text:
I believe that a photograph needs to be printed to come to it’s full right!
Various different printed products are available from the studio, ranging from the traditional printed photograph, to canvas prints and books. Products can be ordered together with your package of choice or individually.
I offer Fine Art Coffee Table Albums and Coffee Table books to my clients. These books and albums have a wheat or light grey coloured linen cover.
THE FINE ART COFFEE TABLE ALBUM
The Fine Art Coffee Table Albums are lay flat albums. These Albums are printed on a luxurious 140 gsm photographic paper and are available in various sizes.
THE SIGNATURE COLLECTION BOX
There is also the famous “Signature Box” which include 10 of your favourite images printed and mounted on white framing board, presented in a beautiful linen box.


text: Find Bathroom Remodelers Near: Lake Geneva Williams Bay Bathroom Remodelers Zenda Bathroom Remodelers Fontana Bathroom Remodelers Genoa City Bathroom Remodelers Walworth Bathroom Remodelers Burlington Bathroom Remodelers Elkhorn Bathroom Remodelers Twin Lakes Bathroom Remodelers Delavan Bathroom Remodelers Sharon Bathroom Remodelers Don't Know Who to Call? Have Bathroom Remodelers Contact You Tell us your need. Local bathroom remodelers will contact you. It's fast, easy and there is absolutely no obligation. Get Started Now Local Results for Bathroom Remodeling Affinity Improvements N1577 Washington AveLake Geneva, WI 53147 Get Quote View Phone Click to View Phone TLC Home Improvements LLC 1238 la Salle StLake Geneva, WI 53147 View Phone Click to View Phone 262-248-0461 Stone Leaf Company 5.5 Miles 185 Third Ave # 113Fontana, WI 53125 Get Quote View Phone Click to View Phone Elite Improvements LLC 10.2 Miles Serving Lake Geneva, WI Get Quote View Phone Click to View Phone 262-716-6663 Southeastern Wisconsin Thielen Construction 10.2 Miles 300 Henry StBurlington, WI 53105 View Phone Click to View Phone 262-208-9221 Don't Know Who to Call? Searching for a qualified bathroom remodeler in your area? Call Us for Assistance: 1-877-759-8938 Return to First-Page Bathroom Remodeling Unfortunately, no results were found for your request. Please try again.

domain: geography


text:
Slip-and-fall lawsuits are difficult to win, but having the right expert can make the difference. With more than 19 years of experience preventing slip, trip and fall accidents, Traction Plus is uniquely qualified to assist you in your case.
Russ Kendzior is the President of Traction Experts, Inc. and founder of the National Floor Safety Institute (NFSI). Internationally recognized as the leading expert in slip-and-fall accident prevention, Mr. Kendzior is a nationally acclaimed lecturer, author and legal consultant.
Author of the best selling book entitled “Slip-and-Fall Prevention Made Easy,” Mr. Kendzior has written a wide range of articles and newsletters for various trade publications and safety journals. Mr. Kendzior has appeared on Inside Edition, ABC News Good Morning America and has been the subject of a PBS documentary on floor safety.
For more information, please contact Russ Kendzior at: email@example.com or visit the Traction Experts website at: http://www.tractionexperts.com
- Slip, Trip & Fall
- Walking & Working Surfaces
References upon request.

domain: law


The past week has brought us some interesting news on the Norfolk Redevelopment and Housing Authority (NR&HA).
First, three developers rented houses in Broad Creek in violation of deed covenants.
Now we learn the NR&HA is in danger of defaulting on the loan it took out for East Beach redevelopment.
Here's the kicker: R&HA advocates in Virginia Beach have pointed to East Beach as a showcase of what a R&HA can do. Beach grassroots activists are now laughing.


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Developing guidelines for professionals to facilitate the best interests of the child during the divorce mediation process
The purpose of the study was to contribute to the improvement of the mediation process and in the process facilitate a better dispensation for children in divorce and a more child-orientated practice. The main aim of this study was to develop substantive guidelines for professionals involved in the divorce mediation process to enable them to apply the best interests of the child principle during the divorce mediation process, as well as the development of parenting plans. The study was conducted within an interpretivist paradigm and a qualitative, descriptive and interpretive design was applied in the study. Data was collected in two consecutive phases. In phase 1, a content analysis of 20 documents, including reports, parenting plans and court orders was conducted. In phase 2, individual interviews were conducted with 12 participants, including lawyers, psychologists and social workers with extensive experience in the field of divorce mediation. The findings from each of these phases were integrated to develop guidelines from a psychological perspective to assist professionals involved in the divorce mediation process in the South African context to adhere to the specifications and ethical standards for parenting plans and to ensure that the best interests of the children involved are served at all times.
- Humanities

domain: psychology


text:
Altynay received a loan from Leelja to buy dairy cows to increase her cattle headcount and sales of organic milk.
Kiva Lending Team: ONE WORLD UNITED
We loan because...
WE ARE ONE FAMILY, ALL COLORS, ALL RACES - ONE WORLD UNITED
Location: PLANET EARTH - ARIZONA
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domain: economics


Apple is set to discontinue iOS upgrade support for the iPhone 5 and iPhone 5C handset devices. Reports suggest that the latest iOS 10 (iOS 10.3.2) will not be supported by devices with less than a 64-bit processor. This includes the iPhone 5 and iPhone 5C in its list. Moreover, the upgrade will only be available for the iPhone 5S and above.
The discontinuation is a bit of an out of character move for Apple due to the fact that the company normally tends to roll out frequent updates to all their latest and older smartphone devices.
In addition, the introduction of a 64-bit exclusive OS is something Apple was always keen on implementing. However, the neglect of older devices could see iPhone users upgrade to a newer device, or even shift to a different smartphone provider completely.
In regards to the iOS 10.3.2 software update, there isn’t anything major or completely innovative. It only introduces new features for its voice AI Siri, and additional minor in-app functionalities.
This also brings to light the fact that 32-bit applications will also soon be discontinued on the App Store, with app developers now focussing on just a 64-bit functioning software. Therefore, users of the iPhone 5 and iPhone 5C, or any other 32-bit processor smartphone device released by Apple, will now not only have an out of date iOS, but will also be limited to older applications, and missing out on the latest application releases on the App Store.


text:
Let's Get your Work Published
So many students, professionals, and aspiring writers harbor hopes of one day publishing a book. Whether you are writing for a conventional academic audience or the broader public, we would be honored to help you make that dream a reality!
Dr. Allen Roda, CEO and Editor in Chief
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If you are looking to self-publish with, e.g., Amazon KDP, we offer the entire suite of typesetting and cover-design services for paperback, hardcover and eBooks.
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When writing my dissertation, I realized that I needed, and wanted, to make my final deliverables the very best representation of me and my work. I employed D|E to review and give suggestions, recommendations, and input to my efforts – and I was not disappointed. D|E’s staff were all very responsive, professional, and qualified.
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Dr. Zelma Talley

domain: literature


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Summer Fashion Styling Courses – My Toronto Student!
It seemed to come and go in a flash but what a week. I feel sometimes I have to pinch myself at how lucky I am to do something I love so much and my one week Personal Styling Diploma course in early August was one of those times. I had one very special lady I want to mention on this course. She came all the way from Toronto to complete my Diploma in Personal Styling to my utter amazement. Carol Takasahi is a legal clerk from Toronto. While travelling in Ireland 5 years ago she stumbled across a Stylefish leaflet about fashion styling courses I was teaching in The Stillorgan Park Hotel and decided while in Dublin to join in!
Carol wasn’t around long enough to complete the course so I ended up sending her course materials and styling ideas in the post to make sure she didn’t miss out! This course was purely for Carol’s enjoyment and for her own style. Since then (2009) Carol has been keeping in touch with Stylefish and me through my newsletter and to my utter amazement called me as soon as she read about my one week Diploma course to sign up! “Are you sure?” was my first response! Through my class Carol had discovered a love of fashion and a passion for wanting to help others with their style. Now, having completed her course is back in Toronto and is going back to work part-time in order to set up her business as a personal stylist and coach.
I am honoured Stylefish has been and is able to inspire and mentor her towards doing something she loves and exploring her love of fashion in Canada! There are moments in life you realise why you do what you do and this is certainly one of those moments for me. Being able to help someone else do something they love is very special
I also don’t want to forget the other fabulous lady in this group! Susan Buckley came from Mallow in Cork to complete my Diploma course. Another lady with a passion for fashion, Susan has worked in HR in a bank for 12 years before leaving in February of this year to follow other passions including fashion! I want to welcome both Susan and Carol to the Stylefish community and am delighted Stylefish has been part of their fashion journey!
Summer Fashion Styling Course Moments!
1. Beautiful course surroundings at 34 Upper Fitzwilliam Street in the heart of georgian Dublin (and a bit too close to Topshop!)
2. Running first mock body shape assessments together before hitting the shops!
3. First experience choosing looks for various body shapes on the shop floor. This picture was taken with both Carol and Susan talking through the looks they picked in the personal shopping suite in House of Fraser.
4. Understanding how colour theory works, how certain colours work well for women and others make us look tired and how to know the difference. We looked at using colour to help women get more creative with their styling, make-up and hair colouring and how to build a great wardrobe with the right mix of neutrals and colour.
5. Susan and Carol styling their first real clients on the final day of their training in Dundrum Town Centre. They had two very different ladies and age groups and they had a lot of fun choosing looks to suit their clients age, budget, needs, shape and personality!
Happy styling ladies!
Click here to view a little video I made with Carol on our final day of training in House of Fraser.

domain: artistic


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The public are demanding that the government puts a halt on fuel prices following a public outcry in relation to the increasing rise of fuel at the pump.
An e-petition signed by more than 100,000 people was flagged up by MP Robert Halfon and supported by more than 100 other MPs.
Plans to increase fuel duty by 3p per litre were put in place by the government for January 2012, but the effect of this rise will see motorists paying an extra £1.50 to fill up the average car.
With petrol prices tripling in price in the past 20 years, it is becoming more and more difficult for motorists to afford to run their vehicles and many are turning to public transport as a cheaper and more efficient way to travel.
Although Chancellor George Orsbourne cut the annual fuel tax escalator whereby fuel duty rose by 1p above inflation each year, he only postponed the duty rise from April 2011 to January 2012 and from April 2012 to August 2012, meaning that a rise in imminent in January 2012.
MP, Mr Halfon has made it clear that high fuel prices are causing “immense difficulties” for small and medium sized businesses, referring to those lower paid workers who are currently paying a tenth of their income just to fill up their cars.
The motion put forward by Mr Halfon urges ministers to look at the impact of fuel tax rates on economic growth and unemployment levels, and suggests the government puts a price stabilisation mechanism in place which will even out fluctuations in fuel prices.
The AA has already emphasised how fuel prices are effecting the public, revealing that the average motorist will spend an extra £38 at the pump if the increased fuel tax goes ahead.
AA president Edmund King told BBC Radio 5Live that price increases were “socially divisive” and said that the nation was being divided into “drives” – people who can afford to drive – and “drive nots”, those that can’t.
Mr King went on to describe how motoring is an essential way of life for some people on low incomes who live in rural areas and need a car to simply get to work.
Fairfuel UK, a pressure group behind the e-petition presented to Parliament have expressed the need for “the whole fuel pricing issue to become open and transparent”, and have added that high fuel duty is “strangling the economy”.
Motoring journalist, Quentin Wilson speaking on behalf of Fairfuel UK said: “There are desperate, desperate people who cannot afford to use the roads. The effect on society at the moment has been absolutely desperate. Fuel duty is strangling us”.
The Treasury has already said it will introduce a “fair fuel stabiliser” to ensure price rises are capped to inflation when oil prices are high, with oil firms filling the gap by paying extra tax.
News of the government’s decision on the 3p tax rise will be revealed later today.


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Stock Exchange China move fails
A PLAN by the London Stock Exchange (LSE) to speed the dual-listing of Chinese companies through a tie-up with the Hong Kong market has fallen through, damaging its much vaunted push into Asia.
The failure of the long-running talks with Hong Kong Exchanges and Clearing (HKEx) is a potential embarrassment just days ahead of the first trip by Clara Furse to Hong Kong since she became chief executive in 2001.
Furse - seeking to drum up Asian interest in the LSE while the New York Stock Exchange grapples with the fallout from the Grasso crisis - arrives in the former British colony next Monday, the first of two trips to China for her this autumn.
Lorraine Chan, spokesman for HKEx, said: 'HKEx is not currently working on rule amendments to facilitate dual-listing China enterprises in Hong Kong and London.'
Asked whether talks on the LSE tieup were effectively dead, Chan said: 'You may take that as your understanding.'
Had the proposed scheme been successful, companies that cleared regulatory hurdles to list in Hong Kong would have automatically qualified for a dual listing on the LSE.
The potential link had been talked up by LSE officials, who saw it as a strong entry point to the booming Chinese economy, the fastest-growing in Asia. Many companies from mainland China already opt to list shares in Hong Kong, although the Shanghai and Shenzhen exchanges are also popular.
A spokesman for the LSE confirmed the details of Furse's Hong Kong trip - including a meeting with her HKEx counterpart Paul Chow - but declined to comment on the status of the fast-track discussions.
At present there are five Chinese companies listed on the LSE out of 445 international groups: Beijing Datang Power Generation, China Petroleum & Chemical Corp, Jiangxi Copper, Zhejiang Expressway, and Zhejiang Southeast Electric Power.
Attracting dual-listings from global firms is a major area of competition for the world's premier exchanges, which view them as key area of growth.
Across the Atlantic, America's largest fund manager, Fidelity Investments, is calling on the New York Stock Exchange to replace its trading floor and the time-honoured method of face-to-face buying and selling with the sort of electronic trading system that has been operating in the City for nearly 20 years.
The demand puts renewed pressure on the NYSE - the only major stock exchange in the world still with a trading floor.
Three top Fidelity officials have put their case to John Reed, the exchange's interim chairman. Scott DeSano, head of Fidelity's global equity trading, issued a thinly veiled accusation that the NYSE's floor traders responsible for making markets bent the rules to profit from trading on their own behalf at the expense of investors.
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domain: news


text:
This morning on KIRO/Seattle, Christine Brennan joined me live from Beijing, where she’s covering the Summer Olympics for USA Today (read her blog).
She told a great story about Dara Torres, the 41-year-old mother of two who is competing in the pool at her fifth Olympics (Christine first met her as a 17-year-old at the 1984 games). We also talked about Michael Phelps’ chances to break Mark Spitz’s record by winning 8 gold medals, and the controversies surrounding the US swimming team in the wake of Jessica Hardy’s disqualification for drugs.
I also asked Christine about the pollution in Beijing and the masks some athletes are wearing to deal with it (the USOC has offered them to everyone on the team, but USA Today apparently hasn’t), and whether she thinks we’ll see any of them wearing the masks at the opening ceremonies on Friday.
As I’ve done before when Christine has been on my show, I’ll recommend her book, “Best Seat In The House,” about growing up with sports as a bond with her father, which led to her becoming one of the country’s top sportswriters. It’s a terrific read and shows why she’s a role model to young women like my daughter.


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Ryanair is to open its 11th base in Germany, with a facility opening at Düsseldorf Airport in June.
The Western German city will be its 86th base in Europe, and will be home to one aircraft, as two new summer routes are launched to Alicante and Malaga.
The routes are in addition to the daily service to Palma which was announced earlier this year and the 12 routes operated on behalf of Laudamotion, Ryanair Chief Marketing Officer Kenny Jacobs said.
The move is the latest challenge by Ryanair to Lufthansa which last year overtook it as Europe’s largest airline group by passenger numbers. Lufthansa’s Eurowings subsidiary is the market leader at Dusseldorf.
Ryanair sees huge potential in Europe’s biggest economy, where low-cost airlines typically account for just 10-20pc of traffic compared to over 50pc in Europe as a whole.


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NEW! B.Sc. Program in Chemical Technology
ONLY for Polish and EU candidates !!!
B.Sc. program in Chemical Technology (Faculty of Chemical Technology), duration: 3,5 years
(7 semesters), 210 ECTS points
The Chemical Technology study course was created for people, who wish to become a bridge between chemists and engineers, combining their skills with an interdisciplinary perspective on industrial processes. The particular advantage of starting the Chemical Technology study course is the unique mixture of theoretical knowledge with laboratory and technological practices.

domain: chemistry


text:
Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.
24 Related JoVE Articles!
Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Institutions: Yale University, Yale University, Yale University, Yale University, Massachusetts General Hospital, University of California, Irvine.
We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7
. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8
that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11
C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7
to a conventional model 9
. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.
Behavior, Issue 78, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Image Processing, Computer-Assisted, Receptors, Dopamine, Dopamine, Functional Neuroimaging, Binding, Competitive, mathematical modeling (systems analysis), Neurotransmission, transient, dopamine release, PET, modeling, linear, time-invariant, smoking, F-test, ventral-striatum, clinical techniques
Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Institutions: IRCCS - Istituto di Ricerche Farmacologiche Mario Negri.
After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.
Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 μm z-axis and a 0.23 μm step size, corresponding to a 180 x 135 x 10 μm volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.
Neurobiology, Issue 79, Neuroscience, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Anatomy, Physiology, Central Nervous System Diseases, Neurodegenerative Diseases, biology (general), immunology, life sciences, animal models, Inflammation, stroke, alternative activation, brain injury, brain, imaging, confocal microscopy, three-dimensional imaging, clinical techniques, mouse, animal model
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
Handwriting Analysis Indicates Spontaneous Dyskinesias in Neuroleptic Naïve Adolescents at High Risk for Psychosis
Institutions: University of Colorado Boulder, NeuroScript LLC, University of California, San Diego.
Growing evidence suggests that movement abnormalities are a core feature of psychosis. One marker of movement abnormality, dyskinesia, is a result of impaired neuromodulation of dopamine in fronto-striatal pathways. The traditional methods for identifying movement abnormalities include observer-based reports and force stability gauges. The drawbacks of these methods are long training times for raters, experimenter bias, large site differences in instrumental apparatus, and suboptimal reliability. Taking these drawbacks into account has guided the development of better standardized and more efficient procedures to examine movement abnormalities through handwriting analysis software and tablet. Individuals at risk for psychosis showed significantly more dysfluent pen movements (a proximal measure for dyskinesia) in a handwriting task. Handwriting kinematics offers a great advance over previous methods of assessing dyskinesia, which could clearly be beneficial for understanding the etiology of psychosis.
Behavior, Issue 81, Schizophrenia, Disorders with Psychotic Features, Psychology, Clinical, Psychopathology, behavioral sciences, Movement abnormalities, Ultra High Risk, psychosis, handwriting, computer tablet, dyskinesia
Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans
Institutions: Harvard Medical School, McLean Hospital.
is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans
genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2
. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.
Neuroscience, Issue 84, antipsychotic drug, Caenorhabditis elegans, clozapine, developmental delay, lethality, nematode, pharmacogenetics, pharyngeal pumping, schizophrenia
Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice
Institutions: University of Münster, Interdisciplinary Center for Clinical Research (IZKF), Münster, University of Münster.
Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55
is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.
Immunology, Issue 86, experimental autoimmune encephalomyelitis, EAE, multiple sclerosis, MS, animal model, Autoimmunity, neuroinflammation, central nervous system, pertussis
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
Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity
Institutions: Alpert Medical School, Brown University, University of Georgia.
Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of psychiatric illness severity and provide added validity to the clinical diagnostic process. Recent neuroimaging research suggests that DMN processes may be disrupted in a number of stress-related psychiatric illnesses, such as posttraumatic stress disorder (PTSD).
Although specific DMN functions remain under investigation, it is generally thought to be involved in introspection and self-processing. In healthy individuals it exhibits greatest activity during periods of rest, with less activity, observed as deactivation, during cognitive tasks, e.g.
, working memory. This network consists of the medial prefrontal cortex, posterior cingulate cortex/precuneus, lateral parietal cortices and medial temporal regions.
Multiple functional and structural imaging approaches have been developed to study the DMN. These have unprecedented potential to further the understanding of the function and dysfunction of this network. Functional approaches, such as the evaluation of resting state connectivity and task-induced deactivation, have excellent potential to identify targeted neurocognitive and neuroaffective (functional) diagnostic markers and may indicate illness severity and prognosis with increased accuracy or specificity. Structural approaches, such as evaluation of morphometry and connectivity, may provide unique markers of etiology and long-term outcomes. Combined, functional and structural methods provide strong multimodal, complementary and synergistic approaches to develop valid DMN-based imaging phenotypes in stress-related psychiatric conditions. This protocol aims to integrate these methods to investigate DMN structure and function in PTSD, relating findings to illness severity and relevant clinical factors.
Medicine, Issue 89, default mode network, neuroimaging, functional magnetic resonance imaging, diffusion tensor imaging, structural connectivity, functional connectivity, posttraumatic stress disorder
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g.
, signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.
The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development
Institutions: University of New Mexico Health Sciences Center.
Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.
Medicine, Issue 89, fetal, ethanol, exposure, paradigm, vapor, development, alcoholism, teratogenic, animal, mouse, 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
P50 Sensory Gating in Infants
Institutions: University of Colorado School of Medicine, Colorado State University.
Attentional deficits are common in a variety of neuropsychiatric disorders including attention deficit-hyperactivity disorder, autism, bipolar mood disorder, and schizophrenia. There has been increasing interest in the neurodevelopmental components of these attentional deficits; neurodevelopmental meaning that while the deficits become clinically prominent in childhood or adulthood, the deficits are the results of problems in brain development that begin in infancy or even prenatally. Despite this interest, there are few methods for assessing attention very early in infancy. This report focuses on one method, infant auditory P50 sensory gating.
Attention has several components. One of the earliest components of attention, termed sensory gating, allows the brain to tune out repetitive, noninformative sensory information. Auditory P50 sensory gating refers to one task designed to measure sensory gating using changes in EEG. When identical auditory stimuli are presented 500 ms apart, the evoked response (change in the EEG associated with the processing of the click) to the second stimulus is generally reduced relative to the response to the first stimulus (i.e.
the response is "gated"). When response to the second stimulus is not reduced, this is considered a poor sensory gating, is reflective of impaired cerebral inhibition, and is correlated with attentional deficits.
Because the auditory P50 sensory gating task is passive, it is of potential utility in the study of young infants and may provide a window into the developmental time course of attentional deficits in a variety of neuropsychiatric disorders. The goal of this presentation is to describe the methodology for assessing infant auditory P50 sensory gating, a methodology adapted from those used in studies of adult populations.
Behavior, Issue 82, Child Development, Psychophysiology, Attention Deficit and Disruptive Behavior Disorders, Evoked Potentials, Auditory, auditory evoked potential, sensory gating, infant, attention, electrophysiology, infants, sensory gating, endophenotype, attention, P50
Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices
Institutions: Purdue University.
Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis
oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice 1
and α6 L9′S mice 2
, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.
Neuroscience, Issue 68, Nicotinic, acetylcholine, neurotransmitter, neuron, patch clamp, brain slice, picospritzer
An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Institutions: University of Oxford.
The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3
. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4
. The primer extension assay is carried out using the MassARRAY (Sequenom)5
platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.
Cellular Biology, Issue 45, Gene expression, regulatory variant, haplotype, association study, primer extension, MALDI-TOF mass spectrometry, single nucleotide polymorphism, allele-specific
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
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
Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats
Institutions: University of New South Wales .
Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2
. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3
). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2
), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4
. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al
(1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6
. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3
). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.
Medicine, Issue 61, Neuropathic pain, sciatic nerve, chronic constriction injury, pain hypersensitivity
Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration
Institutions: University of Victoria .
Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1
. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3
. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6
Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons.
Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7
.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10
. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using Förster Resonance Energy Transfer (FRET) in transfected cultured cells11
.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged α4 nAChR subunits (α4YFP), enabling precise quantification of the receptor ex vivo
at submicrometer resolution in CNS neurons via spectral confocal microscopy12
. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.
In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from α4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain α4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of α4YFP receptors in the medial perforant path of the hippocampus.
Neuroscience, Issue 60, nicotine addiction, knock-in mice, spectral confocal imaging, yellow fluorescent protein, nicotinic acetylcholine receptors
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
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
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
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research
Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment
Institutions: Cornell University.
Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.
Plant Biology, Issue 15, Annual Review, Nicotine, Aphids, Plant Feeding Resistance, Tobacco

domain: medical


text:
The Ineseño language was traditionally spoken along middle part of the Santa Ynez River. During the mission period, the language was spoken at Mission Santa Inés. Based on archaeological evidence and the testimony of early observers, it has been estimated that speakers of all Chumashan languages together numbered between 10,700 and 17,250 in pre-contact times (King 1969). Today, there are no first-language speakers of any Chumashan language.
Selected archival materials at Berkeley
- Applegate, Richard B. 1972. Ineseño Chumash grammar. Ph.D. dissertation, University of California, Berkeley.
- Heizer R. F., ed. 1952. California Indian linguistic records: The Mission Indian vocabularies of Alphonse Pinart. University of California Anthropological Records 15:1-84. [PDF]
- Heizer, R.F., ed. 1955. California Indian linguistic records: The Mission Indian vocabularies of H. W. Henshaw. University of California Anthropological Records 15:85-202. [PDF]
- Santa Ynez Band of Chumash Indians. 2007. Samala-English dictionary: A guide to the Samala language of the Ineseño Chumash people. Santa Ynez, CA: Santa Ynez Band of Chumash Indians.

domain: history


text:
Big Ernie's New Home: A Story For Children Who Are Moving
ISBN : 9781591473824
Availability: Only 5 Left In-stock.
Domestic Shipping: $3.99
Internation Shipping (Except United states): $16
Shipping & Tax will be calculated at Checkout.
Estimated delivery time 7-14 days.
International delivery time 2 to 4 weeks.
Big Ernie's New Home: A Story for Children Who Are Moving
In this story, Big Ernie learns to deal with being worried, mad, and a little sad about being uprooted and moved, and he begins to feel at home in his new house.
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domain: library


- Is ps5 still coming out in 2020?
- Will ps5 have better graphics?
- What does the ps5 do?
- How can I get ps5?
- Will ps5 have its own games?
- Can I play ps3 games on ps5?
- Will ps5 have discs?
- What games are going to be on ps5?
- Is ps5 8k?
- Will ps4 controllers work on ps5?
- Are Consoles dying?
- Is ps5 a controller?
- How much will ps5 games be?
- What does the ps5 look like?
- Will there be ps6?
Is ps5 still coming out in 2020?
PlayStation 5 release date While a specific date is yet to be revealed, Sony has confirmed that the PS5 release date is Holiday 2020, setting up a battle royale between the PS5 and new Xbox Series X for Christmas 2020.
Most recently, a retail source told VGC that Sony could release the PS5 on 13th November..
Will ps5 have better graphics?
The PS5 and Xbox Series X Won’t Look That Much Better Than the PS4 and Xbox One. Back in the early days of gaming, graphical improvements were massive. … While the PS5 and Xbox Series X might not look better graphically, that doesn’t mean things, in general, won’t improve.
What does the ps5 do?
The PS5 supports up to 8K output and promises 4K graphics at up to a 120Hz refresh rate. It will also support 3D audio, which Sony said will let you “see with sound” in a commercial shared in August.
How can I get ps5?
Where to Buy a PS5PS5 at Amazon – $499.99.PS5 Digital Edition at Amazon – $399.99.PS5 at Best Buy – $499.99.PS5 Digital Edition at Best Buy – $399.99.PS5 at GameStop – $499.99.PS5 Digital at GameStop – $399.99.PS5 at Walmart – $499.99.PS5 Digital Edition at Walmart – $399.99.More items…•
Will ps5 have its own games?
Other key games arriving in the PS5’s launch window include Assassin’s Creed Valhalla, Mortal Kombat 11 Ultimate, Dirt 5 and NBA 2K21. … While you don’t necessarily need a PS5 to play some of the biggest new games from both Sony and third parties, you will enjoy much better versions of them on the new system.
Can I play ps3 games on ps5?
PS5 is not backwards compatible with PS3 games, although it is backwards compatible with PS4 games, meaning some remasters are playable on PlayStation 5. … It’s worth noting that PS3 Blu-ray discs and PS3 PS Store purchases will not be compatible with the PS5, however.
Will ps5 have discs?
From what Sony has announced, only the regular PS5 will be able to play discs. As opposed to its slightly slimmer digital counterpart, the PS5 does have a disc drive for games, 4K Blu-ray, and DVDs.
What games are going to be on ps5?
All PlayStation 5 launch games on releaseAstro’s Playroom (coming pre-loaded on PS5)Bugsnax.Demon’s Souls.Fortnite.Devil May Cry 5: Special Edition.Dirt 5.Godfall.Marvel’s Spider-Man: Miles Morales.More items…•
Is ps5 8k?
PS5 and Xbox Series X can game in 8K resolution.
Will ps4 controllers work on ps5?
Sony has confirmed that PS4 controllers will not work with PS5 games. However, DualShock 4 will work with PlayStation 4 games via backwards compatibility, it said in a blog post. Speciality peripherals, such as officially licensed racing wheels, arcade sticks, and flight sticks, will work with PS5 games.
Are Consoles dying?
Video game consoles don’t appear to be dying in 2018, with the PlayStation 4 and Xbox One combining to sell more than 100 million systems and the Switch quickly racking up a nice user-base of its own. According to Ubisoft CEO Yves Guillemot, however, dedicated game consoles may not be long for this world.
Is ps5 a controller?
The DualSense PS5 controller is the new gamepad we’ll be using with Sony’s PlayStation 5 console, when it releases in late 2020. First revealed earlier this year, the DualSense is a break in tradition for Sony, dropping the old DualShock name while sporting a futuristic two-tone design and curved shape.
How much will ps5 games be?
Not only will PS5 controllers be getting a price hike to coincide with the fact PS4 controllers aren’t compatible with the PS5, but Sony is dropping a price hike on its games, jumping from $60 to $70 in the U.S., and often even more in Europe, the UK, and other markets.
What does the ps5 look like?
What will the PS5 look like? The PS5 design is a bold step away from the traditional black box, with both the digital-only and disc drive versions boasting a white and black color scheme and serious curves.
Will there be ps6?
Get your pre-orders ready because the PS6 could be out as early as 2026, with a PS5 Pro model perhaps as early as 2023. By this time next year the PlayStation 5 should be out, but despite some rumours to the contrary there’ll only be one model, with Sony saying it has no plans for a PS5 Pro until a few years later.


High production, fully loaded system
The EcoQuip 2 EQs Elite is built for contractors that want the best.
Equipped with a stainless steel crash frame and enclosure, hose rack, step, media strainer, water dose valve and a media shelf, this fully-loaded system makes it easy to get setup faster and start making money.
• Fits inside a 6 ft truck bed for easy transport
• Built to handle the toughest work conditions
• Crash frame supports 5x lift rating of machine
• 6.5 cubic ft (184 L) pot – holds approximately (16) 55 lb bags of media
Expand your business
With EcoQuip 2, contractors are no longer limited to just one or two types of jobs. Concrete repair, coating removal, steel surface preparation and graffiti removal are all potential applications of EcoQuip 2. The versatility of EcoQuip 2 is one of the characteristics that makes this equipment so remarkable. The best part is how easy it is to switch from one application to another. EcoQuip 2 is designed to accept any media heavier than water and has a blast rating of up to 175 psi.
– 50% faster start-up*
– 50% faster media reload process from empty*
– Faster production rates than previous Graco models and equipment with similar technology
– Set-up EcoQuip 2 with just five simple steps
– No longer need to set pot pressure relative to blast pressure
– Easily adjust blast pressure and use media valve to regulate media
– Every unit is tested on state-of-the art test bench before it ships
– Unmatched customer and technical support to assist with equipment needs
– Components tested to the toughest Graco standards – Made in U.S.A.
Built with you in mind
The Graco EcoQuip 2 Vapor Abrasive Blast Equipment is engineered with the kind of features that will make your work day more efficient and productive. It’s exhausively tested and re-tested to make sure every detail works to Graco’s industry leading standards of excellence.


text:
This decision signifies a critical advancement in Japan's pursuit to augment its military preparedness in the face of shifting global security threats.
The U.S. Defense Security Cooperation Agency underscored the strategic value of the sale in a recent proclamation. "The proposed sale will improve Japan’s capability to meet current and future threats by providing a long-range, conventional surface-to-surface missile with significant standoff range that can neutralize growing threats. Japan will have no difficulty absorbing these articles into its armed forces," the agency declared.
In a press briefing, Japanese Defense Minister Minoru Kihara illuminated the importance of the deal, stating that the procurement of the missiles "will contribute to strengthening stand-off defense capabilities of our country."
Produced by Raytheon Technologies, these advanced missiles are equipped to strike targets up to 1,000 miles away. They form a crucial part of the U.S. Navy's arsenal and can be deployed from Aegis destroyers operated by the Japanese Maritime Self-Defense Force.
As reported by Stars and Stripes, Japan's defense expenditure for this fiscal year stands at $51.4 billion, indicating the nation's emphasis on bolstering its military prowess. The Tomahawk missile acquisition, initially slated for 2026, has been brought forward to 2025 in response to what Japan's Defense Ministry characterizes as an increasingly severe regional security environment.
The anticipated purchase of the 400 Tomahawk missiles occurs amidst regional tensions. A 2022 Pentagon report reveals that China possesses over 1,900 ballistic missiles capable of reaching Japan. Additionally, North Korea's recent escalation in ballistic missile testing, with 21 missiles launched over 14 days of testing, intensifies the need to enhance Japan's defensive capabilities, according to Stars and Stripes.
However, the Tomahawk sale has elicited criticism from North Korea, with a defense ministry official cautioning about increased regional tensions and potential escalation of an arms race. The official, as reported by Japanese public broadcaster NHK, branded the missiles as "lethal war equipment" and pledged to bolster North Korea's deterrence in response.
Japan's resolution to proceed with the Tomahawk missile purchase signifies a strategic maneuver to safeguard its national defense amidst an increasingly intricate geopolitical landscape, thereby reinforcing its role as a crucial actor in upholding regional stability.


By Anthony Caruso III | Publisher
The Golden State Warriors had one of its stars back in the lineup on Saturday night. With Stephen Curry back, the Warriors rolled 141-128 over the Memphis Grizzlies at Oracle Arena in Oakland, California.
The Warriors improved to 29-8 on the season. Golden State is now on a one-game win streak after losing to the New Orleans Pelicans on Friday night.
The Grizzlies fell to 11-25 on the season. They are on a one-game losing streak after defeating the Los Angeles Lakers on Friday night.
Stephen Curry, who missed nearly a month with an ankle injury, had a game-high 38 points. He also had four rebounds and three assists.
Curry recorded his ninth career game with 10 three-pointers. No other player in NBA history has more than three games, where they made 10 or more three-pointers.
The Warriors, who were 9-2 without Curry during his absence, had four of its five starters in double-figures. Rookie Jordan Bell also scored in double-figures, as he had 11 points off the bench.
The Big Three, which includes Curry, Klay Thompson, and Kevin Durant, combined for 79 points.
The Warriors had 42 points off the bench.
The Grizzlies were led by Marc Gasol, who had 27 points. He also had six rebounds and an assist.
Gasol was one of three starters in double-figures. They also had three players off the bench, who scored in double-figures, including 17 points from Kobi Simmons.
The Grizzlies had 57 points off the bench.
Any Corrections?. You can contact Anthony Caruso III, Publisher at firstname.lastname@example.org.
©2007-2019 The Capital Sports Report. Please honor copyright! Piracy hurts writers, devalues their works, and puts you and your employer at risk of lawsuits. All original materials contained on this website are protected by the United States copyright law and may not be reproduced, distributed, transmitted, displayed, published or broadcasted without the prior written permission.


text:
Saw this cool idea on Twitter. He is using a diode laser to selectively etched off layers of paint on a tile. The tile is white and he added 3 layers of spray paint; black, rose gold and green. He then varied the power/speed to selectively etch off the layers.
Not sure if a CO2 laser would be able to do this because of the higher power.

domain: design


Silicon carbide (SiC) is generally used in a range of fractions as a grinding and blasting abrasive, and for the manufacture of ceramic materials. Exposure of employees to SiC can be anticipated during the manufacture of SiC itself, during its use for the manufacture of various products, and in various applications of the material. No recognized method has been available to date for measurement of workplace air SiC concentration. A method was to be developed by means of which the SiC concentration can be measured in combination with fine dust sampling on membrane filters.
X-ray diffraction was employed for measurement of the silicon carbide. Analyses were performed with thin surface samples in order to minimize the influence of different mass absorption coefficients of other sample components. A suitable quantity of dust sample was charged for this purpose on a silver membrane filter, which is used for powder diffraction analysis. SiC is a polytypic substance, which may occur in different crystal structures or structure variations with the same chemical composition. This had to be taken into consideration in the selection of suitable standards. For this purpose, different SiC products were examined by means of x-ray diffraction for differences in the characteristic x-ray reflections. Calibration series were produced by means of representative SiC samples. Test measurements were performed in the development phase on samples obtained from practical applications in order to identify disturbance factors or cross-sensitivities.
X-ray diffraction is a suitable method for the measurement of SiC in respirable dusts. Cellulose membrane filters are employed for sampling. Following measurement of the respirable dust concentration, the samples can be prepared in the same way for quartz and SiC measurements, permitting measurement in parallel. A selection of industrial SiC materials should be available in respirable form for calibration purposes, since analysis of different SiC variants can be expected to yield differences in the position and intensity of the peaks in the x-ray diffractogram owing to the polytypic properties of SiC. For SiC in typical industrial form, the absolute detection limit is 50 µg. For two-hour sampling with a personal sampler (volumetric flow of 10 l/min), this results in a relative detection limit of 0.04 mg/m³. A material sample should be supplied of the SiC employed in the working area. Should the diffractogram of this material show it to differ in terms of the polytypic variants which it contains, the SiC standard provided should be used in place of the standard calibration. The method has been employed as a standard method in the BGMG hazardous substance measurement system since 15 June 2004.
-cross sectoral-Type of hazard:
Gefahrstoffe, Arbeitsbedingte GesundheitsgefahrenCatchwords:
Messverfahren, Chemische Arbeitsstoffe, ExpositionDescription, key words:
Silicon carbide (SiC), abrasive, abrasive agent, measurement method, respirable dust fraction


text:
PixWords APK 2.41 Free Download
Updated: August 8, 2018
You are about to download the PixWords 2.41 apk file for Android 4.0.3 and up: The new wonderfully interesting word puzzle challenge ...
Please note that the PixWords apk file v2.41 is the original apk file obtained from the official Google Play server. It DOES NOT contain any mod, cheat, crack or unlimited gold patch. You could now read more about PixWords app, review app permissions or choose a server to download it.
PixWords version 2.41 release notes >
- Various minor bug fixes and improvements
Check out apk file info before download >
Download PixWords apk 2.41 for Android 4.0.3 and up >
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After you get the PixWords 2.41 apk file (com.dekovir.pixwords-2.41-marcosvisoilustracion.com.apk), move it to your Android phone's SD card and then your file manager to browse, install & enjoy it.
Google Play and the Google Play logo are trademarks of Google Inc. PixWords is the trademark of the developer Black Maple Games. All apk files on APK4Fun are only for home or personal use.
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domain: hobby