Electric spark discharge processing is a common process for mold processing. Do you really know the electricity processing?
Maybe you are in contact with electric spark processing every day during production, but do you really know the electric spark processing?
First of all, do you know how the electric spark technology came from? In 1943, when the academician of the former Soviet Academy of Sciences Lazarian Coo and his wife’s research switch contact with the phenomenon and cause of spark discharge corrosion, it was found that the instantaneous high temperature of the electric spark could melt, gasification and gasification of the local metal, pioneering and invention. Electric spark processing.
Electric spark processing is a high temperature generated during the micro of the pulse discharge (the instantaneous temperature is as high as 10000 ° C). The processing method of melting materials and eroding materials is eroded.
Micro process of pulse discharge
Electric spark processing is the process of continuously discharging erosion. Although the time of one pulse is very short, it is a process of comprehensive effects such as electromagnetics, thermodynamics, and fluid mechanics, which is quite complicated. In summary, the process of pulse electricity can be divided into the following stages:
1) Electricity, breakdown and discharge channels of polar media
When the pulse voltage is applied between the tool electrode and the workpiece, a electric field is formed immediately between the poles. The strength of the electric field is proportional to the voltage, which is inversely proportional to the distance. As the polar voltage increases or the polar distance decreases, the strength of the polar electric field will also increase with. Because the micro -surface of the tool electrode and the workpiece is uneven, and the distance is very small, the strength of the polar electric field is very uneven. The strength of the nearest point or the tip of the tip of the poles is generally maximum. When the strength of the electric field increases to a certain amount, the medium is cut, and the gap resistance of the discharge is rapidly decreased from the insulation state to a quarter of Ohm, and the gap current rises to the maximum value. Because the diameter of the channel is small, the current density in the channel is very high. The gap voltage drops rapidly from the breakdown voltage to the spark maintenance voltage (generally about 20 ~ 30V), and the current rises from 0 to a peak current.
2) Medium heat decomposition, electrode material melting, vaporization thermal expansion
Once the polar medium is ionized and breakdown, after forming a discharge channel, the pulse power supplys the electron at high speed between the passage to the positive electrode, and the positive ion runs towards the negative electrode. Electric energy becomes kinetic energy, and kinetic energy transforms into heat energy through collisions. So the positive and negative surfaces in the channel become instantaneous heat sources, reaching a high temperature. The channel high temperature vaporizes the working liquid medium, and then heats up to break the vaporization. These vaporized working liquids and metal steam instantly increased the volume, and became bubbles in the gap between the discharge, which quickly expanded and had the characteristics of explosion. Observing the electric spark processing process, you can see that bubbles emerge from the gap between the discharge, the working liquid gradually turns black, and hears a slight and crisp explosion sound. Electric spark processing mainly relies on thermal expansion and local micro -explosion, so that the melting and vaporized electrode materials are thrown out.
3) Throw the electrode material
The instantaneous high temperature of the channel and the positive and negative surface discharge points make the work liquid and melt and vaporize the metal material, and the heat expansion produces high instantaneous pressure. The pressure in the center of the channel is the highest, which keeps the vaporized gas expand outward. The melting metal liquid and steam at the high pressure of the pressure is excluded and thrown into the work liquid. Due to the role of surface tension and internal agglomeration, the throwing materials have the smallest surface area, and it is condensed into small ball particles during condensation.
When the melting and vaporized metals are splashing around the surface of the electrode, in addition to most of them throw into the work solution and shrink into small particles, there are a small part of the electrode surface that splashes, plated, and adsorbed on the opposite side. This phenomenon of splashing, plating, and adsorption can be used under certain conditions to reduce or compensate the loss of tool electrodes during processing.
In fact, the erosion and throwing process of metal materials are more complicated. At present, people’s understanding of this complex mechanism is still deepening.
4) EDC of polar media
With the end of the pulse voltage, the pulse current has also rapidly dropped to zero, but there should still be a interval time since then, so that the gap medium is eliminated, that is, the tape particles in the discharge channel are composed of neutral particles, and the medium at the discharge channel is restored to the medium at the discharge channel. The insulation strength and the temperature of the surface of the electrode, so as not to repeat the same discharge in the same place next time, cause the arc discharge, thereby ensuring that the next breakdown and discharge channel is formed at the latest or minimum resistance between the two poles.
It can be seen that in order to ensure that the electric spark processing process is carried out normally, there is generally enough pulse interval between two pulse discharge. In addition, there should be room to leave room for breakdown and discharge points. Otherwise, it will only discharge nearby, which is easy to form an arc.
Five different discharge states
The discharge state refers to the basic state of each pulse in the discharge gap during the electric spark processing. It is generally divided into five discharge states, as shown in the figure below.
Pulse parameter and pulse voltage and current waveform
1) Open path (empty pulse)
There is no breakdown in the gap between the discharge, and there is a voltage of greater than 50V on the gap, but there is no current flow in the gap, which is a state of air.
2) Spark discharge (working pulse, or effective pulse)
The insulation performance of the gap is good. After the working liquid medium is penetrated, it can be effectively thrown out and eroded metal. Its waveform characteristics are: small oscobies with high frequency oscillating on the voltage and waveforms.
3) Short circuit (short -distance pulse)
The discharge gap is directly short -circuited. The current is large during the short circuit, but the voltage at both ends of the gap is very small, and it does not erode the processing effect.
4) Arc discharge (stable arc discharge)
Due to poor scandal, the discharge point is concentrated in a certain part without dispersion, local heat accumulation, increased temperature, and vicious cycle. At this time, spark discharge becomes an arc discharge. Because the discharge point is fixed at a certain point or a part. Therefore, it is called a stable arc, which often makes the electrode surface charcoal and burns. The waveform of the arc discharge is characterized by the small jaggedness of the high -frequency oscillation.
5) Transition arc discharge (unstable arc discharge, or unstable spark discharge)
The transition arc discharge is the transition state of normal spark discharge and stable arc discharge, and is a precursor of stable arc discharge. The waveform characteristics are that the breakdown delay is very small or close to zero, and only becomes a spike. The high frequency component on the voltage current meter becomes lower or becomes a sparse sawtooth.
The above -mentioned discharge status is alternately and probably appearing in actual processing (related to processing regulations and feed, use of oil, pollution, etc.), and even in the process of single pulse power release, there may be two types of alternation alternation. The above discharge status.
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Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.
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Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.
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Institutions: Tulane National Primate Research Center, Tulane National Primate Research Center.
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The family Poxviridae
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Institutions: Northwestern University.
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Institutions: Wistar Institute, University of Pennsylvania, Geisel School of Medicine at Dartmouth, University of Pennsylvania, University of Pennsylvania, University of Pennsylvania.
Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αβ and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.
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Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
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Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis
Institutions: Purdue University.
embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila
tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila
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Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR
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Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4
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RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma
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The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ
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analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.
Medicine, Issue 85, RNAscope, Head and Neck Squamous Cell Carcinoma (HNSCC), Oropharyngeal Squamous Cell Carcinoma (OPSCC), Human Papillomavirus (HPV), E6/ E7 mRNA, in situ hybridization, tumor
Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA
Institutions: Morgridge Institute for Research, University of Wisconsin, University of California.
Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina’s genome analyzer performs sequencing of a large number (> 107
) of relatively short sequence reads (< 150 bp).The "paired end" approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2
but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4
but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5
, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined "T7LA" (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6
and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7
Molecular Biology, Issue 56, Genetics, mRNA-Seq, Illumina-Seq, gene expression profiling, high throughput sequencing
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord
Institutions: Universidade de São Paulo.
electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.
Developmental Biology, Issue 93, chicken embryo, in ovo electroporation, spinal cord, RNA-Seq, transcriptome profiling, Galaxy workflow
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
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
Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells
Institutions: Diagenode S.A., Diagenode Inc..
Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.
Molecular Biology, Issue 94, Automation, chromatin immunoprecipitation, low DNA amounts, histone antibodies, sequencing, library preparation
Purification of Transcripts and Metabolites from Drosophila Heads
Institutions: University of Florida , University of Florida , University of Florida , University of Florida .
For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila
as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila
are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.
Genetics, Issue 73, Biochemistry, Molecular Biology, Neurobiology, Neuroscience, Bioengineering, Cellular Biology, Anatomy, Neurodegenerative Diseases, Biological Assay, Drosophila, fruit fly, head separation, purification, mRNA, RNA, cDNA, DNA, transcripts, metabolites, replicates, SCA3, neurodegeneration, NMR, gene expression, animal model
An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
Institutions: Georgetown University, The Ohio State University.
Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus
, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus.
Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus
due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.
Genetics, Issue 93, Aedes albopictus Asian tiger mosquito, photoperiodic diapause, RNA-Seq de novo transcriptome assembly, mosquito husbandry
Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library
The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3΄TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3
), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).
Genetics, Issue 62, Target ID, miRNA, ncRNA, RNAi, genomics

domain: medical


Cindy Cashman and Mitch Walling are planning to get married at an altitude of approximately 300,000 feet in 2009. Rocketplane, the space tourism operator that will take them there won't begin test flights until at least 2008.
Other issues potentially threaten their big day. Austin lawyer Mike Slack suggested that getting married on the trip would not create a legal marriage because nobody has jurisdiction to confer recognition.
"It's a big deal to us that it's legal. I went through a lot, my attorney went through a lot, and in my opinion, it'll be legal. This is history. This is the ultimate adventure... Is this the American dream or what?" said Cashman.


text:
OAKLAND – Skyline’s storied football program tabbed a longtime Oakland fixture as its next head football coach.
Virdell Larkins III announced on social media that he will coach at Skyline a few months after departing from Oakland Tech.
“It’s an unbelievable feeling,” Larkins, 37, told the Bay Area News Group on Monday afternoon. “I feel like someone who played for the Raiders, and then just signed with the 49ers … It was a hard decision, but I sat down and talked to my family about it.”
The Tech alum spent 11 years as the Bulldogs’ head coach, where he went 53-54 according to MaxPreps. Larkins’ Tech team reached the past two Oakland Athletic League titles, also known as the Silver Bowl.
So why did Larkins leave the successful Tech program, which replaced him with another Tech alum in Anthony McCrady?
“The administration wanted to move in a different direction,” said Larkins, who added that he harbors no ill will toward his alma mater.
Larkins, alongside cousins Josh Johnson and future NFL All-Pro Marshawn Lynch and coached by Delton Edwards, won the 2003 Silver Bowl against Skyline as a player.
That was legendary coach John Beam’s last game in charge of the Titans, who moved on to Laney College, where he remains to this day. Skyline was the premier team in the OAL under Beam from 1987-2003, winning 15 league championships and 160 games overall.
Larkins said he regularly speaks with Beam, his former coach at Laney, and considers him a mentor that he shares an “unbreakable bond” with.
“My dad being a coach at Oakland Tech growing up, I saw the dominance of Skyline, and I understand their prestigious football history Skyline has,” Larkins said.
Two decades after beating Skyline in the Silver Bowl, Larkins will be the program’s third head coach in as many seasons. KC Adams coached the team in 2022, and Jonathan Carroll led the program in 2023. Skyline went 2-7 last season and forfeited its playoff game against McClymonds.
So what are the expectations for his first season?
“I don’t want to put a number on it, but you can quote me when I say that we’re going to win right now,” Larkins said. “We’re not winning tomorrow, we’re not winning later, we’re winning right now.”
Larkins stopped short of predicting Skyline would reach the heights it did in the 1990s. After all, McClymonds, even without retired coach Michael Peters, still has not lost a game to an OAL opponent since 2009.
“I feel like it still goes through Mack until we beat them in a Silver Bowl,” Larkins said.

domain: sports


text:
Plasma cells, also called plasmocytes, are a type of white blood cell, or leukocyte. These cells play a role in the production of antibodies, virus-neutralizing proteins, bacteria, parasites, and other foreign bodies in the system. Plasma cells can be found in the blood, but only temporarily when the body is under attack. Most of the time, they remain in the lymphatic organs, particularly the many lymph nodes distributed around the body, but also in the spleen.
Published by Jeff
Latest update on October 29, 2013 at 07:16 AM by Jeff.


text:
Discover King Arthur's Death here, Michael Smith’s new translation of the vibrant fourteenth century poem.
This book is now in production but you still have the chance to get the strictly limited collector's hardback edition with its coloured endpapers, embossed boards and spot laminate cover
Written in the North West of England towards the end of the fourteenth century, Sir Gawain and the Green Knight is a masterpiece of mediaeval alliterative poetry. Comprising over 2500 lines, it draws on a rich vocabulary with ancient roots, including many dialect words still in use in Lancashire and Cheshire today. It is a magnificent work which rivals even Chaucer in the beauty and complexity of its language.
As a north-westerner and mediaevalist myself, I have been attracted to this exquisite work like Tristan to Yseult – bewitched by its power. Despite its age, the story and its characters are as fresh and vibrant as when the anonymous poet first put quill to paper over 600 years ago. It blends temptation and erotica with horror and suspense. It is exciting and funny yet melancholic and existential. Its descriptions of the passing seasons, the mediaeval hunt and the wintry landscape of Cheshire and Staffordshire are quite simply astounding.
As a writer, I wanted to capture the poet’s courtly style and translate his work in such a way that if the Gawain poet were to come back today he would feel at home reading it in modern English. And of course I was determined to maintain the wonderful alliteration, with its fabulous “bob and wheel” device at the end of each stanza.
But Sir Gawain and the Green Knight is also fabulous journey into the mediaeval world. When you look carefully there is, behind the narrative, a vivid description of courtly ritual, contemporary fashion, hunting techniques and so much more. There are also, I think, some coded references to the dark events surrounding the death of Richard II. So as a historian, I wanted not only to translate the work but to help provide a key to understand the social and political landscape within which the poem was set. Hence, I have also included detailed supplementary notes about words used by the poet and the references he makes to the world he knew.
An illuminated manuscript for today.
The result, I hope, is what might be described as a new courtly edition of this fabulous masterpiece which also enables the reader to get a flavour of the poet’s life and times. But I also wanted to make the book so much more than this, something really special. So, as an artist, I have created a collection of linocut prints especially for it.
Every print has been meticulously researched to reflect the style of the 1390s. I have also created illuminated letters replicating those in the original manuscript (known as Cotton Nero A.x in the British Library). Each print you will see in the book has taken at least 20 hours to cut before printing on a Victorian Albion press in the depths of Cambridgeshire.
The result is a beautiful volume to treasure and enjoy – like a really good book should be. It is like a mediaeval illuminated manuscript for the modern age, to be enjoyed again and again and passed down, like an heirloom, through the ages.
But this new illuminated manuscript cannot exist without your help. Please pledge your support and let the Gawain Poet speak to you anew!
Michael Smith comes from Cheshire and read history at the University of York, specialising in English and European mediaeval history. In later years, he studied as a printmaker at the Curwen Print Study Centre near Cambridge; you can find out more about his mediaeval-themed printmaking at www.mythicalbritain.co.uk.
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17th December 2022The Wintry Wonderland of Sir Gawain and the Green Knight - a short film for you to enjoy.
Dear Gawain reader,
With the recent cold snap I was reminded of the fabulous descriptions of winter in Sir Gawain and the Green Knight and I have produced a film featuring readings from various parts of the poem.
In the film, I dip in and out of the original Middle English poem while subtitles, featuring my translation of the work, keep you on track with the narrative.
I hope you enjoy the…
9th March 2021Was Sir Gawain and the Green Knight written in York?
In my introduction to Sir Gawain and the Green Knight I stated that the unknown Gawain-poet was "most likely a north-westerner"; a view consistently held by scholars for many years. The locations for the story, the use of the North-west Midlands dialect and a strong sense of either Ricardian or Henrican courtly regionalism all support this view. However, in recent years, new studies have emerged…
10th July 2018Green Man or Green Knight in the story of Sir Gawain?
One of the recurring themes in the discussion about the Green Knight in Sir Gawain and the Green Knight is that he represents the Green Man of folklore. While it is possible that the poet may have drawn from this legend in the creation of his “enker-grene” monster, an association of the character with the legend is much more problematic.
The folklore of the Green Man
The Green Man in…
2nd May 2018How the Gawain-poet employs Nature in Sir Gawain and the Green Knight
A common technique employed by the Gawain Poet - and others in the Alliterative Revival of the fourteenth century - is the use of descriptions of bucolic idylls as a contrast to horror to create astonishing tension. In Sir Gawain and the Green Knight, we are introduced to a range of different uses of this technique - a range which makes him a master of the art. Let's take a look...
7th April 2018Two Gawain Appearances for your Diary!
As my translation of Sir Gawain and the Green Knight gets ever closer to publication (and your deluxe copies being sent at the beginning of July), I have a brief update about two events where I am appearing and which may be of interest: a stage performance based on the book, and an appearance at the Bradford Literature Festival.
Sir Gawain and the Green Knight - a staged production with authentic…
4th March 2018Four Fitts or Nine Parts? Issues of layout in Sir Gawain and the Green Knight
In all the many translations there have been of Sir Gawain and the Green Knight, it has long been accepted that the work breaks down into four fitts, or parts. Does this still hold true today?
Recent analysis has attempted to change this way of thinking by examining the role played in the original manuscript by the series of illuminated letters which occur within it; indicating that the work…
8th February 2018Sir Gawain and the Green Knight - what you can look forward to when the book arrives.
As we approach 11th February, after which no more pledgers' names can be entered in the back of Sir Gawain and the Green Knight, I wanted to take a brief moment to write to you to thank you for pledging and making this book a reality. I could not have achieved this without the kind and generous support of each and every supporter, each and every patron.
So what is there to look…
1st February 2018The sexuality of Lady Bertilak – empowered or manipulated?
The character of Lady Bertilak in Sir Gawain and the Green Knight is one of the most astonishing creations of the Middle English period. But what was the Gawain-poet’s intention in creating a woman of enormous sexual power and confident in her own needs?
Lady Bertilak, or more simply “the Lady”, is characterised by the poet as someone who is eye-catchingly attractive, articulate and artful…
29th January 2018Exciting News!
Dear supporters of Sir Gawain and the Green Knight,
Exciting news! I am posting this brief update to tell you all that last week I submitted the amended page proofs of the book to Unbound; very shortly the book itself will be ready to go to print. Publication is drawing ever closer!
Still time for friends to have their names in the back:
Unbound tells me that the book will go to print…
2nd January 2018New Year arrives for Sir Gawain – but what message does it bring?
New Year is a time when we all try to change our ways and perhaps seek a greater virtue in the year ahead. Yet for Sir Gawain on his quest, New Year is a time of dread and foreboding. What was the poet’s real intention?
Image: Temptation and renewal for Sir Gawain at new year?
In setting his poem in the holidays of Christmas and New Year, the Gawain-poet knew exactly what he was doing…
14th December 2017The Spirit of Christmas comes to Camelot
In many mediaeval romances, the “holiday” of Christmas features strongly in the story line – particularly, as is usual, if there is an “adventure” which takes place. Sir Gawain and the Green Knight is no different; certainly, the festive season provides a backdrop to the story which, helped by its religious significance, works in placing Gawain’s personal struggle right at its heart.
26th November 2017Something a little different - a colourful knight emerges in linocut!
As readers will know, as well as translating Sir Gawain and the Green Knight I have also produced all the illustrations for the book using the linocut process. For this update, I thought I'd share with you some work I have been doing on a range of new prints featuring mediaeval knights from the time of the Gawain-poet. In this case, a Cheshire knight, Sir Hugh Calveley who may well have known…
23rd November 2017How pure is Sir Gawain? The role of the five-pointed star in Gawain and the Green Knight
Before Sir Gawain sets off on his quest to find the Green Knight, he prepares dutifully for his journey, being ritually armed on a carpet of the finest Toulouse tapestry. It is here that we are introduced to his emblem – the five pointed star –which would mean much to the mediaeval reader, and which serves to explain Gawain’s seeming fall from grace as the poem progresses.
A crucial component…
11th November 2017The Terrifying Journey of Gawain
Image: The uncertainty of travelling alone is writ large in Gawain's first journey.
One of the astonishing elements of the sheer genius of the Gawain-poet is his ability to place the reader/listener in the mind of the hero, preparing us for what is to come. This is no more so than when Gawain first travels across the land of Logres in search of the Green Knight.
The dangers of travel…
2nd November 2017Cover design for Sir Gawain and the Green Knight!
It feels strange, after five years of work, seeing a book come to life which I first started to work on many moons ago while waiting for my son learning to play the accordion!
But now things are really starting to move forward - Sir Gawain and the Green Knight is beginning to emerge as a tangible entity. A book. It is leaving its own Camelot. It is journeying into a new realm. Like Gawain himself…
22nd October 2017Researching Sir Gawain and the Green Knight
This week I learned the great news that my illustrated translation of Sir Gawain and the Green Knight will be published in July 2018. I thought I’d reveal a little bit about what you’ll see in the book when it’s published…
(Above: a selection of versions of the poem consulted - plus a sneaky peak of the cover of the new edition!)
Over the last seven days, I have been going through the…
11th October 2017The Secrets told by Landscape and Geography in Sir Gawain and the Green Knight
Image: St Winefride's Well - the "Holy Head" mentioned by the Gawain-poet
One of the bewitching elements of the masterpiece which is Sir Gawain and the Green Knight is that its mediaeval writer is completely unknown. Yet, occasionally, the poet gives intriguing insights into who he was and where he lived – none more so than his descriptions of the landscape in which the poem is set; in…
23rd September 2017Sir Gawain goes to the publishers!
It's been a long time in coming - five years in fact - but at long last the manuscript and illustrations for Sir Gawain and the Green Knight have been delivered to the team at Unbound for the next step in its story. Five years ago, I could little have imagined it getting this far - here's how it all came about...
Back in 2012, I was busy producing a small range of greetings cards featuring scenes…
10th September 2017The Romance of Castles in the Landscape of Sir Gawain and the Green Knight
In the last decade or so, there has been a significant change in the understanding of castles in Britain. In addition to sophisticated architectural reconstructions and the innovative use of techniques such as lidar (light detection and ranging) in showing the lie of the land, much work has also been done into the concept of the castle as statement.
In truth, this should not come as a surprise…
4th September 2017Help Gawain reach 130% funding - special competition and rewards!
Thanks to you all for your support and helping us to spread Sir Gawain's epic story to a new audience. We're now at an incredible 117%. But Sir Gawain and our journey isn't over yet.
I've discussed the campaign with Unbound and we've decided to not only upload some exciting new pledge levels (check out the new giclee "dedicated print" options featuring some of my linocuts of castles and ancient…
29th August 2017Sir Gawain meets two ladies at Castle Hautdesert. Now let the story really begin...
After his journey through the land of Logres, Sir Gawain arrives at last at Hautdesert where he meets Lord Bertilak. Then, as shown here in part of my new translation, he is introduced to two women who, it emerges later, yield more power than might be supposed...
(Image: Lord Bertilak and the ladies. The original print of this illustration, framed and signed by me, is available to one lucky…
19th August 2017Gawain arrives at the Green Chapel - an excerpt from my translation of Sir Gawain and the Green Knight
One of the most haunting sections in Sir Gawain and the Green Knight is when Gawain comes across the Green Chapel for the first time in his quest. What follows is the part of my new translation which relates to his arrival here.
(above: the cave in its mound at Wetton Mill on the Manifold valley in Derbyshire; one of the candidates for the actual Green Chapel referred to by the Gawain-poet…
9th August 2017Is there a secret message buried in Gawain and the Green Knight?
One of the strange features of Sir Gawain and the Green Knight is the set of nine crudely drawn illuminated letters which the poet originally produced to accompany his work. Do they constitute a hidden message?
Is there some form of invocation contained in the Gawain manuscript?
There is a main letter for each of the four fitts (parts) and another five elsewhere in the poem (each of…
31st July 2017In search of the Green Chapel in Sir Gawain and the Green Knight
One of the many wonders of Sir Gawain and the Green Knight is its magnificent setting; thought by many to be the Staffordshire Roaches The evidence is compelling...
In translatiing and illustrating my telling of Sir Gawain and the Green Knight, I've been fascinated by trying to understand the references made by the (unknown) poet and to grasp not just who he was but also where he was living and…
14th July 2017Now publishing soon - a mediaeval illuminated manuscript for the modern age!
How wonderful it was to learn last week that Sir Gawain had managed to achieve 100% funding. I want to thank everyone who has pledged to help make this new translation of Sir Gawain and the Green Knight a reality - I could not have done this without you. But the story's not over yet - this is where the real hard work begins!
Revisions and accuracy
Since I began work on the translation…
24th June 2017The fabulous hunting scenes in Gawain and the Green Knight
Sir Gawain and the Green Knight is rightly famous for its depiction of hunting – a favourite pastime of Richard II himself, in whose reign this fabulous work is thought to have been written. The inclusion of such sport is a masterstroke by the Gawain Poet in support of the main narrative: while Lady Bertilak hunts Gawain in the castle, her husband, Lord Bertilak, is away, hunting in the fields.
17th June 2017The Part Played by Chivalry in Sir Gawain and the Green Knight
Chivalric test - Lady Bertilak tests the limits of knightly virtue to bring Gawain down
Sir Gawain and the Green Knight is justifiably famous for many things; one of the most unusual is the way that it ends. Here, written in another hand, is the famous motto of the Knights of the Garter, Honi Soit Qui Mal Pense (missing the “y” of the full version). Founded by Edward III, the Order of the…
10th June 2017The place of nature in Sir Gawain and the Green Knight
One of the most dramatic contrasts in Sir Gawain and the Green Knight is that between the action (in Camelot, Hautdesert and the Green Chapel) and the outside world. There is no doubt that the Gawain (or Pearl) Poet was a man deeply in touch with nature. Little wonder because the natural world, the seasons and their effects meant life or death in equal measure in a society where crop failure, famine…
7th June 2017The complex character of Sir Gawain revealed
The Arthurian canon is rich with many characters, many of whom have become known to us all: King Arthur, Guinevere, Lancelot, Galahad and of course Gawain. Sir Gawain, the subject ofSir Gawain and the Green Knight, is himself a curious character who varies in personality depending on which of the stories one reads. He has certainly been a constant figure throughout the histories of Arthur.
26th May 2017Printing linocuts for Sir Gawain and the Green Knight - film
As well as my translation, the linocut prints I have produced for Sir Gawain and the Green Knight lie at the heart of my work. The film above shows you the actual printing process for each end every image in the book and below I tell you a little more about the research and production process.
We know from clues within the original manuscript that, despite the anonymity of the Gawain…
21st May 2017Green Knight book plus greetings cards
One of the pledge options for my translation of Gawain and the Green Knight enables backers to receive 12 mediaeval greetings cards plus a signed and personally dedicated copy of the book.Here's more information...
As you'll know from my other updates, and from reading about my book on Unbound, much of my work in recent years has centred around the creation of a range of linocut prints. In particular…
13th May 2017Sir Gawain and the Green Knight - a wonderful story of pride and its downfall
Sir Gawain and the Green Knight is a wonderful story set in a fabulous landscape of chivalric rules and the ever-present grip of nature and the seasons. It is also a masterpiece of what is now referred to as the Alliterative Revival of the fourteenth century, a form which achieved particular popularity in the north of England and parts of Scotland. Here, for those unfamiliar with the story, I give…
7th May 2017Researching and creating the back cover image for Sir Gawain and the Green Knight.
Among the different pledge reward options for supporters of my new translation of Sir Gawain and the Green Knightare those which include an Artiists Guild quality giclee print of my linocut print of the Green Knight himself, the same image which will also feature on the back of the book. Here I give an insight into how the image was created - and how you might acquire a signed and numbered limited…
23rd April 2017Sir Gawain says thank you!
Dear pledgers, fellow travellers, poets, artists, mediaevalists and lovers of literature, I wanted to thank all of you so far who have helped this edition of Sir Gawain and the Green Knight reach pledging of 40% after just three weeks. I am honoured by the reception to my work and by the many positive comments I have received.
40% is a great landmark; the equivalent of funding this translation…
16th April 2017How I make the illuminated letters for this edition of Sir Gawain and the Green Knight
I have been overwhelmed by the support shown for my work on Sir Gawain and the Green Knight. As well as the four years spent researching and translating my edition, the artwork itself has been a highly involved process. I thought I'd show supporters some of the methods involved in making the artwork itself. In this case, the illuminated letters which are to introduce each of the four Fitts of the…
Hi, Sir Gawain was featured in Prowling Dog: Cool Things: http://www.prowlingdog.com/prowling-dog-cool-things-issue-130/ Kindest regards, Alex
Thank you for the update Alexander. Good to see the title page of Cotton Nero A.x in there; the writing is exceptional. Very best Michael
Hi Michael, How will you be binding your work? The leather-bound volume/folder you have in the video looks great. Might it look something like that? Best, Ben.
Hi Ben, I don't think it will be leather bound, unfortunately (nor, indeed, in old pages from one of William Caxton's lost tomes as per today's announcement from Reading University) - I dare say the tooling and vellum might indeed be prohibitive. However, everything I have seen of Unbound's work is impressive and very high in quality. We intend I think to use the image you can see on my latest update (from my print, The Green Knight in the Woods) although at this stage I've no idea about how the final design will look. I think we'll know more once we get closer to our pledging target. Incidentally, the versions you saw in the video are not actually leather bound and date from the 1930s & 40s. You are right though, they do look most handsome and are amongst my favourites on my bookshelves. Kind regards Michael
Is it possible to buy an edition, and a pack of the cards by Michael within the one transaction? thanks, clare
Hello Clare, yes the Greetings Cards Pledge Option is the one to go for (6th one down on the list). The cards and book will be sent out together on publication. Details about the cards in the set are included in one of my "updates" (click on updates and then search for the update about the greetings cards) Kind regards Michael
These look stunning, Michael. What size are the three-colour prints? Would there be a future opportunity to buy a print of one of the other inages (I love the stag...) Thank you so much! Victoria.
Dear Victoria, Thank you for your interest in my work and thank you too for your lovely comments about the book. The three-colour prints are in one of two sizes depending on the image itself. The larger ones (for example the Green Knight you can see on this page) are 12 inches (30cm) square (printed area); the smaller ones (the deer you mention) are 6 inches (15cm) square (printed area); the sheet of (Somerset Velvet) paper on which each is printed is much larger to enable you to frame accordingly. In terms of purchasing the original prints themselves, they are available via my printmaking site (www.mythicalbritain.co.uk). Kind regards Michael
This book looks like a real stunner! I can't wait to see the final version! :) Where could I purchase the cards?
Hi Kim - Thank you for your kind comments about the book - the manuscript is nearly ready for the Unbound team as we speak. In terms of the cards, you can purchase these as part of a cards pledge option with the book or just take a look at www.mythicalbritain.co.uk and click on the greetings cards section of my site. There's a variety of cards there for sale. Kind regards Michael
Thank you so much for your quick reply! I have a friend that would swoon over this edition and the cards, it's going to make an excellent Xmas gift for her :) BTW: Are you available for interviews the press regarding the print process that you are using for the book/cards and the translation of the book itself?
HI Kim. Wonderful that your friend would like my work; happy to oblige. In terms of the press, yes I'd be happy to talk about all aspects of this project. Kind regards Michael

domain: library


text:
Master Movie’s videos (Some pre-release scenes of the movie) leaked online
Outrage sparked on social media after few videos (Master movie videos) emerged on social media. The leaked videos are said to be the scenes of Thalapathy VIJAY’S upcoming film “Master”
According to numerous reports, Few scenes from Vijay’s Master have been leaked by some miscreants and these scenes (known as Master leaked videos) have been spread by many netizens. Following the leaked videos, Master production house XB Film Creators have made a request not to spread the links and report them.
The production house tweeted Team #Master requests you all not to forward/share any leaked content and if you come across anything of these sorts, please share it with us at [email protected]
In the meantime, Fans of Thalapathy Vijay came forward and asking their fellow social media users to not spread “leaked videos of Film master.
“Kind Request To All @Kavin_m_0431 Fans.
Please Dont Share Any Leaked Scenes Of #Master. Every Single Techician Have Worked Day & Night To Make A Movie.Please Respect The Hardwork Behind It,” A Fan wrote on Twitter.
Another Fan added:
“Please Dont Share Any Leaked Scene of #MasterAd
So Much Of HardWork Behind The Movie
When #Kaala , #Kabali And More Movies Teaser, Trailer Leaked We Felt So Bad
Please Dont Share…! #Annaatthe #MasterFilm.”Ad
Meanwhile many fantasy seekers, who can’t wait for the movie, to be released officially, are hunting for the leaked videos of Master.
Upon looking at search queries on Twitter and Facebook search bars, we found that the following queries were among most searched during past few hours.
master leaked video, Master leaked videos, master full movie leaked, master film leaked, master videos, Master scene leaked, master scene leak, master scene, master leaked scenes, master leaked scene, Master leaked videos, master leak, leaked master movie, leaked master, master intro leaked
Although it is against the piracy laws to leak and spread these videos, who is going to watch the Master movie in theater?
Master is slated to release on January 13 in theatres.

domain: entertainment