystopia of a never-ending pressure to work.
Confusing activity with achievement
I recognise the trend Tim Wu has identified. When I started work, all correspondence was received on paper, and if I wanted to send anything other than a hand written letter it had to be sent through a typing pool. The process of issuing a single typed letter could take two to three days. Now, I can deal with dozens of small, focused pieces of correspondence by email every day. Because I can, I do. With 24/7 access to information through my mobile devices, there is no physical factor which will protect me from this tyranny of tiny tasks. The worst of it is falling into the trap of thinking that because I am being active, my time is, therefore, well-spent – of confusing activity with achievement. I need to create a new eco-system to protect me from this plague, to start a revolution against my ubiquitous and incessantly demanding tyrant.
Coaching in action
Every revolution needs its thought leaders, and for one of mine I’ve turned to Dan Sullivan, Co-founder and President of The Strategic Coach Inc. Dan has developed a model that he calls The Entrepreneurial Time System, built around three types of days.
The first is free days – a 24 hour period when you completely switch off from business-related activity and thinking. It’s the principle of the Sabbath – a day of rest and recuperation. I have one of these a week (in my case usually Sunday) in which I have disciplined myself to ignore the temptation to have a look at my emails and detach myself completely from work for the day. Apart from hitting my Mondays with more energy and a clearer head, I’m sending myself a message – time to care for myself, my family and my friends is as important as the demands made by people in my working life.
Second we have focus days when we spend 80% of time on activities that get results for your business or, if you work for an employer, activities which deliver results that have the most impact on your top priority work objectives.
This is in contrast to our third day – buffer days in which we clear the decks of detailed routine tasks, spend some time on a new skill we want to learn, and plan for the week ahead. I have my own weekly “half hour of power” when I mind-map my week ahead to give myself a picture of the kind of balance I want to achieve between my various areas of focus and responsibility. It’s a great way of getting a sense of what kind of return I am getting for my efforts, where I need to re-prioritise, and when priorities change during the week with new unexpected demands, having a basis for re-focusing to ensure that the most important activities still get done.
Take a few minutes to think about the rhythm of your own working week. And if you’re enslaved by the tiny task-masker, take a stand and join the revolution.


Tyre Changing Service
The most important reason to worry about tread wear is safety. When your tire treads are worn, your car may respond poorly in adverse weather conditions like rain.With good treads your car will grip the road better. Also, having insufficient tread is considered illegal in many states. And finally, worn treads can make other parts of your car wear prematurely.When it’s time to change your tires, your Era Maju Automobil Sdn Bhd dealer wants to make purchasing a new set as simple as possible. We have the best selection of quality tires to fit your vehicle, your budget and your driving style.


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Political cartoonist and artist David G. Brown brought great basic tips to a small audience in the city of La Verne Public Library through a Motion Picture and Animation Workshop on Saturday.
Brown encouraged everyone who showed up for the weekend workshop up to sit at two tables and draw whatever they wanted.
“Art is as individual as you are,” Brown told his audience. “It is just a way for you to express yourself.”
The audience seemed to be engaged in Brown’s step-by-step explanation of how to draw a face.
Brown also showed the audience his self-published works including cartoons on the Los Angeles riots, as well anti-drug, and anti-gang messages.
Brown worked with the City of Los Angeles Cultural Affairs Department to create a book, along with a group of students, which was meant to show children and teenagers the negative effects of drugs and gangs.
Brown has been doing library presentations such as these since the mid-1990s.
“I enjoy working with young people and helping them express what they see in the world,” Brown said.
As a self-publisher, Brown explained, you have to pay for the cost of the material used to make your book.
“L.A. Phoenix” was the first book Brown created from the inspiration he got from the Los Angeles riots.
After they made some basic drawings, Brown gave his audience idea of creating a character and making a story out of it.
Brown also described the process of making a basic storyboard as he brought the new character to life by creating a storyboard.
“This workshop is a good thing for the community,” said Alexander Lorenzen, a Citrus College student attending the workshop said, “It’s a good thing to show comics as a part of literature.”
Also in the audience was 12-year-old Jayden Lebran, an aspiring artist. He brought his own drawings of characters to show Brown, who applauded his creativity and gave him some tips on how to make his characters more exciting.
By the end of the workshop, the audience stayed to keep drawing and talking with Brown.
“I’m glad I had the opportunity to attend this workshop, it was very informative” teacher Linda Long of Los Angeles said. “I would attend more of his workshops.”
Brown ended the workshop by giving away comic books, his autographed books, and the drawings created in the workshop.
Genesis Miranda can be reached at firstname.lastname@example.org.

domain: news


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Casino Royale: Recreating James Bond’s Favorite Games
In present-day interconnected entire world, the allure of on the web gambling has taken the globe by storm. With the click of a button, players can immerse themselves in a digital realm of betting and chance. Regardless of whether it really is the thrill of spinning the reels on a slot machine or outsmarting opponents in a game of poker, online gambling offers countless amusement appropriate at our fingertips.
One particular of the most well-liked aspects of on the web gambling is the wide assortment of video games obtainable. From traditional casino online games like blackjack and roulette to reducing-edge slot equipment, there is some thing for everybody. The introduction of engineering has also paved the way for modern platforms this kind of as pg slot, bringing the excitement of land-based casinos right into our houses.
The ease of on the internet gambling cannot be overstated. Absent are the days of organizing journeys to actual physical casinos, as gamers can now obtain their favorite games anytime and anyplace. The seamless integration of virtual wallets and safe payment techniques guarantees a secure and problem-totally free knowledge, allowing players to concentrate on the thrill of the sport.
Exploring the world of on the web gambling opens up a realm of possibilities. No matter whether you happen to be a seasoned poker professional or a newbie seeking to try out your luck, the on the internet casino landscape delivers one thing for everybody. So, buckle up and get prepared to bet on bytes as we delve into the exhilarating entire world of online gambling.
The Increase of Online Gambling
Online gambling has witnessed a meteoric rise in reputation in modern many years, fascinating hundreds of thousands of players about the planet. With the introduction of technological innovation and the common availability of substantial-pace internet, the attract of on-line casinos, slots, poker, and other types of on the internet gambling has grow to be irresistible for many. The comfort and exhilaration provided by on-line gambling platforms have revolutionized the gambling business, generating a new period of leisure.
One of the most substantial aspects driving the development of on the internet gambling is the accessibility it provides. As opposed to traditional brick-and-mortar casinos, online gambling enables fans to indulge in their favorite video games from the comfort and ease of their own residences. The usefulness of becoming in a position to accessibility a myriad of thrilling video games at any time, without having the need for travel or costume codes, has captivated a huge viewers to the on-line gambling realm.
The extensive variety of choices available in on the internet gambling platforms has also contributed to its surge in reputation. Virtual casinos offer you an extensive assortment of online games, from classic slot machines to modern and immersive on the web casino ordeals. This range caters to the preferences and passions of gamers, making sure that there is one thing for every person. Furthermore, the integration of superior graphics and audio consequences in online video games further boosts the total gaming experience, captivating and retaining players in this digital world of exhilaration.
In latest a long time, the rise of on-line gambling has been especially marked by the emergence of well-known platforms these kinds of as PG Slot. This on-line casino hub has obtained a popularity for its user-pleasant interface, extensive game library, and secure gambling environment. The usefulness and reliability offered by platforms like PG Slot have cemented on-line gambling as a mainstream type of amusement, attracting equally seasoned gamblers and newcomers alike.
In summary, the rise of on-line gambling can be attributed to various elements, which includes the accessibility, range of online games, and the emergence of reliable platforms. The on-line gambling market has experienced unparalleled expansion, attractive to men and women seeking comfort, thrills, and the chance of winning huge from the comfort of their own homes. As technological innovation carries on to advance, the entire world of on-line gambling is probably to broaden additional, fascinating an even larger viewers and providing at any time much more exciting and immersive activities.
Well-liked Video games in the On the internet Gambling Planet
In the thrilling realm of on the web gambling, there are numerous popular games that capture the interest and creativeness of gamers throughout the world. From thrilling slots to basic casino games and immersive poker ordeals, there is no lack of alternatives for these looking for some on the web gambling enjoyable.
A single of the most sought-after video games in on the internet gambling is slots. These mesmerizing games offer you an array of themes, captivating visuals, and thrilling gameplay. Whether or not it truly is classic fruit slots or contemporary video clip slots with intricate storylines, the attract of hitting that lucky mixture keeps players coming again for a lot more.
An additional staple in the online gambling entire world is the standard on line casino games. With on the web casinos giving a large choice of desk video games this kind of as blackjack, roulette, and baccarat, players can encounter the thrill of the casino from the convenience of their personal properties. The comfort and accessibility of these games make them a popular decision for equally newcomers and seasoned gamblers alike.
If strategizing and tests your capabilities against opponents is more your type, then on the web poker is the sport for you. Poker has knowledgeable a surge in reputation in latest years, and the on the web gambling entire world gives the excellent system for fanatics to showcase their talents. No matter whether it really is Texas Hold’em or Omaha, on the internet poker rooms offer you a variety of possibilities to cater to different ability levels and preferences.
So, no matter whether you are into the enticing globe of slots, the timeless allure of on line casino classics, or the strategic struggle of on the web poker, the online gambling entire world has something for everyone. With the ease of enjoying from anyplace at any time, it really is no ponder that these games have turn into a favourite pastime for a lot of thrill-looking for people.
The Long term of On the web Gambling
The speedy development of on the web gambling has paved the way for an thrilling potential in the world of enjoyment. As engineering carries on to progress, the on the web gambling sector is poised to revolutionize the way we experience the thrill of positioning bets from the convenience of our properties.
One particular element that retains enormous likely is the growth of digital fact (VR) in on the web casinos. With VR technological innovation, players can now immerse themselves in a sensible casino setting without having at any time leaving their dwelling rooms. Envision sitting down at a digital slot equipment, pulling the lever, and viewing the reels spin before your eyes. The incorporation of VR into online gambling brings a total new level of exhilaration and engagement to the gameplay.
Another promising avenue for the long term of online gambling lies in the increase of cell gaming. With judi pulsa online of smartphones, much more and far more folks have entry to online gambling platforms at their fingertips. The usefulness of gambling on the go has attracted a greater audience, and this trend is only predicted to develop. Builders are constantly optimizing their platforms for cell gadgets, making sure that gamers can take pleasure in seamless and large-top quality gaming encounters where ever they are.
Additionally, the integration of synthetic intelligence (AI) technological innovation is established to revolutionize the online gambling business. AI algorithms can examine vast amounts of knowledge, enabling platforms to provide personalised tips customized to each and every player’s preferences. This not only boosts the overall person encounter but also makes certain that players have obtain to the online games they take pleasure in most.
In conclusion, the potential of on the web gambling holds excellent guarantee. With developments in VR technologies, the growing popularity of mobile gaming, and the integration of AI, gamers can anticipate a more immersive, hassle-free, and individualized gambling encounter. As the industry proceeds to evolve, on the web gambling is poised to turn out to be a mainstay in the planet of amusement.

domain: entertainment


Landscape design festival MAGENTA aims at actualizing landscape design and environmental art, enhancing the role of green spaces in shaping our urban environment, and promoting interdisciplinary public art. The 2022 edition of the festival is dedicated to tactical urbanism.
Tactical urbanism means short-term green interventions into urban life and environment that invite us to consider long-term effects and a need for complex and socially significant actions. Tactical urbanism interventions can creatively and playfully enliven urban spaces, while actively involving citizens in various activities related to environment, plants and urban elements. It’s one of the most effective ways to accommodate new and revitalize old public spaces, to regenerate them or to reveal their potential.
This is even more relevant in current COVID-19 situation as the viral pandemic and mobility restrictions made global urban population strive for more fresh air and open, spacious and healthy public zones in densely urbanized areas.
Read more information and apply here. Deadline for submission of proposals: April 18th 2021
The MAGENTA Landscape Design Festival, launched in the autumn of 2019, is dedicated to actualizing urban nature design and strengthening the role of landscape design in shaping our better future. The festival is about urban design, the post-industrial environment and interdisciplinary [history, philosophy, architecture, art history, street art, etc.] creation.


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The stock market meandered on Tuesday, ending the day with little change, although the Dow Jones industrial average crept up to another nominal high.
The Dow, which has risen for eight consecutive trading sessions, added 2.77 points to close at 14,450.06 after wavering between small gains and losses for most of the day.
The broader Standard & Poor’s 500-stock index ended down 3.74 points, or 0.2 percent, at 1,552.48. The Nasdaq composite index dropped 10.55 points, or 0.3 percent, to 3,242.32.
Stocks have surged this year as investors became encouraged by a recovery in the housing market and a pickup in hiring. Strong corporate earnings and continuing economic stimulus from the Federal Reserve are also supporting demand for stocks.
The Dow has gained 10.3 percent in 2013. Last week it surpassed its previous nominal high of 14,164.53. The S.& P. 500 has risen 8.9 percent this year and is less than one percentage point from its nominal high close of 1,565.15, set in October 2007.
David Bianco, chief United States equity strategist at Deutsche Bank, said the S.& P. 500 index would probably maintain its momentum in the coming weeks and surpass its nominal high. Strong first-quarter corporate earnings reports could also push the market higher.
“I wouldn’t be surprised if the market has a typical 5 percent pullback in the summer,” Mr. Bianco said. “But I think we go higher before that happens.”
The last significant downturn for stocks began before the presidential election in November, when the Dow fell 8 percent from Oct. 5 to Nov. 15 on investors’ concerns that a divided government in Washington might not be able to reach a budget deal to avoid sweeping tax increases and deep spending cuts.
Stocks have not had a correction, typically defined as a decline of 10 to 20 percent, since November 2011.
Peter Cardillo, chief market economist at Rockwell Global Capital, was among those saying investors should expect a pause in the market’s advance.
“Nothing goes up forever,” Mr. Cardillo said. “We will be headed for a correction somewhere along the line.”
Merck was the biggest gainer in the Dow, advancing $1.38, or 3.2 percent, to $45.04 after it said that a data safety monitoring board had recommended that a study of its cholesterol drug Vytorin should continue.
Among the stocks on the move, Yum Brands rose 89 cents, or 1.3 percent, to $68.73 after the company, which owns KFC, Pizza Hut and Taco Bell, announced a smaller-than-expected drop in its sales in China for January and February after a food scare over its chicken suppliers.
Diamond Foods slumped $1.71, or 9.7 percent, to $15.89 after the company reported disappointing second-quarter sales and offered an estimate for the rest of the fiscal year that also fell short of Wall Street estimates.
VeriFone Systems gained $1.22, or 6 percent, to $21.68 after the company, a leading maker of terminals for electronic payments, said late Monday that its chief executive would step down after recent stumbles cut the company’s stock price nearly in half.
Costco Wholesale rose $1.31, or 1.3 percent, to $103.75 after reporting that its fiscal second-quarter net income climbed 39 percent. Costco pulled in more money from membership fees, its sales improved and it recorded a large tax benefit. Even without the tax benefit, the results were better than analysts had expected.
Cabela’s, an outdoor retailer, gained $6.75, or 12.5 percent, to $60.65 after it said that it expected its first-quarter profit to exceed market expectations.
In the bond market, interest rates declined for the first time in seven trading sessions. The price of the Treasury’s 10-year note rose 12/32, to 99 27/32, while its yield dropped to 2.02 percent from 2.06 percent late on Monday.


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A total abortion ban criminalizing the procedure and classifying it as a homicide cleared a committee Tuesday and will now be debated at the Texas House of Representatives. If House Bill 896 becomes law, abortion could carry penalties ranging from a state jail felony to a capital felony, and patients who seek the procedure could in theory be punished with the death penalty.
The legislation, also known as the Abolition of Abortion Act, was introduced by Republican state Rep. Tony Tinderholt. The measure, which he also sponsored in 2017 before it died in committee, stipulates that embryos have the same rights as a "human child" starting at the moment of conception.
The bill alters Texas' penal code, eliminating the exception for abortions from the definition of criminal homicide. Therefore, everyone involved in providing abortion care — from physicians and nurses to patients seeking this type of care — would face murder charges. HB896 makes no exceptions for rape, incest, or cases where the women's life is in danger. Tinderholt also proposed the legislation would be enforced "regardless of any contrary federal law, executive order, or court decision," which would go directly against Roe v. Wade.
Texas has historically been ground zero for attacks on access to abortion care. The state already bans abortions after 21.6 weeks, requires women to wait 24 hours between receiving mandatory counseling on abortion care and undergoing the procedure, restricts the use of telemedicine to obtain abortion medication, among several other regulations. Texas was also at the center of the U.S. Supreme Court battle over House Bill 2 (HB2), a measure which led to the closure of about two dozen of the 44 clinics in the state. The court struck down the law in 2016, but the damage was done.
HB896 follows a troubling trend of total abortion bans introduced in states such as Alabama, Oklahoma, and Ohio — all which could potentially make the procedure punishable by life in prison or the death penalty. This type of extreme anti-abortion legislation is becoming increasingly common across the nation, since anti-choice lawmakers and activists are betting on the conservative majority of the Supreme Court to undermine or overturn Roe. This year alone, over a dozen of states have introduced measures to ban abortion once a fetal heartbeat is detected — which usually occurs at six weeks of pregnancy. In the past, these bans, which are unconstitutional due to Planned Parenthood v. Casey, mostly existed on the fringes. Now, they are being introduced and considered at an unprecedented rate. In total, more than 250 abortion restrictions aimed at limiting access to this type of reproductive care have been introduced at the state level since January.
"States ... are primed to pursue abortion bans since they have already adopted a slew of abortion restrictions designed to make it hard for patients to access services and for providers to keep their doors open," Elizabeth Nash, senior state issues manager at Guttmacher Institute, previously told Refinery29. "The only restrictions left to enact are bans."

domain: sociology


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EETimes: "In an attempt to get out in front of emerging I/O standards for
networking, Xilinx Inc. has rolled out 10-Gbit Ethernet, PCI-X and Common Switch
Interface (CSIX) products. Hypertransport and Infiniband offerings are in the works,
The San Jose, Calif., company said it has begun delivering a 10-Gbit Ethernet
media-access controller (MAC) core; a 100-MHz version of its PCI-X core; and a
reference design for the CSIX interface. The offerings are intended to push Xilinx's
high-end Virtex 2 FPGA deeper into networking applications, where I/O has become a
dominant design issue."


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- What color is the sun?
- Who is Dennis in the color of water?
- Who is mameh in color of water?
- What is the purpose of the color of water?
- What is the weirdest color name?
- What is Colour of the water?
- What kind of text is the color of water?
- Who wrote the color of water?
- Who is Tateh in the color of water?
- What is the conflict in the color of water?
- Is clear Pee good?
- Is clear a Colour?
- What makes water blue or green?
- Who named water?
- What is the mood of the color of water?
- Which Colour is Colourless?
- What does the color of water tell you?
- Is the color of water a movie?
What color is the sun?
whiteThe color of the sun is white.
The sun emits all colors of the rainbow more or less evenly and in physics, we call this combination “white”.
That is why we can see so many different colors in the natural world under the illumination of sunlight..
Who is Dennis in the color of water?
Andrew Dennis McBrideDennis was a violinist from North Carolina who Ruth met while working at her aunt’s leather factory. He was gentle and strong, and fathered eight of Ruth’s twelve children. He died from lung cancer at a young age. Read an in-depth analysis of Andrew Dennis McBride.
Who is mameh in color of water?
Hudis ShilskyRuth’s mother, whose full name is Hudis Shilsky. She was born in 1896 in Poland to a wealthy Orthodox Jewish family. Polio paralyzed half of her body, and left her in poor health for the rest of her life.
What is the purpose of the color of water?
Hover for more information. The author’s purpose in writing this novel is to explore and connect with his own sense of identity. This book is a tribute to the author’s mother, but in being that, it is also an autobiography.
What is the weirdest color name?
13 Incredibly Obscure Colors You’ve Never Heard of BeforeAmaranth. This red-pink hue is based off the color of the flowers on the amaranth plant. … Vermilion. … Coquelicot. … Gamboge. … Burlywood. … Aureolin. … Celadon. … Glaucous.More items…•Feb 27, 2020
What is Colour of the water?
The water is in fact not colorless; even pure water is not colorless, but has a slight blue tint to it, best seen when looking through a long column of water. The blueness in water is not caused by the scattering of light, which is responsible for the sky being blue.
What kind of text is the color of water?
The Color of WaterAuthorJames McBrideLanguageEnglishGenreMemoirPublisherPenguin GroupPublication date19965 more rows
Who wrote the color of water?
James McBrideThe Color of Water: A Black Man’s Tribute to His White Mother/Authors
Who is Tateh in the color of water?
Tateh is an old Jewish Man, “short, dark, hairy, and gruff” (McBride 2) and a strong racist. He is the Father of Ruth Shilsky, and thus grandfather of James McBride. He ran a general store in Suffolk, Virginia, where he employed his family and made as much money as possible.
What is the conflict in the color of water?
The major conflict in The Color of Water has to do with racism in America and how it affects all the characters as they attempt to go about their lives. James McBride’s book is about the life of his family, including his white mother and black father.
Is clear Pee good?
Clear urine is a sign of good hydration and a healthy urinary tract. However, if they consistently notice clear urine and also have extreme or unusual thirst, it is best to speak to a doctor.
Is clear a Colour?
If something is clear it is known as transparent. This measn that instead of reflecting light light just passes straight through it. … Clear isn’t really a colour – if something is clear it doesn’t absorb or reflect any light – for something to have a colour it must absorb and/or reflect light.
What makes water blue or green?
The ocean is blue because water absorbs colors in the red part of the light spectrum. Like a filter, this leaves behind colors in the blue part of the light spectrum for us to see. The ocean may also take on green, red, or other hues as light bounces off of floating sediments and particles in the water.
Who named water?
water (n. 1) Old English wæter, from Proto-Germanic *watr- (source also of Old Saxon watar, Old Frisian wetir, Dutch water, Old High German wazzar, German Wasser, Old Norse vatn, Gothic wato “water”), from PIE *wod-or, suffixed form of root *wed- (1) “water; wet.”
What is the mood of the color of water?
Tone. The tone of this book is congratulatory to Ruth and her life. She had been through so many struggles, but the book is almost a way to cheer her on and show that James was very grateful of the things she did for him, even if he didn’t show it at the time.
Which Colour is Colourless?
White is the blending of all colors and is a color. Explanation: Light appears colorless or white. Sunlight is white light that is composed of all the colors of the spectrum.
What does the color of water tell you?
Perhaps the most common cause of water color is the presence minerals. Red and brown colors are due to iron; black to manganese or organic matter; and yellow to dissolved organic matter such as tannins. Iron and manganese are common, at least in small amounts, in most rocks and sediments.
Is the color of water a movie?
A wonderfully eccentric woman who stays around after her death and meddles in her niece’s love life in a hilarious and often touching duel of wills.

domain: artistic


text:
Get the latest 「ワンピース ネタバレ」 ONE PIECE 603 spoiler pics and summaries at MangaSpoiler.com. You can also read previous ONE PIECE 602 manga spoiler. Please leave a comment below if you would like to discuss the spoilers or have any predictions.
ONE PIECE 603 Spoiler Pictures
Trivia: Fishman Island is an underwater island which dwells 10,000 meters under the sea level, in a giant hole that goes under the Red Line. It serves as an underwater gateway to the New World for those who do not wish to cross over the Red Line above. It is the home of the Fishmen and some Merfolk.

domain: geography


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Ukrainians warned of a sharp cold.
Ukrainians warned of a sharp cold
In Ukraine in the coming days will decrease the temperature.
About this on his page in Facebook said the forecaster Natalia Didenko.
According to her, on Friday, October 19, will be almost the last warm day this autumn.
“Tomorrow is the last day this warm and Sunny air. No, the “twenties” in degrees still enough and the sun will be a lot. However, in the West of Ukraine is already possible to smell the first true air of Mature autumn – colder, higher clouds, and even in the Carpathian region and around the mountains possible modest first rains,” wrote forecaster, reports Rus.Media.
The rest of the territory of Ukraine will be warm to +23 degrees.
“During the Saturday-Sunday cold rush of warm air. 21 Oct in the Western and Northern regions will be a little rain,” warned Didenko.
According to her, the next week it is already possible to contact the utility about the health of the batteries.
“Next week you can safely disturb the public utilities to have at least started rattling battery and pull the air from the upper floors. Or where need. Because at least since Tuesday and next, according to preliminary estimates, the norms for the start of the heating season for sure will. A necessary condition for the start of the heat in the room – three days with average daily air temperature is +8 degrees and below,” – said Didenko.
Note, in Kiev on 16 October, set a new temperature record – the thermometer this day has risen to 23 degrees.


January 4, 2008
Stop the deployment of laser weapons to Afghanistan
Dear Ceasefire.ca supporter,
The Harper government has bought new laser weapons to deploy in Afghanistan.
These laser weapons, sometimes called laser dazzlers, are mounted on rifles, and are intended to temporarily blind, or “dazzle” people. General Rick Hillier says he wants soldiers to use them to keep Afghans away from military checkpoints and vehicles. But the truth is, these weapons may be more dangerous than General Hillier thinks, and could inadvertently blind innocent civilians – permanently! That’s why we need to be sure laser weapons are being passed through a rigorous, transparent testing mechanism.
I ask that you please join me in sending a letter to Stephen Harper, demanding that he take the following steps:
1) Place a moratorium on the deployment of laser weapons;
2) Undertake the development and implementation of a transparent review process for assessing whether new weapons violate international law;
3) Test any new weapons currently under consideration for purchase by Canada.
All the best,
Military sets out plan to test high-tech weapons
Monday, December 24, 2007
Byline: David Pugliese
OTTAWA – The Canadian military has submitted details to the International Committee of the Red Cross in Geneva about how it will test new weapons such as laser “dazzler” systems for use in Afghanistan.
But an Ottawa think-tank is pushing for greater scrutiny of such purchases and how new high-tech weapons are used in the field. The Rideau Institute argues that the policy is simply a draft document and shows that Canada hasn’t moved forward in meeting its obligation under the Geneva Conventions to put protocols in place governing the fielding of new arms.
The Defence Department has set aside a little more than $10-million for the purchase of laser dazzlers for use in Afghanistan.
The Canadian Forces is looking at buying the devices designed to temporarily blind individuals as part of its efforts to reduce the number of innocent Afghans killed or wounded by troops for failing to heed warnings not to approach military convoys. The systems are capable of disrupting the vision of a person 50 to 500 metres away, depending on the specific type of model used.
Chief of the Defence Staff General Rick Hillier said the military has put the dazzlers through some tests but no decision has yet been made on whether to purchase them for the Afghanistan mission. But the Defence Department has submitted to the Red Cross a draft of a policy it would follow for the fielding of such new weapons.
In meetings in 2003 and 2005, officials with the Defence Department and Foreign Affairs met with the Red Cross to discuss improving procedures for examining the capabilities of weapons at the time of purchase. This was being done to comply with a protocol of the Geneva Conventions meant to ensure that a nation can show that a new weapon does not violate treaties the country has signed.
But Anthony Salloum, program director of the left-leaning Rideau Institute, said the draft policy the Defence Department produced shows little progress has been made on the issue of testing new weapons.
“All they’ve come up with in the last two years is a draft policy,” he said. “It’s not much and considering the promises the government made in 2005 it seems like they’ve gone backwards on this issue.”
Defence officials declined to be interviewed on the laser dazzler issue and did not answer questions about testing procedures. The department instead e-mailed a transcript of questions and answers raised about the systems during a recent Commons defence committee meeting.
The Foreign Affairs department has not responded to requests for comment made a month ago on the subject of weapons testing and the Geneva Conventions.
Mr. Salloum said the laser dazzlers fall into the same category as Tasers, as they are untested and could harm innocent people.
Canada has ratified a treaty that prevents the use of weapons that cause permanent blindness. The manufacturers of the dazzlers acknowledge they can damage a person’s eyes if improperly used at close ranges. Being hit by a dazzler is like looking directly into the sun.
Gen. Hillier said medical reports on the health effects of the dazzlers are being examined. “We are not going to be using something that clearly would be against international conventions that we’ve signed on to,” he told the Commons committee.
But if tests show that the dazzlers can do the job, the general said, the systems will be purchased.
The military wants to mount the dazzlers on rifles and vehicles, mainly for use in protecting convoys. It’s hoped the systems can reduce the number of Afghan civilians killed or injured by soldiers after failing to heed warnings to stop at checkpoints or to not approach convoys. At least 22 Afghans have been wounded and another nine killed in such incidents.


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Justin Verlander allowed five runs over six innings, but went home with a win as the Tigers demolished the Dodgers by a 14-5 margin on Tuesday.
Justin Verlander limited the A's to two runs over six innings Wednesday in a 9-3 Tigers victory.
Justin Verlander delivered a quality start in a no-decision against the Astros on Friday, allowing three runs on six hits over his six frames.
Justin Verlander limited the Indians to one earned run over seven innings Saturday in a Tigers victory.
Tigers manager Brad Ausmus said Tuesday that Justin Verlander has some "easily correctable" mechanical adjustments that he needs to make.
Justin Verlander was tagged for seven runs in six innings by the Royals in another loss Monday.
Justin Verlander yielded seven runs in 5 2/3 innings Wednesday in a loss to the White Sox.
Justin Verlander was tagged for six runs (five earned) in seven innings Thursday in a loss to the Blue Jays.
Justin Verlander beat the Mariners by allowing three runs in 7 2/3 innings on Friday.
Justin Verlander gave up five runs in six innings and took the loss on Tuesday against the Indians.
Justin Verlander survived a rough fifth inning to win his fifth game Wednesday, allowing five runs over six frames in a 7-5 victory over the Orioles.
Justin Verlander pitched well in a losing effort against the Twins on Friday, allowing just two runs on seven hits over seven frames.
Justin Verlander struck out seven while allowing three runs over seven innings in Sunday's win over the Royals.
Justin Verlander struggled early but got better as the game went on, allowing three runs over seven innings in a no-decision against the White Sox.

domain: game


For already five years Tautumeitas have given audiences a peek into a young woman’s thoughts and dreams. With the album Songs from Auleja released earlier this year, they introduced the world to sixteen songs from the village of Auleja in Latvia. These songs, based on recordings found in archives, were also unfamiliar to most Latvians. In fact, it is this repertoire of songs that brought Tautumeitas together in the fi rst place. The traditional form of a cappella multipart singing on this album, known as bolsi, is linked with specifi c times of the year or special celebrations. The texts tell about various natural phenomena as well as events and activities in people’s lives. The springtime bolss is about nature waking up aſt er a cold winter. It is a time of great potential and aspiration. These multipart songs were traditionally sung outdoors, the singers actively interacting with the environment. “Panama” is from Tautumeitas’ upcoming album, which, like the group’s self-titled debut album, features vocal-instrumental music. According to this song, a young woman’s most beautiful and powerful giſt is her voice.


By Hileni Nembwaya
DURING the 2015/16 financial year, the green scheme farms that produced maize accounted for 24.9 percent of national maize production, while over 70 percent was imported.
According to the Managing Director of agricultural business development (AGRIBUSDEV), Petrus Uugwanga, during the year under review the green scheme projects produced a total output of 17 829 metric tonnes of maize, which represented a 24.5 percent increase compared to the 2014/15 results, to the domestic production of maize.
“We can equally note with pride that over 95 percent of maize that was delivered to the national strategic food reserves under the auspices of agro-marketing and trade agency (AMTA) was procured via the green scheme projects,” said Uugwanga.
A total of 3 006 hectares of land were planted with maize, which produced a total yield of 17 829 metric tonnes. In addition the harvest, compared to baseline year yield, showed an increase of 3 052 metric tonnes, representing a 21,7 percent increase.
Production records of 2013/14 and 2014/15 are also said to have exceeded that of the baseline year by 2 522 metric tonnes and 272 metric tonnes respectively.
According to the 2014/15 Namibia agronomic board data, Etunda irrigation farm, which is one of the largest green schemes, planted maize on a total of 738 hectares planted and 3 103 metric tonnes were harvested.
Musese irrigation project also recorded a bumper harvest during the year under review with a total of 4 084 metric tonnes harvested from 430 hectares.
The report further stated that Namibia consumed a total of 151 960 metric tonnes of maize during the financial year 2014/15 and out of the total national consumption, 71 305 metric tonnes were produced locally and 82 527 metric tonnes were imported.
Of the total national production, 36 244 metric tonnes were produced under rain-fed and 37 213 metric tonnes were produced under irrigation, including green scheme farms.
Based on the AGRIBUSDEV set standards on maize production, maize yield per hectare is set above nine metric tonnes.
However, most of the farms failed to meet this internal standard due to various reasons such as climate change, power shortage or interruptions particularly at Ndonga Linena Irrigation Farm.
Confidente. Lifting the Lid. Copyright © 2015


NVIDIA GeForce GTX 285M SLI vs NVIDIA GeForce GTX 285M
NVIDIA GeForce GTX 285M SLI► remove from comparison
The Nvidia GeForce GTX 285M SLI is a graphics solution that combines two Nvidia GeForce GTX 285M with an SLI connection. As not every game supports SLI efficiently, the performance increase compared to a single card is only about 0-40%. The most frequently used technique is Alternate Frame Rendering (AFR), where each card take turns rendering a frame in an alternating manner. Therefore, the SLI solution may suffer from noticeable micro stuttering at frame-rates between 20-30 fps (due to display irregularities between each rendered frame).
Each of the two GeForce GTX 285M is based on the G92b core and is therefore produced in 55nm. It features the full 128 pipelines of the G92b core and is therefore comparable to the desktop 9800M GTX+ and not the desktop GTX 280.
The Nvidia GeForce GTX 285M SLI in laptops with Core 2 Duo CPUs is bundled with the GeForce 9400M G chipset and therefore supports HybridPower (if the manufacturer enables it). With HybridPower, both GTX cards can be disabled and only the chipset graphics core can run, leading to improved battery life (and possibly less fan noise). Modern Core i7 laptops are using an Intel chipset and do not support HybridPower.
Since there are two GTX 285M cards active in SLI, the electrical current consumption and exhumed heat are about twice as much as what a single GTX 285M can produce. Due to the large power and cooling requirements, this SLI combination can only be found in large DTR laptops (like the Alienware M17x).
NVIDIA GeForce GTX 285M► remove from comparison
The NVIDIA GeForce GTX 285M is a high-end graphic card of the 200M series based on the G92b core and most similar to the Desktop GeForce 9800 GTX+. Therefore the 285M cannot be directly compared to the Desktop GTX 285. The chip is produced in a 55nm fabrication process, which is a step up from the 65nm process of the GTX 9800M. Its 128 pipelines are all enabled as well, as opposed to only 112 pipelines of the GTX 9800M. Compared to the GTX 280M, the GTX 285M features a slightly higher clock speed and is therefore only marginally faster (3-6% on average).
An innovation in the 200M series is the much accelerated switch times if using Hybrid Power (lower than 1 second compared to 7 seconds at the 9800M GTX).
Similar to all other cards with native DirectX 10 capabilities, the GeForce GTX 285M renders 3D images using "Unified Shaders". In other words, there are no longer any more dedicated pixel shaders or vertex shaders. Instead, new stream processors (128 of them in the 285M) now process most of the heavy graphics loading that would have otherwise been done by dedicated pixel and vertex shaders. In fact, the shader units are clocked higher than the core chip itself.
As previously mentioned, the GTX 285M is about 3-6% faster than the GTX280M due to slightly higher clock speeds. This means that notebooks equipped with the GTX 285M should run all modern and demanding games (as of 2009) in high details and resolutions. Only very demanding games, like Crysis Warhead or Metro 2033, may become unplayable if at maximum graphical settings.
Games with PhysX support (e.g., Mirror's Edge) may benefit from improved performance out of the 285M. Still, the Mobility Radeon HD 5870 is arguably a faster and better value single core GPU for laptops.
An advantage of the GeForce GTX 285M is the integrated PureVideo HD video processor (VP2). With this software feature, the 285M can assist the CPU in the decoding of H.264-, VC-1-, MPEG2- or WMV9 videos.
HybridPower is an Nvidia power-saving technology for Windows Vista used for switching between integrated and dedicated graphics cards. In the future, Nvidia wants this switch to occur automatically with drivers (now known as Optimus). GeForceBoost is not supported with the 285M, as there would be no performance gain in combining the integrated GPU with the dedicated video card.
The power consumption can be up to 75 Watts (if including the MXM board and VRAM). As a result, the 285M is usually reserved for larger desktop replacement (DTR) laptops with powerful cooling solutions.
In June 2010, the GeForce GTX 480M (mobile Fermi) was announced with a revamped architecture compared to the GTX 285M. Compared to the 285M, the Fermi core will support DirectX 11 and offer better performance at the cost of higher power consumption levels and possibly higher heat output.
If compared to desktop graphics cards, the performance of the 285M can be considered somewhere in between the 9800 GT and the 9800 GTX, the latter of which is clocked considerably higher (675/1675/1100 MHz) in comparison.
|NVIDIA GeForce GTX 285M SLI||NVIDIA GeForce GTX 285M|
|GeForce GTX 200M Series|
|Pipelines||256 - unified||128 - unified|
|Core Speed||576 MHz||576 MHz|
|Shader Speed||1500 MHz||1500 MHz|
|Memory Speed||1020 MHz||1020 MHz|
|Memory Bus Width||256 Bit||256 Bit|
|Max. Amount of Memory||2048 (2x1024) MB||1024 MB|
|API||DirectX 10, Shader 4.0||DirectX 10, Shader 4.0|
|Power Consumption||150 Watt|
|Transistors||1.5 Billion||754 Million|
|technology||55 nm||55 nm|
|Features||HybridPower, PureVideo HD, CUDA, PhysX ready||HybridPower, PureVideo HD (VP2), CUDA, PhysX ready|
|Date of Announcement||02.03.2009||02.03.2009|
|Information||MXM 3||MXM 3|
|Link to Manufacturer Page||http://www.nvidia.com/object/product_gef...||http://www.nvidia.com/object/product_gef...|
3DM Vant. Perf. total + NVIDIA GeForce GTX 285M SLI