text:
Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.
23 Related JoVE Articles!
A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Waterloo.
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The β-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the β-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on β-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased β-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H
]-thymidine incorporation, protein abundance, and mRNA expression.
Physiology, Issue 88, islet, isolation, insulin secretion, β-cell, diabetes, cAMP production, mouse
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Infinium Assay for Large-scale SNP Genotyping Applications
Institutions: Oklahoma Medical Research Foundation.
Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina's panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.
Basic Protocol, Issue 81, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
Training Synesthetic Letter-color Associations by Reading in Color
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy
Institutions: University of Southampton.
Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.
Chemistry, Issue 85, gas chromatography, fatty acid, pregnancy, cholesteryl ester, solid phase extraction, polyunsaturated fatty acids
Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
Institutions: Hunter College, CUNY.
Efficient expression of transgenes in vivo
is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).
Genetics, Issue 87, hydrodynamic gene delivery, hydrodynamics-based transfection, mouse, gene therapy, plasmid DNA, transient gene expression, tail vein injection
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
Fat Preference: A Novel Model of Eating Behavior in Rats
Institutions: University of Texas Medical Branch.
Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.
Behavior, Issue 88, obesity, fat, preference, choice, diet, macronutrient, animal model
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
Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
Institutions: Children's Hospital of Philadelphia Research Institute, University of Pennsylvania .
ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo
. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.
Molecular Biology, Issue 74, Genetics, Biochemistry, Microbiology, Medicine, Proteins, DNA-Binding Proteins, Transcription Factors, Chromatin Immunoprecipitation, Genes, chromatin, immunoprecipitation, ChIP, DNA, PCR, sequencing, antibody, cross-link, cell culture, assay
A Model of Chronic Nutrient Infusion in the Rat
Institutions: CRCHUM, University of Montreal.
Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.
Biomedical Engineering, Issue 78, Medicine, Anatomy, Physiology, Basic Protocols, Surgery, Metabolic Diseases, Infusions, Intravenous, Infusion Pumps, Glucolipotoxicity, Rat, Infusion, Glucose, Intralipid, Catheter, canulation, canula, diabetes, animal model
Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells
Institutions: University of California, San Francisco , University of Copenhagen, Denmark, National Institute of Nutrition and Seafood Research, Bergen, Norway.
Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPARγ agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.
Cellular Biology, Issue 73, Medicine, Anatomy, Physiology, Molecular Biology, Surgery, Adipose Tissue, Adipocytes, Transcription Factors, Cell Differentiation, Obesity, Diabetes, brown adipose tissue, beige/brite cells, primary adipocytes, stromal-vascular fraction, differentiation, uncoupling protein 1, rosiglitazone, differentiation, cells, isolation, fat, animal model
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts Institute of Technology.
The nematode C. elegans
has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans
triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans
. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans
Genetics, Issue 73, Biochemistry, Cellular Biology, Molecular Biology, Developmental Biology, Physiology, Anatomy, Caenorhabditis elegans, Obesity, Energy Metabolism, Lipid Metabolism, C. elegans, fluorescent lipid staining, lipids, Nile red, fat, high throughput screening, obesity, gas chromatography, mass spectrometry, GC/MS, animal model
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3
. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
Institutions: University of California, Los Angeles.
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
Cellular Biology, Issue 5, mosquito, malaria, popuulation, replacement, modeling, infectious disease
Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes
Institutions: University of California, San Francisco - UCSF.
Issue 7, Immunology, Pancreatic Islets, Cell Culture, Diabetes, Ficoll Gradient, Translational Research
In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts
Institutions: Emory University.
Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.
Cellular Biology, Issue 19, Current Protocols Wiley, Xenopus Egg Extracts, Nuclear Assembly, Nuclear Membrane
The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Institutions: GE Healthcare Bio-Sciences AB.
In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280
. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka
) and affinity (KD
), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.
Cellular Biology, Issue 37, Protein interaction, Surface Plasmon Resonance, Biacore X100, CFCA, Cystatin B, Papain
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing
Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells
Institutions: University of California, San Diego, Janssen Research & Development, University of California, San Diego.
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo
necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
Medicine, Issue 76, Cellular Biology, Molecular Biology, Biomedical Engineering, Immunology, Hepatology, Islets of Langerhans, islet, Insulin, Coculture, pancreatic beta cells, INS-1 cells, extracellular contact, transmembrane protein, transcellular interactions, insulin secretion, diabetes, cell culture
Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive
Institutions: Johns Hopkins University.
In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.
Cellular Biology, Issue 5, mosquito, malaria, genetics, infectious disease, Wolbachia


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Dumbbell Piston Row – Exercise Library
• Stand with knees bent, back straight and flat,
• Hold dumbbells in each hand.
• Squeeze your abs and maintain your body position.
• Row one dumbbell up to your body.
• Lower one dumbbell start rowing the other dumbbell up to your body.
• Repeat, being in constant motion, one after another.
• Use a weight that works for your so you don’t lose form.
Disclaimer: Make sure to contact a Registered Exercise Professional with Fitness Australia or Physical Activity Australia or an Accredited Exercise Physiologist with ESSA to provide you with instructions on correct form for all exercises.
Dumbbell Piston Row Video – Exercise Video Library
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domain: sports


text:
Background: It is difficult to determine whether a second high-risk lesion, including pancreatic ductal adenocarcinoma or high-grade pancreatic intraepithelial neoplasm, is a metachronous multifocal lesion or represents local recurrence after resection of the first high-risk lesion. This study attempts to clarify the characteristics of second high-risk lesions in the remnant pancreas using genetic analyses. Methods: Clinicopathologic data were collected from 12 patients who underwent pancreatectomy for a second high-risk lesion in the remnant pancreas. We performed mutational and immunohistochemical analyses of 4 major genes—KRAS, TP53, CDKN2A, and SMAD4—associated with pancreatic ductal adenocarcinoma progression, as well as targeted next-generation sequencing. Results: Mutations in the four genes in the second high-risk lesion were consistent with the first lesion in four patients but were inconsistent in the remaining eight patients, and thus we considered that the latter eight patients likely had metachronous multifocal high-risk lesions and the other four patients had local recurrence. The estimated cumulative recurrence rate after resection of the second high-risk lesion was greater in the local recurrence group compared with the metachronous multifocal group, and the estimated cumulative disease-specific survival rate was greater in the metachronous multifocal group. Targeted next-generation sequencing demonstrated that the second lesions in the metachronous multifocal high-risk lesion group showed differences in founder mutations compared with the first lesion. In the local recurrence group, the founder mutations in the second lesion were common with those in the first lesion. Conclusion: Genetic assessment might help discriminate metachronous multifocal high-risk lesions from local recurrence.
All Science Journal Classification (ASJC) codes

domain: biology


On Thursday morning, a stabbing along the Las Vegas Strip resulted in two fatalities and six injuries. A suspect has reportedly been arrested, according to police.
The Las Vegas Metropolitan Police Department released a statement stating that one of the victims was declared dead at the scene and another at the University Medical Center. In a news conference on Thursday, LVMPD Captain Dori Koren said that three of the victims were in severe condition.
The deceased were identified as Las Vegas residents Maris Mareen Digiovanni, 30, and Brent Allan Hallett, 47, according to the Clark County coroner’s office.
According to Koren, the other victims “looked to be stable.” At a second press conference later in the day, LVMPD Sheriff Joseph Lombardo stated that the victims seemed to be a mix of locals and visitors. According to Koren, police removed a “large kitchen knife” from the scene.
A Similar Post:
According to authorities, the stabbings started at about 11:40 a.m. local time. According to LVMPD Deputy Chief James LaRochelle, the first incident looked to be unprovoked. After stabbing the first victim, the perpetrator continued south along Las Vegas Boulevard and stabbed numerous more people, according to LaRochelle.
Several “concerned bystanders” pursued the suspect as he rounded a corner and headed east, according to LaRochelle. A combination of hotel security and LVMPD officers then arrested the suspect.
According to court records, Yoni Barrios, 32, was arrested and placed in the Clark County Detention Center on two counts of open murder and six counts of attempted murder. His first court date was set for Friday in the afternoon.
The suspect didn’t seem to be a local, according to LaRochelle. The situation had reportedly been secured by 1:50 p.m. local time, according to police. In order for police to continue gathering evidence, a section of Las Vegas Boulevard remained closed, according to Koren.


BREAKING NEWS: The U.S. Senate will be taking a vote on SJ Res 4 THIS WEEK
Senate Majority Leader Schumer announced yesterday that the Senate will debate and hold a vote on SJ Res 4, the resolution that affirms the validity of the EQUAL RIGHTS AMENDMENT, this week.
We expect the floor debate on Wednesday and then the vote on Thursday, likely followed by a press conference. Between now and this vote, it is crucial that you contact both Senators Marco Rubio and Rick Scott and your US House Rep to tell them to VOTE FOR EQUALITY.
A non-partisan, national petition campaign called Sign-4-ERA to support the Equal Rights Amendment was started at the time of the announcement. Senate Majority Leader Chuck Schumer, former Congresswoman Carolyn Maloney, and feminist leader Gloria Steinem were the first signers.
Please make sure you SIGN and SHARE this petition immediately:
This is the first time in 40 years that the ERA will be voted on the Floor of the U.S. Senate.
To contact your representatives, CLICK HERE.


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Optoelectronic oscillators (OEOs) are hybrid RF-photonic devices that promise to be environmentally robust high-frequency RF sources with very low phase noise. Previously, we showed that Rayleigh-scattering-induced noise in optical fibers coupled with amplitude-to-phase noise conversion in photodetectors and amplifiers leads to fiber-length-dependent noise in OEOs. In this work, we report on two methods for the suppression of this fiber-length-dependent noise: altering the amplitude-dependent phase delay of the OEO loops and suppressing the Rayleigh-scattering-induced noise in optical fibers. We report a 20 dB reduction in the flicker phase noise of a 6 km OEO via these suppression techniques.
© 2013 Optical Society of America
Optoelectronic oscillators (OEOs) are ring resonators that utilize the low loss-per-unit-length of optical fibers to generate ultra-high Q cavities . The high Q of the OEO cavity results in low phase noise RF signals. Theoretical models of the OEO predict that the phase noise of the OEO at frequencies within 100 kHz of the nominal carrier frequency (hereafter referred to as the “close-in” phase noise) should decrease quadratically with fiber length . However, this expected relationship between phase noise and fiber length has not been observed at low offset frequencies . Figure 1 shows the phase noise of 10 GHz OEOs with various fiber lengths. The phase noise of the 6 km OEO is higher than what is predicted by the theory, which indicates that there is a fiber-length-dependent noise source in the OEO. Similar fiber-length-dependent noise has been observed in the duplex transfer of frequencies over optical fiber . The low-offset-frequency phase noise is a critical measure of the stability of an oscillator; therefore, identifying and suppressing such flicker phase noise sources is a crucial step towards optimizing OEOs and other RF-photonic systems.
Sources of fiber-length-dependent phase noise in OEOs have been studied extensively [5, 6]. Volyanskiy et al. showed that in some configurations the OEO flicker phase noise is dominated by the combination of laser frequency noise and chromatic dispersion in optical fibers . In our OEOs, however, the fiber-length-dependent noise is dominated instead by Rayleigh scattering in optical fibers . Rayleigh scattering in the optical domain leads to intensity noise in the RF domain. We have also shown that the nonlinearities in the photodetectors and RF amplifiers convert amplitude noise in OEOs to phase noise . It is this combination of Rayleigh scattering and amplitude-to-phase noise conversion that we will focus on in this work. In the rest of this work, we will refer to “our OEOs”. We do so to distinguish between OEOs like ours where Rayleigh scattering coupled with amplitude-to-phase noise conversion are the dominant fiber length dependent noise mechanisms and OEOs like those of Volyanskiy et al. where laser frequency noise and chromatic dispersion dominate. For a given OEO, the dominant fiber-length-dependent noise source will depend on such parameters as: the laser frequency noise, laser power level, the OEO’s oscillating frequency, and the amount of amplitude-to-phase noise conversion in the amplifiers and photodetectors.
This paper is organized in the following fashion. In Section 2, we briefly review Rayleigh scattering noise in optical fibers and amplitude-to-phase noise conversion in photodetectors and amplifiers. In Section 3, we present the results of suppression experiments designed to reduce the amplitude-to-phase noise conversion and Rayleigh scattering in the OEO. Finally, in Section 4, we provide an analysis of our results and their potential applicability to other RF-photonic systems.
2. Fiber-length-dependent phase noise sources
In this section, we will briefly describe the combination of phenomena that leads to length-dependent noise in our OEOs. Together, amplitude noise induced by Rayleigh scattering in optical fibers and amplitude to phase noise conversion in nonlinear elements — in particular the amplifiers and photodetectors — cause length-dependent flicker phase noise in the OEO’s microwave signal. In the following subsections, we will present experimental data demonstrating both phenomena.
2.1 Guided entropy mode Rayleigh scattering in optical fibers
The refractive index of a dielectric material such as fused silica depends, in part, on the density or strain of the dielectric . Density or strain fluctuations lead to fluctuations of the dielectric susceptibility which cause optical scattering. Density or strain fluctuations caused by pressure or stress lead to Brillouin scattering while density or strain fluctuations due to temperature lead to Rayleigh scattering. We have shown previously that Guided Entropy Mode Rayleigh Scattering (GEMRS) – that is, scattering due to transverse temperature gradients in the fiber – leads to fiber-length-dependent intensity noise at offset frequencies below 100 kHz, which corresponds to the flicker noise region of the OEO . We measured the GEMRS-induced intensity noise in our OEOs by employing the forward scattering measurement system used in ref. 9. Figure 2 shows plots of the forward-scattered optical intensity noise in various lengths of single-mode optical fiber. The data show that increasing the fiber length increases the intensity of the noise due to GEMRS in the relevant frequency range.
2.2 Amplitude-to-phase noise conversion
The GEMRS effect is not sufficient to explain the increased phase noise of the OEO. Intensity noise due to GEMRS will lead to optical intensity noise, yet, to first order, optical intensity noise has no effect on the RF phase noise of an amplitude-modulated OEO with direct detection . A second mechanism is required to convert the optical intensity noise to RF phase noise. That mechanism is intensity-dependent phase delay in the nonlinear photodetectors and RF amplifiers in the OEO. Such AM-to-PM conversion has also been reported in mode-locked laser systems used to generate and transmit RF signals . In order to measure the AM-to-PM conversion factor for each component, we connected an RF-photonic link to a network analyzer and varied the optical power into the photodetector. We then recorded the phase delay experienced by the RF signals at 10 GHz passed through the link.
Figure 3 shows the plots of the phase delay and output intensity versus input optical power for the photodetector used in our OEOs. Our data show that the phase delay is a nonlinear function of the input optical power. Therefore, fluctuations in the optical intensity will lead to fluctuations in the RF phase of the OEO signal. The magnitude of the AM-to-PM conversion factor is proportional to the slope of the phase curve in Fig. 3 at the mean optical power level into the photodetector of the OEO.
We used a similar experiment to measure the intensity-dependent phase delay of the amplifiers in the OEO. Figure 4 shows the resulting phase-delay and output intensity plots versus input RF power to the amplifier block. Again, we note that the magnitude of the AM-to-PM conversion factor is given by the slope of the phase curve at the steady-state mean RF power level into the amplifier block of the OEO.
3. Noise suppression techniques
In the following subsections, we present data from experiments designed to verify our theories about the two phenomena responsible for the fiber-length-dependent phase noise in OEOs. This verification is a necessary step towards our long-term goal of counter-acting the Rayleigh noise effect in OEOs and other RF-photonic systems.
3.1 Suppression of AM-to-PM conversion
From the phase plots in Figs. 3 and 4, we can see that the magnitude of the AM-to-PM conversion terms depend on the input powers into the nonlinear components (photodiodes and RF amplifiers) of the OEO. We showed previously that by introducing a nonlinear gain element with the proper phase relationship, we could eliminate the AM-to-PM effect . In lieu of using such a device, we instead attenuated the optical power into the photodetector and the RF power into the final stage of the amplifier block in the OEO to alter the magnitude of the AM-to-PM conversion in these devices. We note that we attenuated the optical power after the fiber spool so as not to change the GEMRS induced in the fiber. In addition, we placed the RF attenuator after the first stage RF amplifier so as not to change the amount of additive noise generated by the amplifiers; this additive noise is dominated by the first amplifier stage.
Figure 5 shows the phase noise of a 6 km OEO with and without optical attenuation before the photodetector. The unattenuated optical power level into the photodetector was 11 dBm. After attenuation, the optical power was 4 dBm. The AM-to-PM conversion factor in the photodetector is proportional to the slope of the amplitude-dependent phase shift shown in Fig. 3. From Fig. 3, we see that changing the input optical power from 11 dBm to 4 dBm decreases the AM-to-PM conversion factor in the photodetector. We observe a commensurate 7 dB reduction in the relative flicker noise at offset frequencies below 10 kHz. Note that if the flicker noise were due to an additive noise source such as shot noise in the photodetectors, then attenuating the optical power would have increased the relative flicker noise level. Therefore, the observed reduction in flicker noise indicates that the induced noise was due to the nonlinear AM-to-PM effect shown in Fig. 3.
Figure 6 shows the phase noise of a 6 km OEO with and without RF attenuation before the second stage of the amplifier block. Without RF attenuation, the input power into the amplifier block is approximately –30 dBm. Again, the amplitude-dependent phase curve in Fig. 4 shows that by reducing the input RF power by 12 dB, we reduced the AM-to-PM conversion factor in the RF amplifiers. As shown in Fig. 6, we observe a commensurate reduction in the OEO phase noise. Both attenuation methods reduced the flicker noise by approximately 7 dB. Constructing a nonlinear gain element with the precise optimal phase slope may have an even greater effect on the OEO’s flicker noise.
3.2 Suppression of Rayleigh scattering
In this subsection, we present the effect of GEMRS suppression on the OEO phase noise. Previously, we showed that laser frequency modulation suppresses GEMRS-induced intensity noise in optical fibers . The GEMRS effect has a gain bandwidth between 10 and 100 kHz in single-mode optical fibers at 1550 nm. By modulating the laser frequency at frequencies between 10 and 100 kHz, we demonstrated up to 30 dB suppression of the GEMRS-induced intensity noise in the optical domain. We now present experimental data showing that laser frequency modulation also reduces the flicker phase noise of the OEO. Figure 7 shows the phase noise of a 6 km OEO with and without laser frequency modulation. The data show that the phase noise of the OEO was reduced by up to 20 dB. In particular, the phase noise reduction was greatest at offset frequencies where the GEMRS-induced intensity noise shown in Fig. 2 was greatest. We note that the effectiveness of laser-frequency-modulation diminishes at frequencies below 100 Hz. The structure of the noise in this frequency range suggests vibrational effects are the principle noise source in this region. The noise spikes in the red curve in Fig. 7 match typical vibration frequencies of various fans in our laboratory.
In this work, we demonstrated that together GEMRS-induced optical intensity noise and AM-to-PM conversion in photodetectors and RF amplifiers comprise the dominant source of fiber-length-dependent flicker phase noise in our OEOs. We demonstrated that suppressing either effect reduced the flicker phase noise of the OEO. Suppressing GEMRS via laser frequency modulation was the most effective means of flicker noise suppression. We observed up to 20 dB reduction of the flicker phase noise at 1 kHz by laser frequency modulation.
We note that these noise phenomena are not unique to OEOs. We expect similar flicker noise in any RF-photonic system with high-Q resonators or long waveguides with significant transverse gradients and nonlinear elements with amplitude-dependent phase such as photodetectors and amplifiers. Systems that include this combination of elements include: mode-locked laser-based RF sources such as coupled OEOs and carrier envelope phase locked lasers [13, 14]; whispering gallery mode optical resonators ; and time and frequency transfer systems that utilize optical fibers. For any of the above systems, the noise suppression techniques presented in this work may prove valuable.
References and links
1. X. S. Yao and L. Maleki, “Optoelectronic microwave oscillator,” J. Opt. Soc. Am. B 13(8), 1725–1735 (1996). [CrossRef]
2. X. S. Yao and L. Maleki, “Optoelectronic oscillator for photonic systems,” IEEE J. Quantum Electron. 32(7), 1141–1149 (1996). [CrossRef]
3. O. Okusaga, W. Zhou, E. Levy, M. Horowitz, G. M. Carter, and C. R. Menyuk, “Non-ideal loop-length-dependence of phase noise in OEOs,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2009), paper CFB3. [CrossRef]
4. P. A. Williams, W. C. Swann, and N. R. Newbury, “High-stability transfer of an optical frequency over long fiber-optic links,” J. Opt. Soc. Am. B 25(8), 1284–1293 (2008). [CrossRef]
5. K. Volyanskiy, Y. K. Chembo, L. Larger, and E. Rubiola, “Contribution of laser frequency and power fluctuations to the microwave phase noise of optoelectronic oscillators,” J. Lightwave Technol. 28(18), 2730–2735 (2010). [CrossRef]
6. O. Okusaga, J. Cahill, W. Zhou, A. Docherty, G. M. Carter, and C. R. Menyuk, “Optical scattering induced noise in in RF-photonic systems,” in Proceedings of IEEE Conference on Frequency Control (Institute of Electrical and Electronics Engineers, New York, 2011), pp. 1−6. [CrossRef]
7. A. Docherty, C. R. Menyuk, J. P. Cahill, O. Okusaga, and W. Zhou, “Rayleigh-scattering-induced RIN and amplitude-to-phase conversion as a source of length-dependent phase noise in OEOs,” IEEE Photon. J. 5(2), 5500514 (2013). [CrossRef]
8. R. W. Boyd, Nonlinear Optics (Elsevier, 2008), Chap. 9.
10. A. Docherty, O. Okusaga, C. R. Menyuk, W. Zhou, and G. M. Carter, “Theoretical investigation of length-dependent noise flicker-phase noise in opto-electronic oscillators,” in Conference on Lasers and Electro-Optics/Quantum Electronics and Laser Science and Photonic Applications Systems Technologies, Technical Digest (CD) (Optical Society of America, 2011), paper CFM1. [CrossRef]
11. J. Taylor, S. Datta, A. Hati, C. Nelson, F. Quinlan, A. Joshi, and S. Diddams, “Characterization of power-to-phase conversion in high-speed P-I-N photodiodes,” IEEE Photon. J. 3(1), 140–151 (2011). [CrossRef]
12. O. Okusaga, W. Zhou, J. Cahill, A. Docherty, and C. R. Menyuk, “Fiber-induced degradation in RF-over-fiber links,” in Proceedings of IEEE Conference on Frequency Control (Institute of Electrical and Electronics Engineers, New York, 2012), pp. 1−5.
13. F. Quinlan, C. Williams, S. Ozharar, S. Gee, and P. J. Delfyett, “Self-stabilization of the optical frequencies and the pulse repetition rate in a coupled optoelectronic oscillator,” J. Lightwave Technol. 26(15), 2571–2577 (2008). [CrossRef]
14. D. J. Jones, S. A. Diddams, J. K. Ranka, A. Stentz, R. S. Windeler, J. L. Hall, and S. T. Cundiff, “Carrier-envelope phase control of femtosecond mode-locked lasers and direct optical frequency synthesis,” Science 288(5466), 635–639 (2000). [CrossRef] [PubMed]
15. P. Del’Haye, A. Schliesser, O. Arcizet, T. Wilken, R. Holzwarth, and T. J. Kippenberg, “Optical frequency comb generation from a monolithic microresonator,” Nature 450(7173), 1214–1217 (2007). [CrossRef] [PubMed]


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Lighthouse Festival Theatre198 Votes Currently Open
- Address: 247 Main St, Port Dover, ON N0A 1N0, Canada Map
- Timings: 10:00 am - 05:00 pm Details
- Phone: +1-5195832221
- Tags: Entertainment, Performance, Theatre, Family And Kids
Are you looking for some qualitative entertainment in Port Dover? Lighthouse Festival Theatre Corp is the place for you. This place offers a variety of entertainment programs all round the year. Most frequently, comedies and musicals are held over here. This place can seat up to 200 people at a time. More than 170 shows have been held here since it was established in the year 1980. Before that, the old town hall building had fallen into disrepair and neglect. However it was revived by a group of budding performing art professionals. This place has provided a great boost to the growth of performing arts in the city.
More about Port Dover
Want to plan a DIY trip? Use Port Dover trip planner for the same.
Book perfect hotels in Port Dover like St. Johns Bed And Breakfast, Normandale Century Inn and Comfort Inn Simcoe.
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How to Reach Lighthouse Festival Theatre
- By taxi
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- Lighthouse Festival Theatre Address: 247 Main St, Port Dover, ON N0A 1N0, Canada
- Lighthouse Festival Theatre Contact Number: +1-5195832221
- Lighthouse Festival Theatre Timing: 10:00 am - 05:00 pm
- Try the best online travel planner to plan your travel itinerary!
95% of people who visit Port Dover include Lighthouse Festival Theatre in their plan
10 AM - 11 AM
76.14% of people start their Lighthouse Festival Theatre visit around 10 AM - 11 AM
People usually take around 30 Minutes to see Lighthouse Festival Theatre
91.21% of people prefer to travel by car while visiting Lighthouse Festival Theatre
People normally club together Brant Conservation Area and Turkey Point Provincial Park while planning their visit to Lighthouse Festival Theatre.
People also prefer to start their day with Lighthouse Festival Theatre.

domain: travel


text:
Ictineo II replica at the harbour of Barcelona- Ictineo II was a pioneering submarine launched in 1864 by engineer Narcís Monturiol i Estarriol of Catalonia, Spain and was the first air independent and combustion powered submarine and was the first submarine to overcome the basic problems of machine powered underwater navigation.
USS Monitor wreck on the ocean floor.
Adastra Trimaran, Future Super Yacht
Launched in 1800, the French 74-gun Duguay-Trouin escaped destruction or capture during the Battle of Trafalgar. Then, in November 1805, she was again fighting a British fleet. In the thick of battle, her captain and all lieutenants fell dead or wounded. An ensign led her to the last, striking the flag as her main mast crashed and further fighting was impossible. She then became HMS Implacable and once repaired, brought into the Royal Navy where she fought in many further campaigns. She…
USS Nautilus (SSN-571) the world's first operational nuclear-powered submarine in New York Harbor.
Which Real-Life Victorian Sub Inspired Jules Verne's Nautilus?
Which Real-Life Victorian Sub Inspired Jules Verne's Nautilus? Le Plongeur ("the diver"), invented by Captain Simeon Bourgeois and launched in 1863, it was the first submarine to be powered by mechanical means: a compressed-air engine operating a reciprocating engine. It was also the largest submarine built up until that time. At 140 feet,

domain: history


text:
One of the major challenges for humans to live on the Moon is finding sufficient amount of oxygen to breathe. It is important as for dreaming of colonising on the Moon, we need a constant source of oxygen. While astronauts wear spacesuits that do have an oxygen supply, carrying oxygen to the moon for future colonies is rather unfeasible. This is why researchers have for years tried to come up with ways oxygen could be produced on the moon, where even water (a potential source) is close to non-existent. Plants could be a method, but they also require sunlight and carbon dioxide. Now, in what could be seen as a breakthrough, the European Space Agency (ESA) claims to have built a solution that enables oxygen production using resources found on the Moon itself, including lunar soil or what is commonly known as moondust.
The ESA has established a prototype oxygen plant that is designed to produce oxygen from moondust. The plant, which has been set up in the Materials and Electrical Components Laboratory of the European Space Research and Technology Centre, (ESTEC) based in Noordwijk in the Netherlands, is touted to convert moondust aka lunar soil into oxygen that could be utilised for air as well as fuel.
Researchers at the ESA have experimented with the solution by using simulated moondust. The process of extracting oxygen is based on a method called molten salt electrolysis that involves placing regolith or lunar soil (presently, simulated moondust) in a metal basket with molten calcium chloride salt to serve as an electrolyte. This is heated to 950-degree Celsius at which the dust particles remains solid. But once a current is passed through the solid lunar soil, it causes oxygen to be extracted from the regolith and migrate across the salt to be collected at the anode. The process is also claimed to convert lunar soil into usable metal alloys.
“Being able to acquire oxygen from resources found on the Moon would obviously be hugely useful for future lunar settlers, both for breathing and in the local production of rocket fuel,” said Beth Lomax of the University of Glasgow in his study.
The ESA has claimed that samples returned from the lunar surface confirm that lunar regolith or moondust is made up of 40-45 percent oxygen by weight. So what researchers need to extract oxygen from dust particles is a technology like what has been prototyped by the space agency.
Initially, the oxygen plant developed by the researchers vents the oxygen produced in the process into an exhaust pipe. It is, however, in the works that the produced oxygen will be stored for future use. This would come into place through future upgrades of the system.
“The production process leaves behind a tangle of different metals,” said ESA research fellow Alexandre Meurisse. “And this is another useful line of research, to see what are the most useful alloys that could be produced from them, and what kind of applications could they be put to.”
A ‘pilot plant’ that could operate sustainably on the Moon is aimed to be demonstrated sometime in the mid-2020s. Its arrival would help various space researchers at the initial stage. However, the ultimate usage would be to let individuals walk on the Moon without getting worried about the lack of oxygen.
“We are working with our colleagues in the Human and Robotics Exploration Directorate, European industry and academia to provide top class scientific approaches and key enabling technologies like this one, towards a sustained human presence on the Moon and maybe one day Mars,” said Tommaso Ghidini, Head of ESA’s Structures, Mechanisms and Materials Division.


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Desperately short of excuses after her apparent refusal to allow Parliament to vote on the deal with the EU regarding Brexit, Theresa May has now put full blame on her twin sister, Meryl.
"No, it wasn't me in the Commons yesterday", puffed an astonished Prime Minister outside her No. 10 door. "Meryl is always up to such pranks. I was varnishing my nails at the time. You should have recognized her because she doesn't wear silly shoes like me. Otherwise she's the spitting image. Certainly fooled Parliament, the rascal!"
Journalists confronted May pointing out that she was in fact an only child.
"Oh no, wrong again. Sounds like fake news. We're monozygotic twins, identical to the last nasal hair. And of course it's too late now. She's ruined it for me. I'll just have to go back to Brussels and all those wonderful cities in Europe again. What a shame. Silly Meryl! Thank you and godbye."
Labour leader Jeremy Corbyn was sceptical. "I must confess, I didn't know that and, well she did take her off extremely well. What a blow for poor Theresa. I guess she did want us to vote, after all. But it's a difficult situation for the British public to handle, isn't it? I mean, they must think they don't know what to believe and what not...Strange".
May's chauffeur hadn't noticed either. "I don't pay attention to be honest. Just open the door and drive. Could be my Aunt Sally for all I know. But she doesn't live in Downing Street, so it's probably not. Hope that helps......"
The plot thickens

domain: politics


text:
By opening a current account and using our domestic payments services, you will be able to:
- Maintain all account types for payment transactions, including trading securities accounts
- Competitive transaction fees for non-cash transactions via eBanking
- Check account balance and turnover
- Receiving account statements on e-mail address
- Online confirmation issuance
- Register Bill of exchange and initiate enforced collection
- Use day-time/night-time safe for daily takings at any time of the day or night, with the possibility of cash limit
- Founding deposits for start-up companies
How do I register a bill of exchange?
To register a bill of exchange, you need to:
- Fill out Bills of exchange registration forms
- Sign the filled in form by electronic signature
- Send your filled in and signed form (along with scanned copies of the bill(s) of exchange you wish to register) to firstname.lastname@example.org
What is my ‘Cash Limit’?
The Cash Limit is the approved ‘indebtedness’ per current account available to you absolutely free of charge, interest and fee-free.
This is a non-credit service provided by the bank and directly linked to your current account, intended for clients who make their daily deposits via day/night safes.
This grants you access to your daily deposit funds even before they are deposited and posted as a paid-in daily deposit.
Once your daily deposit is posted, the amount you withdrew from your Cash Limit is automatically covered
Submit payment order
Deposit or withdraw RSD or EUR amounts
Print or send via e-mail account statements
Print or send via e-mail credit card invoice
Print or send via e-mail repayment plan/deposit payment plan
Deposit daily takings, or cash via deposit safe

domain: economics